Activity of 2-aryl-2-(3-indolyl)acetohydroxamates against drug-resistant cancer cells

Article abstract of DOI:10.1021/jm501518y

Many types of tumor, including glioma, melanoma, non-small cell lung, esophageal, and head and neck cancer, among others, are intrinsically resistant to apoptosis induction and poorly responsive to current therapies with proapoptotic agents. In addition, tumors often develop multidrug resistance based on the cellular efflux of chemotherapeutic agents. Thus, novel anticancer agents capable of overcoming these intrinsic or developed tumor resistance mechanisms are urgently needed. We describe a series of 2-aryl-2-(3-indolyl)acetohydroxamic acids that are active against apoptosis-and multidrug-resistant cancer cells as well as glioblastoma neurosphere stemlike cell cultures derived from patients. Thus, the described compounds serve as a novel chemical scaffold for the development of potentially highly effective clinical cancer drugs.

Full text of DOI:10.1021/jm501518y

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Article
Activity of 2-Aryl-2-(3-indolyl)acetohydroxamates
Against Drug-Resistant Cancer Cells
Alexander V. Aksenov, Alexander Smirnov, Igor V. Magedov, Mary Reisenauer, Nicolai
Aksenov, Inna Aksenova, Alexander Pendleton, Gina Nguyen, Robert Johnston, Michael Rubin,
Annelise De Carvalho, Robert Kiss, Véronique Mathieu, Florence Lefranc, Jaime Correa, David
Cavazos, Andrew Brenner, Brad Bryan, Snezna Rogelj, Alexander Kornienko, and Liliya Frolova
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Feb 2015
Downloaded from http://pubs.acs.org on February 12, 2015
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Activity of 2-Aryl-2-(3-indolyl)acetohydroxamates Against
Drug-Resistant Cancer Cells
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†
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Alexander V. Aksenov, Alexander N. Smirnov, Igor V. Magedov, Mary R.
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Reisenauer, Nicolai A. Aksenov, Inna V. Aksenova, Alexander L. Pendleton, Gina
‡
‡
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Nguyen , Robert K. Johnston, Michael Rubin, Annelise De Carvalho, Robert Kiss,
¶
∞
∆
#
Véronique Mathieu, Florence Lefranc, Jaime Correa, David A. Cavazos, Andrew J.
#
£
‡,*
∆,*
Brenner, Brad A. Bryan, Snezna Rogelj, Alexander Kornienko, and Liliya V.
‡
,*
Frolova
Department of Chemistry, North Caucasus Federal University, 1a Pushkin St., Stavropol 355009,
Russian Federation; Departments of Chemistry and Biology, New Mexico Institute of Mining and
Technology, Socorro, NM 87801, USA; Department of Chemistry, University of Kansas, 1251 Wescoe
Hall Dr., Lawrence, KS 66045, USA; Laboratoire de Cancérologie et de Toxicologie Expérimentale,
Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, CP205/1,
Boulevard du Triomphe, Brussels, Belgium; Service de Neurochirurgie, Hôpital Erasme, ULB, 808
route de Lennik, 1070 Brussels, Belgium ; Department of Chemistry and Biochemistry, Texas State
University, San Marcos, TX 78666, USA; Cancer Therapy and Research Center, The University of
Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229, USA;
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Department of Biomedical Sciences, Texas Tech University Health Sciences Center, Center of
Excellence in Cancer Research, Paul Foster School of Medicine, 5001 El Paso Drive, El Paso,
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Université Libre de Bruxelles
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Texas State University
#
University of Texas Health Science Center at San Antonio
£
Texas Tech University
Abstract: Many types of tumor, including glioma, melanoma, non-small cell lung, esophageal, head
and neck cancer, among others, are intrinsically resistant to apoptosis induction and poorly responsive
to current therapies with proapoptotic agents. In addition, tumors often develop multi-drug resistance
based on the cellular efflux of chemotherapeutic agents. Thus, novel anticancer agents capable of
overcoming these intrinsic or developed tumor resistance mechanisms are urgently needed. We
describe a series of 2-aryl-2-(3-indolyl)acetohydroxamic acids, which are active against apoptosis- and
multidrug-resistant cancer cells as well as glioblastoma neurosphere stem-like cell cultures derived
from patients. Thus, the described compounds serve as a novel chemical scaffold for the development
of potentially highly effective clinical cancer drugs.
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Introduction
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Apoptosis-resistant cancers represent a major challenge in the clinic as most of the currently
available chemotherapeutic agents work through the induction of apoptosis and, therefore, provide
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limited therapeutic benefits for the patients affected by these malignancies. Cancers with such
intrinsic resistance to proapoptotic stimuli include the tumors of the lung, liver, stomach, esophagus,
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pancreas as well as melanomas and gliomas. For example, patients afflicted by a type of gliomas,
4,5
known as glioblastoma multiforme, have a median survival expectancy of less than 14 months when
treated with a standard protocol of surgical resection, radiotherapy and chemotherapy with
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temozolomide, carmustine or cisplatin. Because glioma cells display resistance to apoptosis, they
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,7
respond poorly to such conventional chemotherapy with proapoptotic agents.
Resistance to apoptosis is also an intrinsic property of tumor metastases. Successful treatment of
metastases remains an important clinical challenge as 90% of cancer patients die from metastastic
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cancer spread. By acquiring resistance to anoikis, a cell death process resulting from the loss of
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contact with extracellular matrix or neighboring cells, metastatic cells display poor sensitivity to
apoptosis induction and are thus poorly responsive to conventional proapoptotic chemotherapeutic
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,9,10
agents.
One solution to apoptosis resistance entails the complementation of cytotoxic therapeutic
regimens with cytostatic agents and thus a search for novel cytostatic anticancer drugs that can
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overcome cancer cell resistance to apoptosis is an important pursuit.
Often, tumors are initially susceptible to cancer agents and patients respond to chemotherapy but
eventually experience a relapse in spite of the continuing treatment. In such instances of acquired
resistance tumors generally become refractory to a broad spectrum of structurally and mechanistically
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diverse antitumor agents and this phenomenon is referred to as multidrug resistance (MDR).
MDR
usually results from upregulation of certain protein pumps, such as P-glycoprotein (P-gp) in cancer
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cells, causing a decreased intracellular drug concentration. MDR is a major factor that contributes to
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alkaloids or the taxanes.
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Our recent studies of a reaction of indole derivatives with β-nitrostyrenes in polyphosphoric acid
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(
PPA) led to the discovery of an efficient synthesis of 2-aryl-2-(3-indolyl)acetohydroxamates.
Although 2,2-diarylacetohydroxamates had been previously synthesized and studied as HDAC
2
1,22
inhibitors,
compounds in which one of the two aromatic rings is an indole moiety had not been
reported in the literature. Thus, 2-aryl-2-(3-indolyl)acetohydroxamate was revealed to be a new
chemotype prompting our thorough investigation of biological properties of compounds incorporating
this structural feature. Although HDAC inhibition was not observed with these compounds (data not
shown), these studies led to the discovery of significant activity associated with a number of
synthesized compounds against cancer cell lines displaying resistance to various types of proapoptotic
stimuli as well as glioblastoma neurosphere stem-like cell cultures derived from patients. It was also
found that the active analogues exhibited their antiproliferative activity through a cytostatic non-
apoptotic mechanism of action and maintained their potency against multi-drug resistant cells, which
are poorly responsive to important clinical cancer drugs taxol and vinblastine. Although the detailed
mechanistic studies aimed at the elucidation of mode(s) of action of the 2-aryl-2-(3-
indolyl)acetohydroxamates are currently pursued in our labs, the compelling evidence for the
effectiveness of these compounds against the apoptosis- and multidrug resistant cancer cells prompts
us to disclose our findings in the present paper.
Results and Discussion
Chemistry
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-Aryl-2-(3-indolyl)acetohydroxamates (3, Figure 1) were identified to be intermediates in our
recently discovered transannulation of indoles to 2-quinolones carried out by reacting 2-substituted
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indoles with β-nitrostyrenes in PPA at 100 C. It was found that if the reaction temperature kept at 70
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C, compounds 3 could be isolated as the main reaction products (Figure 1A, Tables 1 and 2). The
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reaction scope was found to allow for the introduction of a variety of substituents R , R , R and R into
the 2-aryl-2-(3-indolyl)acetohydroxamate scaffold 3. In addition, the recognition of limited access to a
number of specific substituted indoles that would be required for systematic structure-activity
relationship (SAR) analyses prompted the development of an alternative route based on an in situ
Fisher indole synthesis utilizing arylhydrazines 4 and ketones 5 (Figure 1B). In this multicomponent
o
variation, compounds 4 and 5 are reacted at 100 C to allow for the indole formation and then the
o
reaction temperature is lowered to 70 C prior to the introduction of β-nitrostyrenes 2. Thus, the
availability of two complementary approaches to compounds 3 permits the synthesis of analogues with
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2
3
4
the desired identity and positioning of substituents R , R , R and R on the 2-aryl-2-(3-
indolyl)acetohydroxamate scaffold facilitating the development of these compounds as medicinal
agents. Since the synthesized compounds have four diversification points, a four-dimensional tagging
system is employed for labeling the products. Thus, the reaction of hydrazine 4aa with ketone 5f
produces indole 1aaf, which in the subsequent reaction with nitrostyrene 2n affords hydroxamic acid
3
aafn.
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R1
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(
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OH
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H
0 oC, 1-2 h
up to 86%
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. 4+5, 100 oC, 0.5-1 h
2. add 2, 70 oC, 1-2 h
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R3
NHNH2
4
5
up to 77%
R4
2
Figure 1. Two synthetic approaches toward 2-aryl-2-(3-indolyl)acetohydroxamates 3
Pharmacology
a) SAR analyses
The evaluation of an initially synthesized series of compounds 3 for a variety of activities led to the
(
identification of double-digit micromolar antiproliferative potencies associated with the parent
acetohydroxamate 3aaaa (Table 1). This finding led to an exploration of the SAR analyses by
synthesizing the first generation compounds 3 containing diverse substituents at different positions in
the 2-aryl-2-(3-indolyl)acetohydroxamate skeleton and testing this series for in vitro growth inhibition
2
3
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay
against two cell lines, human HeLa cervical and MCF-7 breast adenocarcinomas (Table 1). It emerged
2
from these experiments that the substitution on the benzene ring of the indole moiety (R ≠ H) was not
1
tolerated (e.g., 3abfa and 3acfa), whereas the nitrogen could be derivatized (R ≠ H) with only a small
activity drop (e.g., 3bafa vs 3aafa). The key SAR finding resulted from the variations of the C2-
3
position of the indole moiety (R ≠ Ph as in 3aaba, 3aaca, 3aada, 3aaea and 3aafa) and identification
of single-digit micromolar potencies associated with compounds containing the β-naphthyl substituent
at this position (as in 3aafa).
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Table 1. Structures, synthetic yields (methods A or B) and antiproliferative activities of the first
generation compounds 3
#
structure
% yield
cell viability
#
structure
% yield
cell viability
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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53
54
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58
59
60
a
a
logP
(method)
GI , µM
logP
(method)
GI , µM
5
0
50
HeLa MCF7
HeLa MCF7
3
aaaa
82 (A)
76 (B)
23.0
31.0
3aaab
73 (A)
68 (B)
>50
>50
4.1
HN
O
± 2.6
± 0.6
4.1
HN
O
OH
OH
N
H
N
H
NO2
3
aaba
68 (A)
61 (B)
>50
>50
3aaca
OMe
43 (A)
35 (B)
>50
>50
O N
2
4.1
HN
O
4.1
HN
OH
O
N
H
OH
N
H
3aada
.7
Me
O
46 (A)
27 (B)
>50
>50
3aaea
76 (A)
70 (B)
25.7
32.0
HN
OH
2
N
H
5.2
± 1.6
± 0.9
HN
O
OH
OH
OH
N
H
3
aafa
.1
85 (A)
73 (B)
3.6
3.4
3abfa
79 (A)
73 (B)
>50
>50
4
± 0.5
± 0.3
5.7
HN
HN
O
O
OH
N
H
N
H
Me
3
acfa
.2
28 (B)
>50
>50
3bafc
54 (A)
36.8
25.7
Me
5
5.5
± 0.4
± 1.4
HN
N
O
O
OH
N
H
N
H
MeO
F
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3
bafa
.4
75 (A)
19.7
10.0
3bafd
36 (A)
24.0
11.2
Me
Me
5
± 1.7
± 0.3
6.2
± 0.2
± 0.9
N
N
O
O
OH
OH
N
H
N
H
Br
0
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a
Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to a
DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay.
Based on the initial SAR in Table 1, the second generation compounds 3 were synthesized and they
2
1
4
all contained an R = β-naphthyl, while R and R remained variable. These experiments led to the
identification of a number of compounds possessing single-digit micromolar (e.g., 3aafe, 3aafk,
3
aafm, 3aafn, 3aafo and 3aafp) or even submicromolar (e.g., 3aafe and 3aafp) activites, all
4
1
containing meta and/or para-positioned R . The addition of an R = alkyl (e.g., 3cafa, 3dafa, 3cafe and
3
eafa) did not appear to be detrimental with GI values still in the single-digit micromolar region.
5
0
Because of the significant lipophilicities associated with our acetohydroxamates and thus the
possibility that the activities were a function of their lipophilic character, logP values were calculated
for each analogue using three different methods, all giving similar results (Tables 1 and 2). The
significant activity was indeed present among both less lipophilic analogues (e.g., 3aafc with logP =
4
.1) and those with higher lipophilicity (e.g., 3cafe with logP = 8.4), thus ruling out such a possibility.
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Table 2. Structures, synthetic yields (methods A or B) and antiproliferative activities of second
generation compounds 3
a
a
#
structure
% yield
cell viability
#
structure
% yield
cell viability
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
logP
(method)
GI , µM
logP
(method)
GI , µM
5
0
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HeLa MCF7
HeLa MCF7
3aafe
73 (A)
61 (B)
0.68
1.4
3aaff
60 (A)
56 (B)
26.9
2.4
6
.7
±0.04
± 0.1
4.9
± 0.3
± 0.1
HN
HN
O
O
OH
OH
N
H
N
H
MeO
OMe
3
aafg
.4
76 (A)
64 (B)
18.2
6.3
3aafh
84 (A)
72 (B)
31.0
4.9
5
± 0.9
± 1.1
5.9
± 0.2
± 0.1
HN
HN
O
O
OH
OH
OH
OH
N
H
N
H
Cl
F
3aafi
.5
45 (A)
43 (B)
31.7
12.2
3aafc
75 (A)
64 (B)
32.9
20.4
6
± 1.7
± 1.0
4.1
± 1.1
± 0.1
HN
Cl
HN
O
O
OH
N
H
N
H
F
Cl
3
aafj
.3
56 (A)
52 (B)
27.4
10.2
3aafk
70 (A)
59 (B)
2.7
2.5
6
± 0.2
± 0.1
6.1
± 0.1
± 0.1
HN
HN
Me
O
O
OH
N
H
N
H
OCF3
Me
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3
aafd
.2
57 (A)
55 (B)
13.0
2.7
3aafl
81 (A)
72 (B)
17.3
8.5
6
± 0.4
± 0.0
5.6
± 0.2
± 0.3
HN
HN
HN
HN
O
O
OH
OH
OH
OH
OH
OH
N
N
H
H
Br
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
OEt
3aafm
.7
80 (A)
72 (B)
6.3
4.9
3aafn
76 (A)
69 (B)
6.9
7.4
5
± 1.3
± 0.2
5.7
± 0.4
± 0.6
HN
Me
O
O
N
H
N
H
Me
3
aafo
.0
53 (A)
50 (B)
4.1
7.9
3aafp
45 (A)
43 (B)
0.60
1.1
6
± 0.2
± 0.7
5.3
±0.02
± 0.0
HN
O
O
N
H
N
H
NEt2
NMe
2
3cafa
.8
83 (A)
5.6
7.8
3dafa
80 (A)
9.8
9.3
6
± 0.3
± 0.6
6.7
± 0.8
± 0.3
N
N
O
O
OH
OH
N
H
N
H
3
cafe
.4
68 (A)
6.6
7.7
3eafa
75 (A)
5.4
5.5
8
± 0.5
± 0.4
7.0
± 0.3
± 0.2
N
O
N
O
OH
N
H
OH
N
H
a
Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to a
DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay.
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Finally, to assess the importance of the hydroxamic acid moiety, 3aafa was converted to nitrile 6 by
treating the former with PCl and further to amide 7 by partial hydration of 6 in 80% PPA (Figure 2).
3
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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2
2
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4
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5
5
5
5
5
5
5
5
5
6
The evaluation of nitrile 6 and amide 7 for antiproliferative activity revealed a 6- and 3-fold lower
potencies associated with these compounds as compared with hydroxamate 3aafa, thus underscoring
the importance of the hydroxamic acid moiety but not its criticality.
0
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2
3
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9
0
HN
HN
O
PCl , EtOAc
3
OH
CN
N
H
reflux, 75%
8
0% PPA
3aafa
GI50
.6 µM (HeLa)
.4 µM (MCF-7)
6
o
8
0 C, 98%
GI50
17.4 µM (HeLa)
20.6 µM (MCF-7)
3
3
HN
O
NH2
7
GI50
.3 µM (HeLa)
1.6 µM (MCF-7)
9
1
Figure 2. Synthesis of non-hydroxamate analogues of 3
(b) Activity against cells exhibiting various types of resistance to proapoptotic stimuli
As part of the ongoing efforts in our lab aimed at identification of compounds active against cancer
2
4-27
cell displaying resistance to proapoptotic agents,
the selected 2-aryl-2-(3-indolyl)-
acetohydroxamates were evaluated for in vitro growth inhibition against a panel of additional cancer
cell lines including those resistant to various proapoptotic stimuli, such as human T98G and U87
2
8,29
30
glioblastoma
and human A549 non-small-cell lung cancer (NSCLC), as well as an apoptosis-
28
sensitive tumor model, such as human Hs683 anaplastic oligodendroglioma, used as reference. The
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obtained GI values associated with potent hydroxamates are shown in Table 3. The data reveal that
50
for the most part these compounds retain the single-digit antiproliferatve GI values in this challenging
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0
cancer cell panel. Further analysis of the results from Tables 2 and 3 combined shows that the
hydroxamates do not discriminate between the cancer cell lines based on the apoptosis sensitivity
criterion and display comparable potencies in both cell types, indicating that apoptosis induction is not
the primary mechanism responsible for antiproliferative activity in this series of compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Antiproliferative properties of potent hydroxamates against cancer cell lines displaying
apoptosis resistance and representing cancers with dismal prognoses
a
GI50 in vitro values (µM)
compound
glioma
U87
lung carcinoma
A549
Hs683
T98G
3
aafa
aafe
8.9 ± 0.4
6.1 ± 1.0
4.7 ± 1.0
10.8 ± 0.5
11.2 ± 0.9
5.1 ± 0.5
9.5 ± 0.3
5.0 ± 0.5
6.7 ± 1.5
6.7 ± 0.3
9.1 ± 0.3
21.3 ± 1.6
36.4 ± 1.9
8.8 ± 0.5
7.5 ± 0.8
12.3 ± 0.8
10.6 ± 0.4
1.9 ± 0.2
2.8 ± 0.4
3.3 ± 0.6
2.9 ± 0.6
5.7 ± 0.5
5.8 ± 0.8
1.5 ± 0.3
3
3
aafk
cafa
3eafa
aafp
3
3
a
Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to a
DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay.
Our previous experience of working with cells resistant to various proapoptotic stimuli shows that
generally a certain population of cells becomes rapidly eliminated with proapoptotic agents used at low
concentrations leading to low GI values. However, these high potencies can be somewhat misleading
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as there often remains a significant portion of cells that resists the effects of the proapoptotic agents
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even at concentrations 100- or 1000-fold of their GI s. It was thus instructive to compare the
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hydroxamates with common proapoptotic agents for their ability to affect such resistant populations.
Indeed, as can be seen in Figure 3, hydroxamates 3aafa and 3aafp have potent growth inhibitory
properties against most of the cells in U87 and A549 cultures and, with increasing concentration,
rapidly reach antiproliferative levels of a non-discriminate cytotoxic agent phenyl arsine oxide (PAO).
In contrast, common proapoptotic agents taxol and podophyllotoxin have no effect on proliferation of
ca. 50% of cells in these cultures at concentrations up to 100 µM.
0
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Activity of 3aafp and 3aafa against cell populations resistant to proapoptotic agents.
(
c) Quantitative videomicroscopy
To obtain insight into the effectiveness of 2-aryl-2-(3-indolyl)acetohydroxamates against apoptosis-
1
2,13,15
resistant cancers, computer-assisted phase-contrast microscopy
(quantitative videomicroscopy)
was employed to observe the phenotypic morphological changes in cancer cells as they are treated with
these compounds. Figure 4 shows that acetohydroxamate 3aafa inhibits cancer cell proliferation
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without inducing cell death when assayed at concentrations slightly exceeding the GI values (25 µM)
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in SKMEL-28 melanoma and U373 glioblastoma cells, both exhibiting resistance to various
2
8,32
proapoptotic stimuli.
Based on the phase contrast pictures obtained by means of quantitative
0
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0
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videomicroscopy, a global growth ratio (GGR) was calculated, which corresponds to the ratio of the
mean number of cells present in a given image captured in the experiment (in this case after 24, 48 and
7
2 h) to the number of cells present in the first image (at 0 h). The ratio obtained in the 3aafa-treated
experiment was then divided by the ratio obtained in the control. The GGR value of ca. 0.3 in both of
these two cell lines indicates that 30% of cells grew in the 3aafa-treated experiment as compared to the
control over a 72 h observation period. Thus, the GGR calculations are consistent with the MTT
colorimetric data and indicate that it is the cytostatic properties associated with the hydroxamates that
are responsible for their antiproliferative effects against apoptosis-resistant cancer cells at least at
relevant concentrations (slightly above the GI values).
5
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Figure 4. Cellular imaging of 3aafa against SKMEL-28 melanoma and U373 glioblastoma cells
illustrating the cytostatic antiproliferative mechanism.
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(
d) Redifferentiation of U87 cells to an astrocytic phenotype
To elucidate the fate of the cells whose growth has been arrested with the hydroxamates, the
phenotypic morphological changes of U87 glioma cells were observed for a period of several weeks
after the treatment with hydroxamate 3aafa at the GI concentration. Interestingly, while untreated
0
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0
cells proliferated rapidly and quickly formed spheroids (Figure 5B), the treated cells ceased to replicate
and appeared to undergo redifferentiation to a non-malignant state resembling a reactive astrocyte
(data not shown) phenotype (Figure 5C). Although such redifferentiation processes are known, there
3
3,34
are only a few small molecule agents reported to induce these epigenetic transformations.
The
literature reports indicate that these redifferentiated cells possess significantly reduced tumorigenicity
3
3
in vivo and, thus, new chemical entities capable of triggering such phenotypic changes hold a
promising but completely unexplored potential as anticancer agents.
Figure 5. Redifferentiation of growth-inhibited malignant U87 cells to an astrocytic phenotype. (A)
Three day old glioblastoma cancer cells. (B) Untreated, these grow into mini-tumors during the
following three days. (C) After a 33-day treatment with 7 µM 3aafa.
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(
e) Activity against MDR cells, glioblastoma neurosphere stem-like cell cultures derived from patients
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
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3
3
4
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4
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6
and normal fibroblasts.
0
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2
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9
0
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9
0
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0
Compared with the intrinsic drug resistance, as described above for such as cancers as glioblastoma
and melanoma, a large variety of tumors can also develop resistance to anticancer drugs resulting in
MDR as explained in the introduction. To assess whether the hydroxamates can overcome this
resistance mechanism, selected hydroxamates were tested against MDR cells (Table 4). The MDR
uterine sarcoma cell line MES-SA/Dx5 was utilized for this experiment. This cell line was established
from the parent uterine sarcoma MES-SA, grown in the presence of increasing concentrations of
3
5
doxorubicin and is known to be resistant to a number of P-gp substrates. Both taxol and vinblastine
displayed more than a thousand fold drop in potency when tested for antiproliferative activity against
the MDR cell line as compared with the parent line (Table 4). In contrast, there was only a small
variation in the sensitivities of the two cell lines towards the hydroxamates indicating their potential to
overcome clinical multi-drug resistance.
Given the ability of the hydroxamates to overcome drug resistance a few select compounds were
further evaluated against glioma cells grown in neurosphere conditions, which are known to promote
the growth of stem-like cells from human glioma tissue. Indeed, the neurospheres show the ability of
self-renewal by regrowing in culture from individual cells, can differentiate into multiple neural
lineages and recapitulate human gliomas on both histological and genetic levels more faithfully than
3
6-39
serum cultured glioma cell lines when injected into the brains of mice.
Because, neurosphere cells
40-43
are generally resistant to radiation and chemotherapy,
the micromolar to submicromolar activity of
of the hydroxamates against the glioma neurosphere cell cultures is noteworthy (Table 4). The glioma
culture 031810 used is derived from a patient with glioblastoma who progressed on temozolomide due
6
to high O -methylguanine-DNA-methyltransferase (MGMT) expression and thus shows high
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2
2
2
2
2
2
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3
3
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3
3
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3
3
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3
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resistance to this agent (Table 4). It is worthy of note, that the unmethylated MGMT promoter leading
to such temozolomide resistance is found in about half of all GBM patients, who respond poorly to
4
4
44
temozolomide chemotherapy. To date, no alternative treatment exists for this group of patients.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Antiproliferative effect of selected compounds against MDR cells and patient-derived GBM
neurosphere cells
compound
GI50 in vitro values (µM)
a
a
b
MES-SA
MES-SA/Dx5
GBM 031810
Taxol
Vinblastine
Temozolomide
3aafa
0.007 ± 0.001
0.006 ± 0.001
9.8 ± 0.3
5.0 ± 1.4
> 1000
0.8 ± 0.6
5.6 ± 0.8
3.4 ± 0.7
2.0 ± 0.2
0.8 ± 0.1
1.7 ± 0.4
1.8 ± 0.4
5.9 ± 1.7
7.1 ± 0.1
4.0 ± 1.1
1.6 ± 0.6
4.9 ± 1.9
2.2 ± 0.8
2.7 ± 0.3
8.5 ± 0.9
3
aafp
aafe
aafk
cafa
3eafa
3
3
3
a
Concentration required to reduce the viability of cells by 50% after a 48 h treatment with the indicated compounds relative to a
b
DMSO control ± SD from two independent experiments, each performed in 4 replicates, as determined by the MTT assay. Average GI
5
0
±
SD from three GI50 determinations.
Finally, selected hydroxamates were tested against the normal human dermal (NHDF) and lung
NHLF) fibroblast cell lines in comparison with the cancerous glioma and NSCLC cells (Figure 6).
(
The compounds displayed a modest but noteworthy selectivity in inhibiting the growth of cancer cells
with 3aafa and 3aafp being particularly ineffective at inhibiting the proliferation of the normal NHDF
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cell line (Figure 6). These results show that the selectivity of the hydroxamates toward cancer cells is
structure-dependent and can be optimized to select the best candidates for the forthcoming in vivo tests
in animal models.
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Figure 6. Activity of selected analogues toward non-cancerous and cancerous cell lines. The results
were obtained using two independent experiments (both shown in Figure) in sextuplicates. Non-
cancerous fibroblast cell lines are presented with open symbols, while cancer cell lines are presented
with filled symbols.
Conclusion
Drug resistance is one of the main causes for the failure of cancer chemotherapy, affecting patients
with a broad variety of tumors. Resistance to chemotherapy can be intrinsic, in which cancers such as
glioma, melanoma or NSCLC, among others, fail to respond to the first chemotherapy given.
Resistance can also be acquired, in which tumors innately respond to chemotherapy but eventually
become refractory to a broad spectrum of structurally and mechanistically diverse antitumor agents.
The results presented herein demonstrate the potential 2-aryl-2-(3-indolyl)acetohydroxamates for the
treatment drug-resistant cancer, regardless of whether the latter harbors intrinsic and acquired
resistance mechanisms. The structural scaffold associated with these compounds represents a new
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chemotype, whose further investigation is warranted by the described findings and should be facilitated
by the straightforward synthetic methodologies developed to accommodate systematic SAR studies as
well as preparation of specific designed analogues. The ongoing work includes further optimization of
compound potency, elucidation of mechanisms responsible for cytostatic and redifferentiation effects
as well as experiments involving animal models of drug-resistant human cancer.
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Experimental Section
General Experimental.
Reagents, solvents and catalysts were purchased from commercial sources (Acros Organics and
Sigma-Aldrich) and used without purification. All reactions were performed in oven-dried flasks open
to the atmosphere and monitored by thin layer chromatography on TLC precoated (250 μm) silica gel
60 F254 glass-backed plates (EMD Chemicals Inc.). Visualization was accomplished with UV light.
1
13
Filtration was performed using silica gel (32-63 μm, 60 Å pore size). H and C NMR spectra were
recorded on Bruker DRX-400 and Bruker DRX-500 spectrometers. Chemical shifts (δ) are reported in
ppm relative to the TMS internal standard. Abbreviations are as follows: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet). Indoles: 2-phenyl-1H-indole (1aaa), 2-(2-nitrophenyl)-1H-indole
(3aab), 2-(4-methoxyphenyl)-1H-indole (3aac), 2-methyl-1H-indole (3aad), 2-(naphthalen-1-yl)-1H-
indole (1aae), 2-(naphthalen-2-yl)-1H-indole (1aaf), 1-methyl-2-(naphthalen-2-yl)-1H-indole (1baf),
and 5-methoxy-2-(naphthalen-2-yl)-1H-indole (1acf) were purchased from commercial sources and
used as received. Procedures for preparation of 5-methyl-2-(naphthalen-2-yl)-1H-indole (1abf), 1-
butyl-2-(naphthalen-2-yl)-1H-indole (1caf), 1-(sec-butyl)-2-(naphthalen-2-yl)-1H-indole (1daf), 1-
benzyl-2-(naphthalen-2-yl)-1H-indole (1eaf) are provided below. Ketones: acetophenone (5a), o-
nitroacetophenone (5b), p-methoxyacetophenone (5c), acetone (5d), 1-acetylnaphalene (5e), and 2-
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acetylnaphalene (5f) were obtained from commercial sources and used as received. Arylhydrazines:
pehylhydrazine (4aa), p-tolylhydrazine (4ab), and p-anisylhydrazine (4ac) were obtained from
commercial sources and used as received. Nitroalkenes: (2-nitrovinyl)benzene (2a), 1-nitro-4-(2-
nitrovinyl)benzene (2b), 1-fluoro-3-(2-nitrovinyl)benzene (2c), 1-bromo-2-(2-nitrovinyl)benzene (2d),
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,2-dimethoxy-4-(2-nitrovinyl)benzene (2f), 1-chloro-2-(2-nitrovinyl)benzene (2h), 1,2-dichloro-4-(2-
nitrovinyl)benzene (2i), 1-(2-nitrovinyl)-4-(trifluoromethoxy)benzene (2j), 1-methyl-4-(2-
nitrovinyl)benzene (2m), N,N-dimethyl-4-(2-nitrovinyl)aniline (2p) were acquired from commercial
sources and used as received. 1-Isopropyl-4-(2-nitrovinyl)benzene (2e), 1-fluoro-4-(2-
nitrovinyl)benzene (2g), 1,2-dimethyl-4-(2-nitrovinyl)benzene (2k), 1-ethoxy-4-(2-nitrovinyl)benzene
4
5
(
2l), 1-methyl-3-(2-nitrovinyl)benzene (2n) were synthesized using a reported procedure, as well as
4
6
N,N-diethyl-4-(2-nitrovinyl)aniline (2o).
Elemental analyses were performed using a CHN-1
analyzer. HRMS analyses were performed on ESI Bruker Maxis. The synthesized compounds were at
least 95% pure according to elemental analyses and/or HPLC chromatograms.
Compound 1abf: A mixture of 4-methylphenylhydrazine (4ab) (1.22 g, 10 mmol) and 2-
acetylnaphthalene (5f) (1.70 g, 10 mmol) was vigorously stirred at 100-110 °C in 80% PPA (3-5 g) for
40 min. When the reaction was complete based on TLC analysis the mixture was cooled down to rt,
poured into water (50 mL), and neutralized with aqueous ammonia. The formed precipitate was
filtered, dried in vacuum, and used without additional purification. Yield 2.44 g (9.5 mmol, 95%);
1
m.p. = 212-213 °C (toluene); H NMR (400 MHz, CDCl ) δ, ppm: 8.43 (br. s, 1H), 8.08 (s, 1H), 7.93-
3
7
2
.86 (m, 4H), 7.56-7.48 (m, 2H), 7.46 (s, 1H), 7.30-7.34 (m, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.9 (s. 1H),
1
3
.49 (s. 3H); C NMR (100 MHz , CDCl ) δ: 133.7, 129.8, 128.9, 128.7, 128.4, 128.1, 127.9, 126.8,
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26.7, 126.4, 126.3, 125.4, 124.9, 124.4, 123.9, 120.6, 118.2, 111.0, 21.6; HRMS calc`d for C H N
19 16
+
(
M+H) : 258.1277, found 258.1276.
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Compound 1caf: To a stirred solution of KOH (2.24 g, 40 mmol) in DMSO (20 mL was added 2-(2-
naphthyl)indole (1aaf) (2.43 g, 10 mmol), and the mixture was stirred for 45 min. Then, n-butyl
bromide (2.7 g, 20 mmol) was added and the stirring was continued for additional 45 min. The
mixture was diluted with water (20 mL) and extracted with benzene (3 x 50 mL). Combined organic
layers were washed with water (3 x 100 mL), dried with CaCl and concentrated in vacuum to obtain
2
1
the titled compound as yellowish oil. Yield 2.60 g (8.7 mmol, 87%); H NMR (400 MHz, CDCl ) δ,
3
ppm: 7.99-7.92 (m, 4H), 7.77 (d, J = 7.8 Hz, 1H), 7.65 (dd, J = 8.4, 1.7 Hz, 1H), 7.58-7.55 (m, 2H),
7
.45 (d, J = 8.2 Hz, 1H), 7. 28 (ddd, J = 7.4, 7.6, 1.1 Hz, 1H), 7.19 (ddd, J = 7.4, 7.4, 0.9 Hz, 1H), 6.65
1
3
(s, 1H), 4.25 (t, J = 7.5 Hz, 2H), 1.74 (m, 2H), 1.21 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H); C NMR (100
MHz , CDCl ) δ: 141.5, 137.7, 133.4, 132.9, 130.9, 128.5, 128.4, 128.3, 128.2, 127.9, 127.4, 126.6,
3
+
1
3
26.5, 121.7, 120.7, 119.9, 110.2, 102.7, 44.1, 32.3, 20.1, 13.8; HRMS calc`d for C H N (M+H) :
22 22
00.1747, found 300.1749.
Compound 1daf: To a stirred solution of KOH (2.24 g, 40 mmol) in DMSO (20 mL was added 2-(2-
naphthyl)indole (1aaf) (2.43 g, 10 mmol), and the mixture was stirred for 45 min. Then, sec-butyl
bromide (2.7 g, 20 mmol) was added and the stirring was continued for additional 60 min. The
mixture was idluted with water (20 mL) and extracted with benzene (3 x 50 mL). Combined organic
layers were washed with water (3 x 100 mL), dried with CaCl and concentrated in vacuum to obtain
2
the titled compound as colorless solid. Yield 2.52 g (8.4 mmol, 84%); m.p. = 103-104 °C (petroleum
1
ether); H NMR (400 MHz, CDCl ) δ, ppm: 7.99-7.91 (m, 4H), 7.69, (d, J = 7.9 Hz, 1H), 7.63 (dd, J =
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.4, 1.5 Hz, 1H), 7.59-7.55 (m, 3H), 7.44 (d, J = 8.2 Hz, 1H), 7.27 (ddd, J = 7.6, 7.5, 0.7 Hz, 1H) 7.18
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(
t, J = 7.3 Hz, 1H), 6.66 (s, 1H), 4.15-4.13 (m, 2H), 2.15-2.04 (m, 1H), 0.69-067 (m, 6H); C NMR
(
100 MHz , CDCl ) δ: 141.8, 138.0, 133.4, 132.9, 131.22, 128.7, 128.4, 128.3, 128.2, 127.9, 127.6,
3
0
1
2
3
4
5
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7
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9
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2
3
4
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6
7
8
9
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126.6, 126.5, 121.6, 120.7, 119.9, 110.7, 103.0, 51.5, 29.1, 22.9, 20.2; HRMS calc`d for C H N
22 22
+
(M+H) : 300.1747, found 300.1750.
Compound 1eaf: To a stirred solution of KOH (2.24 g, 40 mmol) in DMSO (20 mL was added 2-(2-
naphthyl)indole (1aaf) (2.43 g, 10 mmol), and the mixture was stirred for 45 min. Then, benzyl
bromide (3.4 g, 20 mmol) was added and the stirring was continued for additional 45 min. The
mixture was diluted with water (20 mL) and extracted with benzene (3 x 50 mL). Combined organic
layers were washed with water (3 x 100 mL), dried with CaCl and concentrated in vacuum to obtain
2
1
the titled compound as colorless solid. Yield 3.07 g (9.2 mmol, 92%); m.p. = 144-146 °C (toluene); H
NMR (400 MHz, CDCl ) δ, ppm: 7.89-7.84 (m, 3H), 7.76-7.70 (m, 2H), 7.57 (dd, J = 8.5, 1.7 Hz, 1H),
3
7
.51-7.49 (m, 2H), 7.32-7.24 (m, 4H), 7.20-7.17 (m, 2H), 7.08 (d, J = 6.8 Hz, 2H), 6.77 (s, 1H), 5.43
1
3
(s, 2H); C NMR (100 MHz , CDCl ) δ: 142.0, 138.4 (2C), 133.3, 133.0, 130.2, 128.9 (2C), 128.5,
3
1
4
28.4, 128.3 (2C), 127.8, 127.4, 127.2, 126.6, 126.5, 126.2 (2C), 122.2, 120.7, 120.4, 110.7, 102.9,
+
8.1; HRMS calc`d for C H N(M+H) : 334.1590, found 334.1595.
25 20
Preparation of 2-aryl-2-(3-indolyl)acetohydroxamates 3. General Method A. A mixture of a
selected indole 1 (1 mmol) and a selected nitrostyrene 2 (1.2 mmol) in 80% PPA (3-4 g) was stirred at
o
6
5-70 C for 1 h. The disappearance of the starting indole was monitored by TLC. After the indole has
reacted completely, the mixture was cooled to rt, poured in water (50 mL) and treated with saturated
NH OH to pH 8. The formed precipitate was filtered and recrystallized from the indicated solvent.
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Preparation of 2-aryl-2-(3-indolyl)acetohydroxamates 3. General Method B. A mixture of a
selected arylhydrazine 4 (1 mmol) and a selected methylaryl ketone 5 (1 mmol) in 80% PPA (2-3 g)
o
was stirred at 100-110 C for 40 min. The disappearance of the starting arylhydrazine was monitored
o
0
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by TLC. After the arylhydrazine has reacted completely, the temperature was decreased to 65-70 C
and a selected nitrostyrene 2 (1.2 mmol) was added. The mixture was stirred at this temperature for 1 h
and the disappearance of the intermediate indole 1 was monitored by TLC. After the indole has reacted
completely, the mixture was cooled to room temperature, poured in water (50 mL) and treated with
saturated NH OH to pH 8. The formed precipitate was filtered and recrystallized from the indicated
4
solvent.
Compound 3aaaa. Synthesized according to the general method A from 2-phenylindole (3aaa) and
(2-nitrovinyl)benzene (2a) in 82% yield; Alternatively prepared according to the general method B
starting from phenylhydrazine (4aa), acetophenone (5a) and (2-nitrovinyl)benzene (2a): 76%; m.p. =
1
2
20-221°C (toluene/petroleum ether); H NMR (500 MHz, CDCl ) δ, ppm: 11.30 (br. s, 1H), 10.75 (br.
3
s, 1H), 8.81 (br. s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.54-7.48 (m, 4H), 7.41 (dd, J = 7.2, 7.1 Hz, 1H),
7
5
1
.35 (d, J = 8.1 Hz, 1H), 7.27-7.16 (m, 5H), 7.06 (dd, J = 7.6, 7.4 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H),
1
3
.10 (s, 1H); C NMR (125 MHz, CDCl ) δ, ppm: 168.6, 140.7, 136.2, 132.5, 128.6 (2С), 128.5 (2С),
3
28.0 (2С), 127.9 (2С), 127.7, 127.6, 126.1, 122.3, 121.1, 118.5, 110.9, 109.2, 46.0; EA: Calcd for
C H N O : C 77.17, H 5.30, N 8.18. Found: C 77.33, H 5.22, N 8.11; HRMS calc`d for C H N O Na
2
2
18
2
2
22 18
2
2
+
(M+Na) : 365.1260, found 365.1272.
Compound 3aaab. According to the method A, starting from 2-phenyl-1H-indole (3aaa) and 1-nitro-
4-(2-nitrovinyl)benzene (2b): 73%; According to the method B, starting from phenylhydrazine (4aa),
1
acetophenone (5a) and 1-nitro-4-(2-nitrovinyl)benzene (2b): 68%; m.p. = 156-157 °C (toluene); H
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NMR (400 MHz, DMSO) δ, ppm: 11.44 (br. s, 1H), 10.89 (br. s, 1H), 8.96 (br. s, 1H), 8.14 (d, J = 8.8
Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.49-7.46 (m, 4H), 7.42-7.36 (m, 5H), 7.09 (t, J = 7.2 Hz, 1H), 6.92
3
4
5
6
7
8
9
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1
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2
2
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3
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4
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5
5
5
5
5
5
5
5
5
5
6
1
3
(
t, J = 7.5 Hz,1H), 5.20 (s, 1H); C NMR (100 MHz , DMSO) δ: 167.8, 148.6, 146.0, 136.7, 136.2,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
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132.2, 129.3 (2C), 128.7 (4C), 127.9, 127.4, 123.3 (2C), 121.6, 121.4, 118.9, 111.2, 108.0, 46.1;
+
HRMS calc`d for C H N O Na(M+Na) : 410.1111, found 410.1111.
2
2
17
3
4
Compound 3aaba. According to the method A, starting from 2-(2-nitrophenyl)-1H-indole (3aab) and
(
2-nitrovinyl)benzene (2a): 68%; According to the method B, starting from phenylhydrazine (4aa), 2-
1
nitroacetophenone (5b) and (2-nitrovinyl)benzene (2a): 61%; m.p. = 118-119 °C (toluene); H NMR
(400 MHz, DMSO) δ, ppm: 11.27 (br. s, 1H), 10.71 (br. s, 1H), 8.87 (br. s, 1H), 8.12 (dd, J = 8.1, 0.9
Hz, 1H), 7.80-7.61 (m, 4H), 7.54 (d, J = 7.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.18-7.06 (m, 5H), 6.91
1
3
(t, J = 7.4 Hz, 1H), 4.77 (s, 1H); C NMR (100 MHz , DMSO) δ: 166.4, 147.6, 139.9, 136.2, 134.2,
1
33.7, 133.1, 129.8, 127.8 (2C), 127.7 (3C), 127.0 (2C), 126.1, 124.5, 121.4, 118.5, 111.2, 110.9, 45.9;
+
HRMS calc`d for C H N O Na(M+Na) : 410.1111, found 410.1109.
22 17
3
4
Compound 3aaca. According to the method A, starting from 2-(4-methoxyphenyl)-1H-indole (3aac)
and (2-nitrovinyl)benzene (2a): 43%; According to the method B, starting from phenylhydrazine (4aa),
1
4
-methoxyacetophenone (5c) and (2-nitrovinyl)benzene (2a): 35%; m.p. = 133-134 °C (toluene); H
NMR (400 MHz, DMSO) δ, ppm: 11.22 (br. s, 1H), 10.73 (br. s, 1H), 8.81 (br. s, 1H), 7.70 (d, J = 8.0
Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.27-7.17 (m, 5H), 7.06-7.01 (m, 3H), 7.86
13
(
t, J = 7.5 Hz, 1H), 5.04 (s, 1H), 3.81 (s, 3H); C NMR (100 MHz, DMSO) δ: 169.1, 159.4, 141.3,
1
36.7, 136.5, 130.4 (2C), 128.5 (2C), 128.4 (2C), 128.3, 126.6, 125.4, 122.6, 121.3, 118.9, 114.6 (2C),
+
111.2, 109.0, 55.7, 46.6; HRMS calc`d for C H N O Na(M+Na) : 395.1373, found 395.1366.
2
3
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Compound 3aada. According to the method A, starting from 2-methyl-1H-indole (3aad) and (2-
nitrovinyl)benzene (2a): 46%; According to the method B, starting from phenylhydrazine (4aa),
1
acetone (5d) and (2-nitrovinyl)benzene (2a): 27%; m.p. = 110-112 °C (toluene); H NMR (400 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
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4
5
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2
3
4
5
6
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3
4
5
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7
8
9
0
DMSO) δ, ppm: 10.86 (br. s, 1H), 10.79 (br. s, 1H), 8.86 (br. s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.37 (s,
1
H), 7.26-7.20 (m, 5H), 6.94 (ddd, J = 7.4, 7.4, 0.6 Hz, 1H), 6.83 (t, J = 7.2 Hz, 1H), 4.93 (s, 1H), 2.30
1
3
(
s, 3H); C NMR (100 MHz , DMSO) δ: 168.7, 140.5, 135.1, 133.2, 128.1 (2C), 127.9 (2C), 127.7,
+
126.1, 119.9, 119.8, 118.0, 110.2, 108.5, 45.3, 11.9; HRMS calc`d for C H N O Na (M+Na) :
2
2
17
3
4
3
03.1104, found 303.1103.
Compound 3aaea. According to the method A, starting from 2-(1-naphthyl)-1H-indole (3aae) and (2-
nitrovinyl)benzene (2a): 76%; According to the method B, starting from phenylhydrazine (4aa), 1-
1
acetylnaphthalene (5e) and (2-nitrovinyl)benzene (2a): 70%; m.p. = 110-112 °C (toluene); H NMR
(400 MHz, DMSO, 338K) δ, ppm: 11.28 (br. s, 1H), 10.41 (br. s, 1H), 8.63 (br. s, 1H), 8.03-7.99 (m,
2
H), 7.79-7.50 (m, 5H), 7.40-7.33 (m, 2H), 7.26-7.07 (m, 6H), 6.93 (t, J = 7.4 Hz, 1H), 4.73 (s, 1H);
C NMR (100 MHz , DMSO) δ: 168.8, 140.3, 136.3, 134.9, 133.2, 132.5, 130.0, 129.2, 128.6, 128.5,
1
3
1
4
28.1, 127.9 (2C), 127.2, 126.4, 126.0 (2C), 125.7, 125.5, 125.2, 122.4, 121.1, 118.4, 111.4, 110.8,
+
6.2; HRMS calc`d for C H N O Na(M+Na) : 415.1417, found 415.1417.
26 20
2
2
Compound 3aafa. According to the method A, starting from 2-(2-naphthyl)-1H-indole (3aaf) and (2-
nitrovinyl)benzene (2a): 85%; According to the method B, starting from phenylhydrazine (4aa), 2-
1
acetylnaphthalene (5f) and (2-nitrovinyl)benzene (2a): 73%; m.p. = 152-154 °C (toluene). H NMR
(
500 MHz, DMSO) δ, ppm: 11.31 (br. s, 1H), 10.76 (br. s, 1H), 8.82 (br. s, 1H), 8.03-7.97 (m, 3H),
7.91 (dd, J = 8.8, 2.1 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.68 (dd, J = 8.5, J = 1.3 Hz, 1H), 7.58-7.56
(m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.29-7.17 (m, 5H), 7.09 (dd, J = 7.8, 7.4 Hz, 1H), 6.91 (dd, J = 7.8,
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.5 Hz, 1H), 5.19 (s, 1H); C NMR (125 MHz, DMSO) δ: 168.5, 140.8, 136.4, 135.9, 132.8, 132.2,
30.0, 128.1, 128.0 (2С), 127.9, 127.8, 127.6, 127.5, 127.4 126.6, 126.4, 126.3, 126.2, 122.3, 121.3,
18.6, 110.9, 109.9, 99.9, 45.8; EA: Calcd for C H N O : C 79.57, H 5.14, N 7.14. Found: C 79.68, H
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
2
6
20
2
2
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
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4
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7
8
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0
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9
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5.09, N 7.16; HRMS calc`d for C H N O Na (M+Na) : 415.1417, found 415.1419.
2
6
20
2
2
Compound 3abfa. According to the method A, starting from 5-methyl-2-(2-naphthyl)-1H-indole
(3abf) and (2-nitrovinyl)benzene (2a): 79%; According to the method B, starting from 4-tolyl-
hydrazine (4ab), 2-acetylnaphthalene (5f) and (2-nitrovinyl)benzene (2a): 73%; m.p. = 133-135 °C
1
(
toluene); H NMR (400 MHz, DMSO) δ, ppm: 11.34 (br. s, 1H), 10.75 (br. s, 1H), 8.82 (br. s, 1H),
8
6
.00-7.87 (m, 5H), 7.65 (dd, J = 8.65, 1.08 Hz, 1H), 7.60 (s, 1H), 7.57-7.54 (m, 2H), 7.29-7.17 (m,
1
3
H), 7.92 (dd, J = 8.2, 0.9 Hz, 1H), 5.17 (s, 1H), 2.31 (s, 3H); C NMR (100 MHz , DMSO) δ: 169.1,
141.4, 136.7, 135.3 (2C), 133.3, 132.7, 130.6, 128.6 (4C), 128.5, 128.4, 128.1, 127.9, 127.3, 127.1,
+
1
4
27.0, 126.8, 126.7, 123.5, 122.2, 111.2, 109.8, 46.7, 22.0; HRMS calc`d for C H N O Na (M+Na) :
2
7
22
2
2
29.1573, found 439.1577.
Compound 3acfa. According to the method B, starting from (4-methoxyphenyl)hydrazine (4ac), 2-
1
acetylnaphthalene (5e) and (2-nitrovinyl)benzene (2a): 28%; m.p. = 128-130 °C (toluene); H NMR
(
400 MHz, DMSO) δ, ppm: 11.31 (br. s, 1H), 10.80 (br. s, 1H), 8.85 (br. s, 1H), 8.01- 7.88 (m, 4H),
7
.65 (d, J =8.59 Hz, 1H), 7.59-7.53 (m, 2H), 7.37 (s, 1H), 7.30-7.17 (m, 5H), 7.75 (dd, J = 8.7, 2.4 Hz,
1
3
1H), 5.16 (s, 1H), 3.64 (s, 3H); C NMR (100 MHz , DMSO) δ: 168.6, 152.8, 140.8, 136.8, 132.8,
1
1
32.2, 131.7, 130.1, 128.4, 128.3, 128.1 (4C), 128.0, 127.6, 127.3, 126.6, 126.5, 126.3, 126.2, 111.5,
+
11.2, 109.6, 104.5, 55.2, 46.3; HRMS calc`d for C H N O Na(M+Na) : 445.1523, found 445.1523
27 22
2
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Compound 3bafc. According to the method A, starting from N-methyl-2-(2-naphthyl)-1H-indole
1
(
3baf) and 3-fluoro(2-nitrovinyl)benzene (2c): 54%; m.p. = 133-134 °C (toluene/petroleum ether); H
NMR (400 MHz, DMSO) δ, ppm: 10.69 (br. s, 1H), 8.89 (br. s, 1H), 8.07-7.92 (m, 4H), 7.73 (d, J =
8.0 Hz, 1H), 7.61 (m, 2H), 7.50 (d, J = 8.2 Hz, 2H), 7.26-7.17 (m, 4H), 7.08 (d, J = 7.3 Hz, 1H), 7.0 (t,
0
1
2
3
4
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6
7
8
9
0
1
2
3
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7
8
9
0
1
2
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5
6
7
8
9
0
1
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0
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2
3
4
5
6
7
8
9
0
13
1
J = 7.3 Hz, 1H), 4.84 (s, 1H), 3.59 (s. 3H); C NMR (100 MHz , DMSO) δ: 167.7, 159.8 (d, J =
CF
3
2
47.2 Hz), 138.8, 136.9, 132.6, 132.5, 130.2, 129.9, 128.4 (d, J = 6.9 Hz), 128.2, 128.1, 127.9,
CF
2
127.8, 127.6, 127.5, 126.7, 126.6, 126.5, 123.8, 121.4, 120.9, 119.2, 114.7 (d, J = 22.3 Hz), 109.8,
CF
+
1
08.9, 40.4, 30.9; HRMS calc`d for C H FN O Na(M+Na) : 447.1479, found 447.1493.
2
7
21
2
2
Compound 3bafa. According to the method A, starting from N-methyl-2-(2-naphthyl)-1H-indole
1
(
3baf) and (2-nitrovinyl)benzene (2a): 75%; m.p. = 114-115 °C (toluene/petroleum ether); H NMR
(400 MHz, DMSO) δ, ppm: 10.66 (br. s, 1H), 8.84 (br. s, 1H), 8.03-7.91 (m, 4H), 7.77 (d, J = 8.1 Hz,
H), 7.62-7.60 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.26-7.14 (m, 7H), 6.98 (dd,
1
1
3
J = 7.8, 7.5 Hz, 1H), 4.85 (s, 1H), 3.60 (s, 3H); C NMR (100 MHz , DMSO) δ: 168.5, 140.7, 138.8,
137.1, 132.6, 132.5, 130.0, 128.4, 128.2, 128.1, 127.9 (4C), 127.6, 126.7, 126.5 (2C), 126.1, 122.3,
1
21.3, 118.8, 110.8, 109.5, 99.5, 46.4, 30.8; EA: Calcd for C H N O : C 79.78, H 5.46, N 6.89.
2
7
22
2
2
+
Found: C 80.03, H 5.39, N 6.81; HRMS calc`d for C H N O Na(M+Na) : 429.2416, found 429.2418.
27 22
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Compound 3bafd. According to the method A, starting from N-methyl-2-(2-naphthyl)-1H-indole
1
(3baf) and 2-bromo(2-nitrovinyl)benzene (2d): 36%; m.p. = 109-113 °C (toluene/petroleum ether); H
NMR (400 MHz, CDCl ) δ, ppm: 7.91 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 7.00 Hz, 1H), 7.69-7.64 (m,
3
1
H), 7.59-7.49 (m, 5H), 7.42 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H),
1
3
7.17-7.13 (m, 2H), 7.09-7.06 (m, 1H), 5.43 (s, 1H), 3.68 (s, 3H); C NMR (100 MHz, DMSO) δ, ppm:
1
67.6, 139.8, 139.0, 137.0, 132.5, 132.4, 132.3, 130.9, 129.8, 128.4, 128.2, 128.1, 127.8, 127.6, 127.5,
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27.2, 126.8, 126.6, 126.4, 123.9, 121.4, 120.2, 119.4, 110.0, 109.5, 47.1, 31.0; HRMS calc`d for
3
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5
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9
1
1
1
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+
C H BrN O Na(M+Na) : 507.0679, found 507.0677.
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0
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Compound 3aafe. According to the method A, starting from 2-(2-naphthyl)-1H-indole (3aaf) and 4-
isopropyl(2-nitrovinyl)benzene (2a): 73%; According to the method B, starting from phenylhydrazine
(
4aa), 2-acetylnaphthalene (5f) and 4-isopropyl(2-nitrovinyl)benzene (2e): 61%; m.p. = 147-148 °C
1
(toluene/petroleum ether). H NMR (400 MHz, DMSO) δ, ppm: 11.44 (br. s, 1H), 10.76 (br. s, 1H),
8
.82 (br. s, 1H), 8.02-7.9 (m, 4H), 7.82 (d, J = 8.1 Hz, 1H), 7.66 (dd, J = 8.5, 1.59 Hz, 1H) 7.60-7.53
(
m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.17-7.06 (m, 5H), 6.92 (ddd, J = 15.0, 7.5, 0.5 Hz, 1H), 5.16 (s,
1
3
1
1
H), 2.86-2.76 (m, 1H), 1.14 (d, J = 6.9 Hz, 6H); C NMR (100 MHz, DMSO) δ, ppm: 168.8, 146.2,
38.1, 136.4, 136.0, 132.8, 132.2, 130.1, 128.1, 128.0 (2C), 127.9, 127.6, 127.5, 126.7, 126.5, 126.3,
125.9 (2C), 122.4, 121.3, 118.6, 111.0, 110.0, 45.9, 39.9, 33.0, 23.9 (2C); Calc`d for C H N O : C
2
9
26
2
2
+
8
4
0.16, H 6.03, N 6.45. Found: C 80.31, H 5.95, N 6.36; HRMS calc`d for C H N O Na (M+Na) :
29 26 2 2
57.1884, found 457.1887.
Compound 3aaff. According to the method A, starting from 2-(2-naphthyl)-1H-indole (3aaf) and 3,4-
dimethoxy(2-nitrovinyl)benzene (2f): 60%; According to the method B, starting from phenylhydrazine
(
(
4aa), 2-acetylnaphthalene (5f) and 3,4-dimethoxy(2-nitrovinyl)benzene (2f): 56%; m.p. = 143-144 °C
1
toluene); H NMR (400 MHz, DMSO) δ, ppm: 11.44 (br. s, 1H), 10.70 (br. s, 1H), 8.81 (br. s, 1H),
8.02-7.89 (m, 4H), 7.85 (d, J = 8.1 Hz, 1H), 7.67 (dd, J = 8.5, 1.4 Hz, 1H), 7.59-7.54 (m, 2H), 7.38 (d,
J = 8.0 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.85-6.83 (m, 2H), 7.76 (dd, J = 8.4,
1
3
1
.5 Hz, 1H), 5.12 (s, 1H), 3.68 (s, 3H), 3.59 (s, 3H); C NMR (100 MHz , DMSO) δ: 168.8, 148.3,
147.4, 136.4, 136.0, 133.1, 132.8, 132.2, 130.1, 128.1, 127.9, 127.8, 127.6, 127.5, 126.7, 126.5, 126.3,
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