Synthesis and antitumor activity of new thiazole nortopsentin analogs

Article abstract of DOI:10.3390/md14120226

New thiazole nortopsentin analogs in which one of the two indole units was replaced by a naphthyl and/or 7-azaindolyl portion, were conveniently synthesized. Among these, three derivatives showed good antiproliferative activity, in particular against MCF7 cell line, with GI50 values in the micromolar range. Their cytotoxic effect on MCF7 cells was further investigated in order to elucidate their mode of action. Results showed that the three compounds act as pro-apoptotic agents inducing a clear shift of viable cells towards early apoptosis, while not exerting necrotic effects. They also caused cell cycle perturbation with significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population.

Full text of DOI:10.3390/md14120226

marine drugs
Article
Synthesis and Antitumor Activity of New Thiazole
Nortopsentin Analogs
Virginia Spanò, Alessandro Attanzio, Stella Cascioferro, Anna Carbone, Alessandra Montalbano,
Paola Barraja, Luisa Tesoriere, Girolamo Cirrincione, Patrizia Diana and Barbara Parrino *
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, STEBICEF, via Archirafi 32,
90123 Palermo, Italy; virginia.spano@unipa.it (V.S.); alessandro.attanzio@unipa.it (A.A.);
stellamaria.cascioferro@unipa.it (S.C.); anna.carbone@unipa.it (A.C.); alessandra.montalbano@unipa.it (A.M.);
paola.barraja@unipa.it (P.B.); luisa.tesoriere@unipa.it (L.T.); girolamo.cirrincione@unipa.it (G.C.);
patrizia.diana@unipa.it (P.D.)
* Correspondence: barbara.parrino@unipa.it; Tel.: +39-91-238-97413
Academic Editor: Orazio Taglialatela-Scafati
Received: 10 October 2016; Accepted: 5 December 2016; Published: 14 December 2016
Abstract: New thiazole nortopsentin analogs in which one of the two indole units was replaced
by a naphthyl and/or 7-azaindolyl portion, were conveniently synthesized. Among these, three
derivatives showed good antiproliferative activity, in particular against MCF7 cell line, with GI50
values in the micromolar range. Their cytotoxic effect on MCF7 cells was further investigated in order
to elucidate their mode of action. Results showed that the three compounds act as pro-apoptotic
agents inducing a clear shift of viable cells towards early apoptosis, while not exerting necrotic effects.
They also caused cell cycle perturbation with significant decrease in the percentage of cells in the
G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase,
and appearance of a subG1-cell population.
Keywords: marine alkaloids; bis-indolyl alkaloids; thiazolyl-indoles; apoptosis; antiproliferative activity
1. Introduction
In the latest decades, marine environment has increasingly provided a huge number of biologically
active molecules. Among marine organisms, deep-sea sponges have contributed with several
compounds endowed with antitumor activity [1–4]. The isolation of such molecules is very important
since cancer is still an important social problem, in fact it is supposed to maintain as causes of death its
primacy after heart and circulatory disorders. This scenario justifies the attention paid by a multitude
of researchers in the individuation and development of natural or synthetic heterocyclic compounds
as scaffold for antitumor agents [
sponge metabolites, useful as leads for antitumor agents. They are characterized by two indole units
linked, through their position 3, by a spacer [ 10]. The spacer can be an acyclic chain such as in
5–8]. Bis-indolyl alkaloids represent an important class of deep-sea
9,
hyrtiosin B, isolated from Hyrtios erecta [11], or a carbocyclic ring as in the case of asterriquinone,
isolated from Aspergillus fungi [12]. Heterocyclic rings can also play as spacer for bis-indolyl alkaloids.
Thus, dragmacidin isolated from the deep water sponges Dragmacidon, Halicortex bears a saturated
six-membered piperazine ring (Chart 1) [13].
Topsentins A, B1 and B2, bearing a 2-acyl imidazole spacer, were isolated from Topsentia genitrix
sponge [14].
Nortopsentins A–C, which exhibit a 2,4-disubstituted imidazole ring as a spacer, were isolated
from Spongosorites ruetzleri, and showed in vitro cytotoxicity against P388 cells [15].
Due to their interesting cytotoxicity, nortopsentins attracted remarkable attention by researchers
and several total syntheses of these natural products were reported [16–19]. Moreover, the synthesis
Mar. Drugs 2016, 14, 226; doi:10.3390/md14120226
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 226
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and antiproliferative evaluation of analogs in which the imidazole ring of the natural compounds was
replaced by several five-membered heterocycles such as bis-indolyl-thiophenes [20], -pyrazoles [21],
-furans [22], [1,2]oxazoles [22], -pyrroles [23], and -1,2,4-thiadiazoles [24] (Chart 1), many of them
showing antiproliferative activity often reaching GI50 values in the low micromolar range or even at
sub-micromolar level, were reported.
N
O
HO
O
O
OH
HO
OH
O
N
H
N
H
N
Hyrtiosin B
Asterriquinone
H
N
O
N
Br
N
R1
N
OH
H
N
H
R
N
H
N
H
Br
N
H
Br
Topsentin A R=R1=H
Topsentin B1 R=H; R1=OH;
Topsentin B2 R=Br; R1=OH;
Dragmacidin
R
R3
Y
Z
X
N
N
R1
R2
Nortopsentin A X = N; Y = CH; Z = NH; R1 = R2 = H; R = R3 = Br; Furans X = O; Y = Z = CH; R = R3 = H, Me, OMe; R1 = R2 = Me
Nortopsentin B X = N; Y = CH; Z = NH; R = R1 = R2 = H; R3 = Br; [1,2]Oxazoles X = CH; Y = N; Z = O; R = R3 = H, Me, OMe, Cl,
Br; R1 = R2 = Me
Nortopsentin C X = N; Y = CH; Z = NH; R1 = R2 = R3 = H; R = Br; Pyrroles X = NH; Y = Z = CH; R1 = R2 = Me; R = R3 = H, Me,
.
OMe, Cl, Br
Thiophenes X = S; Y = Z = CH; R = R3 = H, Me, OMe, Cl, Br;
1,2,4-Thiadiazoles X = Z = N; Y = S; R or R3 = H, OMe, NO2,
R1 = R2 =H, Me, SO2Ph
Br; R1 or R2 = H
Thiazoles X = N; Y = CH; Z = S; R1 or R2 = H, Me; R or R3 = H, OMe, Br
Pyrazoles X = CH; Y = N; Z = NH, NMe; R = R3 = H, Me, OMe, Cl, Br;
R1 = R2 = Me
Chart 1. Bis-indolyl alkaloids.
Other structural manipulation of the natural product also involved one or both indole
units producing 3-[(2-indolyl)-5-phenyl]pyridine and 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindole
derivatives, which showed significant antiproliferative activity and inhibited CDK1 (Chart 2) [25,26].
The interesting results obtained by the aza-substitution of the indole moiety, led to the
synthesis and biological evaluation of 3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-4-azaindoles and the
corresponding 1H-7-azaindole derivatives (Chart 2) [27,28]. Both series showed potent antiproliferative
activity against a wide range of cell lines, including diffuse malignant peritoneal mesothelioma
(DMPM), a fatal disease, poorly responsive to conventional therapies, and acted as CDK1 inhibitors.
Moreover, a derivative belonging to the 7-aza series, in the mouse model, by intraperitoneal
administration was effective in a significant reduction of the DMPM at well tolerated doses.

Mar. Drugs 2016, 14, 226
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R
R
S
R1
R2
OR2
N
N
N
N
N
R3
R1
3-(2-Phenyl-1,3-thiazol-4-yl)-1H-7-azaindole
3-[(2-Indolyl)-5-phenyl]pyridine
S
R2
S
X
R
N
N
N
N
Y
N
R
N
N
N
R1
R1
R2
4-Aza series: X = N; Y = CH
7-Aza series: X = CH; Y = N
1
R
S
S
R2
N
N
N
N
N
N
N
N
Cl
R1
R2
R
R1
2
3
Chart 2. Nortopsentin analogs.
Lately, three new series of nortopsentin analogs of type
1
,
2
and 3 (Chart 2) were efficiently
synthesized and exhibited remarkable antiproliferative activity against several human tumor cell
lines [29,30].
Interestingly, a derivative of the series
2 at low concentrations (GI30) caused morphological
changes typical of autophagic death with massive formation of cytoplasmic acid vacuoles without
apparent loss of nuclear material, and with arrest of cell cycle at the G1 phase, whereas higher
concentrations (GI70) induced apoptosis with arrest of cell cycle at the G1 phase [29].
Considering the interesting biological activity of nortopsentin analogs and in particular of
3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindole derivatives previously reported by us [26], herein we
report the synthesis of new derivatives of type 4, 5 (Scheme 1) and 6 (Scheme 2), in which one of the
two indole units was replaced by a naphthyl portion, to further investigate the contribution of the aryl
moiety on biological activity. The antiproliferative activity of the novel compounds was evaluated in
different human cancer cell lines and further studies were performed on the most active derivatives,
in order to clarify their mechanism of action.
2. Results and Discussion
2.1. Chemistry
3-[2-(Naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles of type
4 and 3-[2-(naphthalen-2-yl)-1,3-
thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridines of type (Table 1) were conveniently synthesized by Hantzsch
5
reaction between naphthalene-2-carbothioamide 17 and 3-haloacetyl compounds of type 11
and 16 (Scheme 1).
, 12, 15
3-Haloacetyl intermediates 11c
9c or 7-azaindoles 13a and 14a
corresponding N-methyl-1-(1H-indol-3-yl)ethanone 7b, as previously reported [29,30].
3-Haloacetyl compounds 11a and 12a were prepared (70%–85% and 60%–70%, respectively),
reacting their corresponding N-methyl or N-SO2Ph derivatives 9a and 10a with chloroacetyl
,
15a
,b and 16a,b were obtained from the corresponding indole
,
b
,b respectively, while compounds 11d was synthesized from the
,b
–c
,
b
–c
chloride (ClCOCH2Cl) in presence of aluminum chloride (AlCl3) in dichloromethane (DCM);
compound 12d was obtained (70%) from the corresponding N-SO2Ph 1-(1H-indol-3-yl)ethanone
7c using bromine in refluxing methanol (MeOH).
Reaction of the synthesized key intermediates 11a
–d, 12a–d, 15a,b and 16a,b with naphthalene-
2-carbothioamide 17 in refluxing ethanol gave the 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles

Mar. Drugs 2016, 14, 226
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4a
–
h
(48%–95%) and 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine 5a–d
(55%–85%), respectively.
O
N
iii
R
S
O
X
7a R = H
7b R = Me
7c R = SO2Ph
S
NH2
i
R1
R1
N
ii
17
vii
N
N
R
Y
R
4a-h
4i-l
5a-d
for 4e-h
iv
R1
11a-d R = Me
12a-d R = SO2Ph
viii
N
R
a R1 = OMe, X = Cl; b R1 = Br, X = Cl;
c R1 = F, X = Cl; d R1 = H, X = Br
S
8a-c R = H
NH2
vii
i
9a-c R = Me
ii
17
10a-c R = SO2Ph
O
a R1 = OMe; b R1 = Br; c R1 = F
Br
R1
R1
vi
N
R
N
R
N
N
13a,b R = H
14a,b R = Me
15a,b R = H
16a,b R = Me
v
a R1 = H; b R1 = Br
Scheme 1. Synthesis of substituted 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles
4
and
) t-BuOK, toluene,
) benzensulphonyl
3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine
TDA-1, rt, 1–24 h; ( ) MeI, rt, 30 min–2 h, 80%–98%; (ii) (
5
. Reagents: (i) (
a
◦
b
a) NaH, THF, 0 C-rt, 1 h; (
b
chloride, rt, 1–24 h, 87%–90%; (iii) Br2, methanol, reflux, 2 h, 40%–70%; (iv) AlCl3, DCM, ClCOCH2Cl,
rt, 1–5 h, 60%–85%; (v) (
DCM, BrCOCH2Br, reflux, 40 min, 70%–92%; (vii) ethanol, 60 C-reflux, 30 min–12 h, 48%–95%; and
(viii) NaOH, water, ethanol, reflux, 5–6 h, 50%–80%.
a
) t-BuOK, toluene, TDA-1, rt, 3 h; (
b
) MeI, rt, 1 h, 96%–99%; (vi) AlCl3,
◦
Table 1. 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles
4-yl]-1H-pyrrolo[2,3-b]pyridines 5.
4 and 3-[2-(naphthalen-2-yl)-1,3-thiazol-
Compound
Substrate
R
R1
Y
Yields (%)
S
R1
N
N
Y
R
4a-l
5a-d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
11a
11b
11c
11d
12a
12b
12c
12d
4e
4f
4g
4h
16a
16b
15a
15b
Me
Me
Me
Me
SO2Ph
SO2Ph
SO2Ph
SO2Ph
H
H
H
H
Me
OMe
Br
F
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
95
72
48
75
90
70
60
94
50
68
75
80
75
55
80
85
H
OMe
Br
F
H
OMe
Br
F
H
H
Br
H
Br
Me
H
H
N
N
N

Mar. Drugs 2016, 14, 226
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N-SO2Ph protected indoles 10a
–c
[31
,
32] and 1-(1H-indol-3-yl)ethanone 7c (90%) were synthesized
from the commercially available indoles 8a–c or 1-(1H-indol-3-yl)ethanone 7a by reaction with
benzenesulphonyl chloride and sodium hydride (NaH), in tetrahydrofuran (THF); whereas methylated
compounds 9a–c and 7b were prepared as previously reported [26,30].
The subsequent deprotection of N-SO2Ph derivatives 1e
under reflux afforded, after neutralization, the corresponding unprotected derivatives 4i
3-[4-(Naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6a were synthesized (Table 2), also in this
case, by Hantzsch reaction between the key intermediates indolo-3-carbothiamides 22d 23a 24a
and naphthalene-2-acetylbromide 25, performed in dimethylformamide (DMF) under reflux (Scheme 2).
In particular, reaction of naphthalene-2-acetylbromide 25 with N-Boc indolo-3-carbothiamides 24a
afforded the corresponding unprotected 3-[4-(naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6e
Indolo-3-carbothiamides 22d 23a and 24a were prepared from the corresponding indoles 8a
–
h
using sodium hydroxide in ethanol
–
l
(50%–80%).
–h
,
–d
,
–c
–c
–h.
,
–d
–c
–d,
9a
–
d
and 18a
–
c
through the formation of amides 19d
,
20a
–
d
and 21a–c as previously reported by
us [28].
S
O
NH2
NH2
R1
R1
R1
iv
N
N
R
N
R
iii
R
22d R = H
23a-d R = Me
24a-c R = Boc
8a-d R = H
9a-d R = Me
18a-c R = Boc
19d R = H
20a-d R = Me
21a-c R = Boc
i
ii
a R1 = OMe, b R1 = Br c R1 = F, d R1 = H
O
Br
v
25
S
R1
N
N
R
6a-h
Scheme 2. Synthesis of 3-[4-(naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6a–h. Reagents: (i) (a) t-BuOK,
toluene, TDA-1, rt, 6 h; (
b
) MeI, rt, 1 h, 96%–98%; (ii) Boc, triethylamine, THF, reflux, 24–48 h,
◦
90%–100%; (iii) (
a
) chlorosulphonyl isocyanide, acetonitrile, 0 C then rt 0.5–2 h or reflux, 15 min;
(b) acetone/water 8:1, KOH 10%, 40%–70%; (iv) Lawesson’s reagent, toluene, reflux, 0.5–24 h, 90%–98%;
◦
(v) DMF, 60 C-reflux, 3–24 h, 48%–99%.
Table 2. 3-[4-(Naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6a–h.
Compound
Substrate
R
R1
Yields (%)
S
R1
N
N
R
6a-h
Me
Me
Me
Me
H
6a
6b
6c
6d
6e
6f
23a
23b
23c
23d
24a
24b
24c
22d
OMe
Br
F
H
OMe
Br
98
98
75
99
48
75
60
60
H
H
H
6g
6h
F
H

Mar. Drugs 2016, 14, 226
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2.2. Biology
2.2.1. Cytotoxic Activity
All synthesized nortopsentin analogs 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles 4a
3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridines 5a–d, and 3-[4-(naphthalene-2-yl)-
1,3-thiazol-2-yl]-1H-indoles 6a
, were tested at a single dose (10−5 M) for cytotoxicity against three
–l
–h
human tumor cell lines, HCT 116 cells (colorectal carcinoma), MDA-MB-435 cells (melanoma) and
MCF-7 cells (breast cancer) by MTT assay. In Table 3 are shown the growth percentages calculated for
some of the nortopsentin analogs since those derivatives for which growth percentages higher than
90 were measured against all the three lines are not reported.
−5
Table 3. One dose (10 M) cytotoxic activity of compounds 4–6.
Growth Percent 1
Compound
HCT116
MCF-7
MDA-MB-435
4a
4c
4i
5b
5d
6a
6c
6d
6g
85.6 ± 4.3
87.6 ± 5.2
86.5 ± 4.8
91.7 ± 5.4
103.5 ± 2.3
87.9 ± 3.8
83.1 ± 4.1
95.3 ± 4.5
91.4 ± 6.4
24.9 ± 1.9
74.5 ± 4.3
84.8 ± 5.4
62.9 ± 4.2
47.1 ± 3.0
30.7 ± 3.1
50.3 ± 4.8
37.5 ± 2.2
41.6 ± 2.9
87.7 ± 4.1
87.9 ± 5.3
94.8 ± 5.5
96.4 ± 4.9
101.4 ± 3.2
70.1 ± 4.2
39.1 ± 2.7
82.2 ± 5.1
98.4 ± 4.6
1
Cells were treated with the compounds for 72 h and cell survival was measured by MTT assay in comparison
to cells treated with vehicle alone (control), as reported in Section 3.2. Values are the mean
experiments carried out in duplicate.
±
SD of two separate
Compounds 4a
their activity was further investigated on MCF-7 cells, which are the most sensitive to the cytotoxic
property of the compounds. When assayed in the concentration range 0.1–100 M, they inhibited the
, 6a and 6d appeared the most active compounds in inhibiting cell growth and
µ
growth of MCF-7 cells in dose-dependent manner (Figure 1) and on the basis of GI50 value, the drug
concentration effective in causing 50% inhibition of cell growth, compound 4a appeared the most
effective (Table 4).
Figure 1. Effect of compounds 4a, 6a and 6d on the growth of MCF-7. Cells were treated with the
compounds for 72 h and cell survival was measured by MTT assay in comparison to cells treated
with vehicle alone (control), as reported in Section 3.2. Values are the mean
experiments carried out in triplicate.
±
SD of three separate

Mar. Drugs 2016, 14, 226
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Table 4. GI50 values of the most active compounds 4a, 6a and 6d.
Compound
GI50 (µM) 1
4a
6a
6d
2.13±0.12
3.26±0.19
5.14±0.34
1
Values were calculated using non-linear regression and are the mean
carried out in triplicate.
±
SD of three separate experiments
2.2.2. Cell Death Mechanism
The mechanism of the most active compounds, 4a, 6a and 6d, in inducing cell death (necrosis
or apoptosis) was investigated by double staining with propidium iodide (PI) and Annexin V-FITC
followed by cytofluorimetric analysis. As shown in Figure 2, all three compounds induced a clear
shift of viable cells towards early apoptosis in MCF-7 cells after 24 h treatment, while did not exert
necrotic effects.
Figure 2. Flow cytometric analysis for the quantification by Annexin V/PI double staining of
compounds 4a, 6a and 6d induced apoptosis in MCF-7 cells. Cell monolayers were incubated in
the absence (control) or in the presence of the compounds at their relevant GI50 values. After 24 h
incubation, cells were submitted to double staining with Annexin V/PI as reported in Section 3.2. U3,
viable cells (Annexin V
apoptosis (Annexin V+/PI+); U1, necrotic cells (Annexin V
experiments with comparable results.
−
/PI
−
); U4, cells in early apoptosis (Annexin V+/PI
−
); U2, cells in tardive
−
/PI+). Representative images of three
2.2.3. Cell Cycle Analysis
The distribution of MCF-7 cells in the cell cycle phases after 24 h treatment with the three
compounds 4a 6a and 6d, was assessed by flow cytometric analysis after staining of DNA with PI. All
,
synthesized compounds caused a significant decrease in the percentage of cells in the G0/G1 and S
phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance
of a subG1-cell population (Figure 3).
Figure 3. Cell cycle analysis of MCF-7 cells treated with compounds 4a, 6a and 6d. Cell monolayers
were incubated in the absence (control) or in the presence of the compounds at their relevant GI50 values.
After 24 h incubation, propidium iodide-stained cells were submitted to flow cytometric analysis as
reported in Section 3.2. Representative images of three experiments with comparable results.

Mar. Drugs 2016, 14, 226
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3. Materials and Methods
3.1. Chemistry
3.1.1. General
All melting points were taken on a Büchi-Tottoly capillary apparatus. IR spectra were determined
in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1H and 13C NMR spectra were
measured at 200 and 50.0 MHz, respectively, in dimethylsulfoxide (DMSO)-d6 solution, using a Bruker
1
Avance II series 200 MHz spectrometer. Compounds 5c
,d
were characterized only by H NMR
spectra because of their poor solubility. Column chromatography was performed with Merk silica gel
230–400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge system).
Elemental analyses (C, H, N) were within
VARIO EL III elemental analyzer. Purity of all the tested compounds was greater than 98%, determined
by HPLC. Compounds 7b 30], 9a 26], 10a 31 32], 11c 30], 14a 15a 16a , [29 18a–c,
±
0.4% of theoretical values and were performed with a
[
–
d
[
–
c
[
,
,d
[
,b,
,b,
,b
]
20a–d, 21a–c, 23a–d and 24a–c [28] were prepared as previously described by us.
3.1.2. Synthesis of 1-[1-(Phenylsulfonyl)-1H-indol-3-yl]ethanone (7c)
To a solution of the 3-acetylindole 7a (12.6 mmol) in anhy◦drous THF (15.0 mL) sodium hydride
(60% dispersion in mineral oil, 0.6 g, 12.6 mmol) was added at 0 C and the mixture was stirred at room
temperature for 1 h. Benzensulfonyl chloride (1.6 mL, 12.6 mmol) was added and the mixture was
stirred at room temperature for 1–24 h. The residue was evaporated under reduced pressure, treated
with water (50 mL) and extracted with EtOAc (3
evaporated under reduced pressure and purified by column chromatography using DCM as eluent.
Yield 90%; analytical and spectroscopic data were previously reported [33].
×
50 mL). The organic phase was dried (Na2SO4),
3.1.3. Synthesis of Substituted 2-Chloro-1-(1-methyl-1H-indol-3-yl)ethanones (11a,b) and
2-Chloro-1-[1-(phenylsulfonyl)-1H-indol-3-yl]ethanones (12a–c)
A solution of the suitable indole 9a,b, 10a–c (3.1 mmol) in anhydrous DCM (12.0 mL) was added
dropwise at 0 ◦C, under nitrogen atmosphere, to a stirred suspension of aluminum chloride (2.9 g,
21.7 mmol) in anhydrous DCM (46.0 mL). Then, chloroacetyl chloride (0.8 mL, 9.3 mmol) was slowly
added to the reaction mixture, which was stirred at room temperature for 1–5 h and then poured in
ice and water (60 mL) and extracted with DCM (3
evaporated under reduced pressure and purified by column chromatography using DCM as eluent.
×
60 mL). The organic phase was dried (Na2SO4),
2-Chloro-1-(5-methoxy-1-methyl-1H-indol-3-yl)ethanone (11a)
◦
−1
;
Conditions: room temperature for 1 h; white solid; yield 85%; mp: 227–228 C; IR: 1653 (CO) cm
1H NMR (200 MHz, DMSO-d6)
: 3.80 (s, 3H, CH3), 3.85 (s, 3H, CH3), 4.80 (s, 2H, CH2), 6.94 (dd,
1H, J = 2.5, 8.9 Hz, H-6), 7.49 (d, 1H, J = 8.9 Hz, H-7), 7.68 (d, 1H, J = 2.5 Hz, H-4), 8.40 (s, 1H, H-2);
13C NMR (50 MHz, DMSO-d6)
: 33.4 (q), 45.9 (t), 55.1 (q), 102.8 (d), 111.6 (d), 112.1 (s), 112.8 (d), 126.5
δ
δ
(s), 132.0 (s), 138.2 (d), 155.9 (s), 185.3 (s). Anal. Calcd. for C12H12ClNO2 C (60.64%) H (5.09%) N
(5.89%) found C (60.32%) H (5.12%) N (5.75%).
1-(5-Bromo-1-methyl-1H-indol-3-yl)-2-chloroethanone (11b)
◦
Conditions: room temperature for 5 h; dark brown solid; yield 70%; mp: 175–176 C; IR 1658 (CO);
1H NMR (200 MHz, DMSO-d6)
δ
: 3.89 (s, 3H, CH3), 4.85 (s, 2H, CH2), 7.46 (dd, J = 2.0, 8.7 Hz, 1H, H-6),
7.59 (d, J = 8.7 Hz, 1H, H-7), 8.30 (d, J = 2.0 Hz, 1H, H-4), 8.51 (s, 1H, H-2); 13C NMR (50 MHz, DMSO)
: 33.6 (q), 46.2 (t), 111.8 (s), 113.2 (d), 115.4 (s), 123.3 (d), 125.8 (d), 127.4 (s), 136.1 (s), 139.3 (d), 186.8 (s).
Anal. Calcd. for C11H9BrClNO C (46.11%) H (3.17%) N (4.89%) found C (46.28%) H (3.54%) N (5.01%).
δ

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2-Chloro-1-[5-methoxy-1-(phenylsulfonyl)-1H-indol-3-yl]ethanone (12a)
Conditions: room temperature for 1 h; dark brown solid; yield 70%; mp: 168–169 ◦C; IR: 1683
(CO), 1448, 1477 (SO2) cm−1; 1H NMR (200 MHz, DMSO)
: 3.79 (s, 3H, CH3), 5.15 (s, 2H, CH2), 7.05
(dd, 1H, J = 2.6, 9.1 Hz, H-6), 7.82–7.58 (m, 4H, ArH), 7.88 (d, 1H, J = 9.1 Hz, H-7), 8.20–8.05 (m, 2H,
ArH), 8.92 (s, 1H, H-2); 13C NMR (50 MHz, DMSO-d6)
: 47.1 (t), 55.4 (q), 104.2 (d), 114.0 (d), 115.0 (d),
δ
δ
117.6 (s), 127.1 (dx2), 128.1 (s), 128.4 (s), 130.1 (dx2), 134.9 (d), 135.3 (d), 136.3 (s), 157.2 (s), 187.4 (s).
Anal. Calcd. for C17H14ClNO4S C (56.12%) H (3.88%) N (3.85%) found C (55.91%) H (3.98%) N (4.07%).
1-[5-Bromo-1-(phenylsulfonyl)-1H-indol-3-yl]-2-chloroethanone (12b)
Conditions: room temperature for 1 h; brown solid; yield 70%; mp: 168–169 ◦C; IR: 1689 (CO),
1
1366, 1442 (SO2) cm−1; H NMR (200 MHz, DMSO-d6)
δ: 5.20 (s, 2H, CH2), 7.63–7.75 (m, 4H, H-6,
ArH), 8.00 (d, 1H, J = 8.9 Hz, H-7), 8.17–8.21 (m, 2H, H-4, ArH), 8.32 (d, 1H, J = 1.8 Hz, ArH), 9.07
(s, 1H, H-2); 13C NMR (50 MHz, DMSO-d6)
δ: 47.1 (t), 115.1 (d), 116.9 (s), 118.0 (s),124.3 (s), 127.2
(dx2), 128.7 (d), 128.8 (d), 130.2 (dx2), 132.8 (s), 134.5 (d), 135.6 (d), 136.0 (s), 187.3 (s). Anal. Calcd. for
C16H11BrClNO3S C (46.57%) H (2.69%) N (3.39%) found C (46.35%) H (2.87%) N (3.25%).
2-Chloro-1-[5-fluoro-1-(phenylsulfonyl)-1H-indol-3-yl]ethanone (12c)
Conditions: room temperature for 1 h; light brown; yield 60%; mp: 126–127 ◦C; IR: 1688 (CO),
1376, 1447 (SO2) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ
: 5.16 (s, 2H, CH2), 7.33 (td, 1H, J = 2.7, 9.2 Hz,
H-6), 7.66 (m, 3H, ArH, H-7), 7.86 (dd, 1H, J = 9.2, 2.6 Hz, ArH), 8.02 (dd, 1H, J = 9.2, 4.3 Hz, ArH),
8.16 (m, 2H, ArH), 9.04 (s, 1H, H-2); 13C NMR (50 MHz, DMSO-d6)
: 47.1 (t), 107.6 (d, JC4-F = 25.1 Hz),
δ
113.9 (d), 114.8 (d, JC7-F = 9.6 Hz), 114.9 (d), 117.4 (s), 117.5 (s), 127.2 (dx2), 130.2 (dx2), 130.4 (s), 135.8
(d, JC6-F = 21.9 Hz), 136.1 (s), 159.2 (s, JC5-F = 299.5 Hz) 187.3 (s); Anal. Calcd. for C16H11ClFNO3S C
(54.63%) H (3.15%) N (3.98%) found C (54.38%) H (2.87%) N (4.22%).
3.1.4. Synthesis of 3-(1-Benzenesulfonyl-1H-indol-3-yl)-2-bromoethanone (12d)
To a stirred solution of 1-[1-(phenylsulfonyl)-1H-indol-3-yl]ethanone 7c (0.5 g, 1.7 mmol) in
ethanol (15.0 mL), bromine (0.1 mL, 2 mmol) was added dropwise under nitrogen atmosphere.
The reaction mixture was heated under reflux for 2 h. After cooling the solvent was evaporated
under reduced pressure. The residue was treated with water (20 mL), made alkaline by adding
sodium hydrogen carbonate (150 mg) and extracted with EtOAc (3
×
50 mL). The organic phase
was dried (Na2SO4), evaporated under reduced pressure and purified by column chromatography
using cycloexane/ethyl acetate 95:5 as eluent. Yield 70%; analytical and spectroscopic data were in
accordance with those previously reported [34].
3.1.5. Synthesis of 5-Substituted-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1-(protected)-1H-indoles (4a–h)
A suspension of the proper 3-haloacetyl derivative 11a–d or 12a–d (0.84 mmol) and
naphthalene-2-carbothioamide 17 (0.16 g, 0.84 mmol), in anhydrous ethanol (5.0 mL), was heated
under reflux for 30 min–6 h or at 60 ◦C for 12 h. The solid formed was filtered, dried, an purified by
column chromatography using cycloexane/ethyl acetate as eluent.
5-Methoxy-1-methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4a)
Conditions: reflux for 1 h; cycloexane/ethyl acetate 7:3; light yellow solid; yield 95%; mp:
◦
168–169 C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.85 (s, 3H, CH3), 3.90 (s, 3H, CH3), 6.99 (dd, 1H, J = 2.4,
8.9 Hz, H-6”), 7.42 (d, 1H, J = 8.9 Hz, H-7”), 7.58–7.63 (m, 1H, ArH), 7.72 (d, 1H, J = 2.4 Hz, H-4”), 7.88
(s, 1H, H-2”), 7.97–8.02 (m, 3H, ArH), 8.06–8.13 (m, 2H, ArH), 8.21 (dd, 1H, J = 1.7, 8.6 Hz, ArH), 8.61
(s, 1H, H-5); 13C NMR (50 MHz, DMSO-d6)
δ
: 32.7 (q), 55.4 (q), 120.2 (d), 109.5 (s), 190.9 (d), 110.0 (s),
111.0 (d), 111.6 (d), 123.6 (d), 125.3 (d), 127.0 (d), 127.1 (d), 127.8 (d), 128.5 (d), 128.9 (d), 129.7 (d), 130.7

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(s), 130.3 (s), 132.9 (s), 133.6 (s), 151.7 (s), 154.3 (s), 165.8 (s). Anal. Calcd. for C23H18N2OS C=C (74.57%)
H (4.90%) N (7.56%) found C (74.85%) H (4.63%) N (7.73%).
5-Bromo-1-methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4b)
Conditions: reflux for 1 h; cycloexane/ethyl acetate 7:3; light brown solid; yield 72%; mp:
124–125 ◦C; 1H NMR (200 MHz, DMSO-d6)
δ: 3.90 (s, 3H, CH3), 7.40 (dd, 1H, J = 1.8, 8.7 Hz, H-6”),
7.50 (d, 1H, J = 8.7, H-7”), 7.60 (m, 2H, ArH), 7.97–8.03 (m, 2H, H-4”, H-2”), 8.08–8.12 (m, 3H, ArH),
8.20 (dd, 1H, J = 1.8, 9.6 Hz, ArH), 8.40 (d, 1H, J = 1.8 Hz; ArH), 8.60 (s, 1H, H-5); 13C NMR (50 MHz,
DMSO-d6) δ: 32.9 (q), 109.6 (s), 111.0 (d), 112.4 (d), 112.9 (s), 112.5 (d), 116.4 (s), 123.5 (d), 124.2 (d),
125.4 (d), 126.5 (s), 127.0 (d), 127.2 (d), 127.8 (d), 128.5 (d), 128.9 (d), 130.6 (d), 132.9 (s), 133.6 (s), 135.7
(s), 150.8 (s), 166.2 (s). Anal. Calcd. for C22H15BrN2S C (63.01%) H (3.61%) N (6.68%) found C (62.89%)
H (3.85%) N (6.44%).
5-Fluoro-1-methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4c)
◦
◦
Conditions: 60 C for 12 h; cycloexane/ethyl acetate 7:3; brown solid; yield 48%; mp: 151–152 C;
1H NMR (200 MHz, DMSO-d6)
: 3.90 (s, 3H, CH3), 7.15 (td, 1H, J = 2.4, 9.1 Hz, H-6”), 7.53–7.65
(m, 3H, ArH, H-7”, H-4”), 7.93 (s, 1H, H-2”), 7.98–8.04 (m, 2H, ArH), 8.07–8.15 (m, 3H, ArH), 8.22
(dd, 1H, J = 1.6, 8.6 Hz, ArH ), 8.61 (s, 1H, H-5); 13C NMR (50 MHz, DMSO-d6)
: 32.9 (q), 105.2 (d,
C-4”-F = 23.9 Hz), 109.6 (d), 110.1 (d), 110.4 (d), 111.4 (d), 111.6 (d), 123.6 (d), 125.0 (s), 125.1 (s), 125.3
δ
δ
J
(d), 127.0 (d), 127.5 (d JC-6”-F = 29.4 Hz), 128.7 (d, JC-7”-F = 16.3 Hz), 131.0 (d), 130.6 (s), 132.9 (s), 133.6
(s), 133.8 (s), 151.1 (s), 166.0 (s). Anal. Calcd. for C22H15FN2S C (73.72%) H (4.22%) N (7.82%) found C
(73.98%) H (4.56%) N (7.631%).
1-Methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4d)
Conditions: reflux for 1 h; cycloexane/ethyl acetate 7:3; brown solid; yield 75%; mp: 174–175 ◦C;
1H NMR (200 MHz, DMSO-d6)
δ
: 3.90 (s, 3H, CH3), 7.19–7.32 (m, 2H, H-6”, H-7”), 7.52–7.65 (m, 2H,
H-4”, ArH), 7.88 (s, 1H, H-2”), 7.98–8.15 (m, 4H, ArH), 8.20–8.28 (m, 2H, ArH), 8.61 (s, 1H, H-5);
13C NMR (50 MHz, DMSO-d6)
: 109.9 (s), 110.2 (d), 110.3 (d), 120.0 (d), 120.3 (d), 121.7(d), 124.9 (s),
δ
125.3 (d), 126.7 (d), 127.0 (d), 127.1 (d), 127.8 (d), 128.6 (d), 128.9 (d), 129.2 (d), 130.7 (s), 132.9 (s), 133.6
(s), 137.0 (s), 151.6 (s), 165.9 (s). Anal. Calcd. for C22H16N2S C (77.62%) H (4.74%) N (8.23%) found C
(77.45%) H (4.79%) N (7.98%).
5-Methoxy-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1-(phenylsulfonyl)-1H-indole (4e)
Conditions: reflux for 1 h; cycloexane/ethyl acetate 9:1; white solid; yield 90%; mp: 168–169 ◦C;
IR: 1451, 1526 (SO2) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ: 3.89 (s, 3H, CH3), 7.06 (dd, 1H, J = 2.5,
9.0 Hz, H-6”), 7.56–7.75 (m, 5H, H-7”, ArH), 7.82 (d; 1H, J = 2.5 Hz, H-4”), 7.92–8.14 (m, 6H, ArH), 8.23
(dd, 1H, J = 1.7, 8.6 Hz, ArH), 8.33 (s, 1H, H-2”), 8.44 (s, 1H, H-5), 8.64 (m, 1H, ArH); 13C NMR (50 MHz,
DMSO-d6) δ: 55.43 (q), 104.3 (d), 110.3 (s), 114.1 (d), 114.2 (d), 115.5 (d), 117.6 (s), 123.5 (d), 125.5 (d),
125.6 (d), 126.7 (d), 127.1 (dx2), 127.3 (d), 127.8 (d), 128.6 (d), 128.9 (d), 129.2 (s), 129.9 (dx2), 130.3 (s),
132.9 (s), 133.7 (s), 134.7 (d), 136.7 (s), 148.6 (s), 156.5 (s), 166.8 (s). Anal. Calcd. for C28H20N2O3S2 C
(67.72%) H (4.06%) N (5.64%) found C (67.55%) H (4.23%) N (5.77%).
5-Bromo-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1-(phenylsulfonyl)-1H-indole (4f)
Conditions: reflux for 6 h; cycloexane/ethyl acetate 8:2; white solid; yield 70%; mp: 216–217 ◦C;
IR: 1451, 1526 (SO2) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ
: 7.57–7.78 (m, 6H, ArH), 7.96–8.04 (m, 2H,
ArH), 8.08–8.14 (m, 4H, ArH), 8.20 (dd, 1H, J = 1.8, 8.1 Hz, ArH), 8.38 (s, 1H, H-2”), 8.52 (d, 1H, J = 1.8,
ArH), 8.56 (s, 1H, H-5), 8.62 (s, 1H, ArH); 13C NMR (50 MHz, DMSO-d6)
: 115.3 (d),116.1 (d), 116.8 (s),
δ
117.0 (s), 123.5 (d), 124.2 (d), 125.7 (d), 126.1 (d), 126.8 (d), 127.1 (dx2), 127.4 (d), 127.8 (d), 128.0 (d),
128.6 (d), 129.0 (d), 129.6 (s), 130.0 (dx2), 130.2 (s), 132.9 (s), 133.5 (s), 133.7 (s), 135.0 (d), 136.5 (s), 147.9

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(s), 167.1 (s). Anal. Calcd. for C27H17BrN2O2S2 C (59.45%) H (3.14%) N (5.14%) found C (59.69%) H
(3.34%) N (5.38%).
5-Fluoro-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1-(phenylsulfonyl)-1H-indole (4g)
Conditions: reflux for 2 h; cycloexane/ethyl acetate 8:2; white solid; yield 60%; mp: 193–194 ◦C;
IR: 1447, 1375 (SO2) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ
: 7.34 (td, J = 2.5, 9.1 Hz, H-6”), 7.57–7.77
(m, 5H, ArH), 7.97–8.04 (m, 1H, ArH), 8.06–8.17 (m, 6H, ArH ), 8.22 (dd, 1H, J = 1.7, 8.6 Hz, ArH)
8.36 (s, 1H, H-2”), 8.59 (s, 1H, H-5), 8.63 (m, 1H, ArH); 13C NMR (50 MHz, DMSO-d6)
: 107.5 (d,
C4”-F = 25.5 Hz), 113.0 (d), 113.6 (d), 114.8 (d, JC7”-F = 9.5 Hz), 115.7 (d), 123.5 (d), 123.6 (d), 125.7 (s),
δ
J
126.6 (d), 126.8 (dx2), 127.0 (d), 127.6 (d, JC6”-F = 21.1 Hz), 128.6 (d), 128.9 (d), 130.0 (dx2), 131.2 (s), 132.8
(s), 133.7 (s), 134.9 (s), 135.0(d), 136.5 (s), 148.2 (s), 165.9 (s) 167.0 (s). Anal. Calcd. for C27H17FN2O2S2
C (66.92%) H (3.54%) N (5.78%) found C (66.73%) H (3.31%) N (5.64%).
3-[2-(Naphthalen-2-yl)-1,3-thiazol-4-yl]-1-(phenylsulfonyl)-1H-indole (4h)
Conditions: reflux for 30 min; cycloexane/ethyl acetate 9:1;yellow solid; yield 94%; mp:
◦
1
199–200 C; IR: 1447, 1373 (SO2) cm−1; H NMR (200 MHz, DMSO-d6)
δ
: 7.41–7.52 (m, 2H, ArH),
7.57–7.75 (m, 5H, ArH), 7.98–8.17 (m, 6H, ArH), 8.23 (dd, 1H, J = 1.8, 8.6 Hz, ArH), 8.33 (s, 1H, H-2”),
8.39 (m, 1H, ArH ), 8.50 (s; 1H, H-5), 8.65 (m, 1H, ArH); 13C NMR (50 MHz, DMSO-d6)
: 113.2 (s),
δ
113.3 (d), 115.6 (d), 117.6 (s), 121.9 (d), 123.6 (d), 124.2 (d), 124.8 (d), 125.4 (d), 125.7 (d), 126.8 (dx2),
127.0 (d), 127.3 (d), 127.8 (d), 128.6 (d), 128.9 (d), 129.9 (dx2), 130.2 (s), 132.9 (s), 133.47 (s), 134.6 (s),
134.8 (d), 136.7 (s), 148.6 (s), 166.9 (s). Anal. Calcd. for C27H18N2O2S2 C (69.50%) H (3.89%) N (6.00%)
found C (69.35%) H (3.98%) N (5.73%).
3.1.6. Synthesis of 5-Substituted-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles (4i–l)
To a suspension of the proper 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1-(phenylsulfonyl)-1H-indole
4e–h (0.3 mmol) in ethanol (6.5 mL), a solution of sodium hydroxide (1.74 mmol, 0.07 g) in water (4.0 mL)
was added. The reaction mixture was heated under reflux for 5–6 h. The solvent was evaporated
under reduced pressure, and the resulting mixture neutralized with HCl 3N (2.0 mL) and extracted in
ethyl acetate (3
×
50 mL). The organic phase was dried (Na2SO4), evaporated under reduced pressure
and purified by column chromatography using cycloexane/ethyl acetate 7:3 as eluent.
5-Methoxy-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (4i)
◦
Conditions: reflux for 6 h; yellow solid; yield 50%; mp: 175–176 C; IR: 3378 (NH) cm−1; 1H NMR
(200 MHz, DMSO-d6) : 3.90 (s, 3H, CH3), 6.85 (dd, 1H, J = 2.3, 8.8 Hz, H-6”), 7.39 (d, 1H, J = 8.8 Hz,
δ
H-7”), 7.58–7.63 (m, 2H, ArH), 7.72 (d, 1H, J = 2.3 Hz, H-4”), 7.89 (s, 1H, H-2”), 7.98–8.01 (m, 2H,
ArH), 8.07–8.14 (m, 2H, ArH), 8.23 (dd, 1H, J = 1.5, 8.6 Hz, ArH), 8.62 (s, 1H, H-5), 11.34 (s, 1H, NH);
13C NMR (50 MHz, DMSO-d6)
δ: 55.4 (q), 102.0 (d), 109.9 (d), 110.6 (s), 111.7 (d), 112.5 (d), 125.6 (s),
123.6 (d), 125.0 (d), 125.3 (d), 127.0 (d), 127.1 (d), 127.8 (d), 128.5 (d), 128.9 (d), 130.8 (s), 131.7 (s), 132.9
(s), 133.6 (s), 152.2 (s), 154.0 (s), 165.7 (s). Anal. Calcd. for C22H16N2OS C (74.13%) H (4.52%) N (7.86%)
found C (73.88%) H (4.71%) N (8.03%).
5-Bromo-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4j)
◦
Conditions: reflux for 6 h; yellow solid; yield 68%; mp: 265–266 C; IR: 3608 (NH) cm−1; 1H NMR
(200 MHz, DMSO-d6) δ: 7.31 (dd, 1H, J = 1.9, 8.6 Hz, H-6”), 7.46–7.50 (m, 1H, H-7”), 7.59–7.64 (m, 2H,
ArH), 7.95 (s, 1H, H-2”), 7.98–8.03 (m, 1H, ArH), 8.08–8.13 (m, 3H, ArH), 8.22 (dd, 1H, J = 1.8, 8.9 Hz,
ArH), 8.40 (d, 1H, J = 1.8 Hz, ArH), 8.60 (s, 1H, H-5), 11.41 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
δ
: 110.8 (d), 110.9 (d), 122.3 (d), 123.5 (d), 124.12 (d), 125.4 (d), 126.9 (d), 127.0 (d), 127.2 (d), 127.8 (d),
128.5 (d), 128.9 (d), 130.7 (s), 132.9 (s), 133.6 (s), 135.5 (s), 141.8 (s), 151.4 (s), 154.2 (s), 161.6 (s), 166.0 (s).
Anal. Calcd. for C21H13BrN2S C (62.23%) H (3.23%) N (6.91%) found C (62.48%) H (3.55%) N (6.75%)

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5-Fluoro-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (4k)
Conditions: reflux for 5 h; white solid; yield 75%; mp: 227–228 ◦C; IR: 3124 (NH) cm−1; 1H NMR
(200 MHz, DMSO-d6) : 7.06 (td, 1H, J = 2.5, 9.2 Hz, H-6”), 7.49 (dd, 1H, J = 4.7, 8.9 Hz, ArH), 7.60 (m,
2H, ArH), 7.95 (s, 1H, H-2”), 7.98–8.14 (m, 5H, ArH), 8.24 (dd, 1H, J = 1.7, 7.8 Hz, ArH), 8.92 (s, 1H,
H-5), 11.62 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
: 105.0 (d, JC4”-F = 24.3 Hz), 109.6 (d), 110.2
δ
δ
(d, JC6”-F = 24.3 Hz) 111.0 (s), 111.1 (s), 112.8 (d), 124.7 (s), 124.9 (s), 123.6 (d), 125.3 (d), 126.9 (s), 127.0
(d, JC7”-F = 3.3 Hz), (d), 127.2 (d), 127.8 (d), 128.6 (d), 128.8 (d), 130.7 (s), 132.9 (s), 133.3 (s), 151.6 (s),
166.0 (s). Anal. Calcd. for C21H13FN2S2 C (73.23%) H (3.80%) N (8.13%) found C (72.91%) H (4.05%)
N (8.44%)
3-[2-(Naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indole (4l)
Conditions: reflux for 5 h; light yellow solid; yield 80%; mp: 260–261 ◦C; IR: 2998 (NH) cm−1
1H NMR (200 MHz, DMSO-d6)
: 7.18–7.22 (m, 2H, ArH), 7.47–7.52 (m, 1H, ArH), 7.58–7.65 (m, 2H,
ArH), 7.90 (s, 1H, H-2”), 7.98–8.16 (m, 4H, ArH), 8.21–8.28 (m, 2H, ArH), 8.62 (s, 1H, H-5), 11.50 (s, 1H,
NH); 13C NMR (50 MHz, DMSO-d6)
: 110.2 (d), 110.8 (s), 111.9 (d), 119.8 (d), 120.2 (d), 121.6 (d), 123.7
;
δ
δ
(d), 124.6 (s), 125.0 (d), 125.3 (d), 127.0 (d), 127.1 (d), 127.8 (d), 128.6 (d), 128.9 (d), 130.7 (s), 132.9 (s),
133.6 (s), 136.3 (s), 152.1 (s), 165.8 (s). Anal. Calcd. for C21H14N2S C (77.27%) H (4.32%) N (8.58%)
found C (77.48%) H (4.25%) N (8.85%).
3.1.7. Synthesis of 3-[2-(Naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridines (5a–d)
To a suspension of naphthalene-2-carbothioamide 17 (0.07 g, 0.4 mmol) in anhydrous ethanol
(15.0 mL), the proper 3-bromo-acetyl-pyrrolo[2,3-b]pyridine 15a,b or 16a,b (0.4 mmol) was added.
The resulting mixture was heated under reflux for 5–6 h. After cooling, the precipitate formed was
filtered off and recrystallized from ethanol.
1-Methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (5a)
◦
Conditions: reflux for 5 h; yellow solid; yield: 75%; mp: 294–295 C; 1H NMR (200 MHz, DMSO-d6)
δ
: 3.96 (s, 3H, CH3), 7.40 (dd, 1H, J = 5.0, 7.9 Hz, H-5”), 7.61 (m, 2H, ArH), 7.98–8.07 (m, 2H, H-2”,
ArH), 8.11–8.15 (m, 2H, ArH), 8.22 (dd, 1H, J = 1.7, 8.6 Hz, ArH ), 8.32 (s, 1H, H-5), 8.46 (dd, 1H, J = 1.3,
5.0 Hz, H-6”), 8.63 (m, 1H, ArH), 8.84 (dd,1H, J = 1.3, 7.9 Hz, H-4”); 13C NMR (50 MHz, DMSO-d6)
δ:
31.6 (q), 109.1 (s), 111.7 (d), 116.3 (d), 118.7 (s), 123.6 (d), 125.4 (d), 127.0 (d), 127.2 (d), 127.8 (d), 128.6
(d), 128.9 (d), 129.7 (d), 130.4 (s), 131.3 (d), 132.9 (s), 133.6 (s), 140.8 (d), 145.3 (s), 150.3 (s), 166.5 (s).
Anal. Calcd per C21H15N3S: C (73.87%) H (4.43%) N (12.31%) found: C (73.62%) H (4.67%); N (12.60%).
5-Bromo-1-methyl-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (5b)
◦
Conditions: reflux for 4 h; yellow solid; yield: 55%; mp 252–253 C; 1H NMR (200 MHz, DMSO-d6)
δ
: 3.90 (s, 3H, CH3), 7.58–8.63 (m, 2H, ArH), 7.96–8.01 (m, 1H, ArH), 8.05–8.12 (m, 3H, H-2”, ArH), 8.18
(dd, 1H, J = 1.7, 8.4 Hz, ArH), 8.28 (s, 1H, H-5), 8.42 (d, 1H, J = 2.1 Hz, ArH), 8.58–8.61 (m, 1H, ArH),
8.78 (d, 1H, J = 2.1 Hz, ArH); 13C NMR (50 MHz, DMSO-d6)
δ: 31.2 (q), 108.2 (s), 111.7 (s), 111.8 (d),
118.8 (s), 123.5 (d), 125.5 (d), 127.0 (d), 127.2 (d), 127.7 (d), 128.5 (d), 128.9 (d), 130.4 (d), 130.5 (s), 130.6
(d), 132.9 (s), 133.6 (s), 143.1 (d), 146.0 (s), 150.0 (s), 166.4 (s). Anal. Calcd per C21H14BrN3S: C (60.01%)
H (3.36%) N (10.00%) found: C (59.85%) H (3.60%) N (10.15%).
3-[2-(Naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (5c)
Conditions: reflux for 4 h; light brown solid; yield: 80%; mp: 277–278 ◦C; IR: 3126 (NH) cm−1
1H NMR (200 MHz, DMSO-d6)
: 7.35–7.43 (m, 1H, ArH), 7.59–7.65 (m, 2H, ArH), 7.99–8.04 (m, 1H,
;
δ
Ar), 8.07 (s, 1H, H-2”), 8.12–8.18 (m, 2H, H-5, ArH), 8.21–8.34 (m, 2H, ArH), 8.41–8.44(m, 1H, ArH), 8.63
(d, 1H, J = 9.5 Hz, ArH), 8.88 (t, 1H, J = 7.7 Hz, ArH), 12.40 (bs, 1H, NH); Anal. Calcd per C20H13N3S:
C (73.37%) H (4.00%) N 12.83 found C (73.39%) H (4.11%) N (12.65%).

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5-Bromo-3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (5d)
Conditions: reflux for 4 h; white solid; yield: 85%; mp 300–301 ◦C; IR: 2906 (NH) cm−1; 1H NMR
(200 MHz, DMSO-d6) : 7.62–7.67 (m, 2H, ArH), 7.58–8.03 (m, 1H, ArH), 8.09–8.13 (m, 3H, H-2”, ArH),
δ
8.20–8.25 (m, 2H, H-5, ArH), 8.39 (bs, 1H, ArH), 8.62 (bs, 1H, Ar), 8.82 (bs, 1H, Ar), 12.30 (bs, 1H,
NH). Anal. Calcd per C20H12BrN3S: C (59.12%) H (2.98%) N (10.34%) found C (59.29%) H (3.15%)
N (10.71%).
3.1.8. Synthesis of 3-[4-(Naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indoles (6a–h)
To a solution of the proper indolo-3-carbothioamide 22d, 23a–d, 24a–c (0.91 mmol) in
dimethylformamide (6.0 mL), naphthalene-2-acetylbromide 25 (0.23 g, 0.91 mmol) was added. The
◦
resulting reaction mixture was heated for 3–6 h at 60 C or for 24 h at reflux. After reaction completion,
monitored by TLC, water (12.0 mL) was added and the formed precipitate was filtered off. The crude
obtained was then purified by column chromatography using cycloexane/ethyl acetate 7:3 as eluent.
5-Methoxy-1-methyl-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6a)
◦
◦
Conditions: 60 C for 6 h; brown solid; yield 98%; mp: 126–127 C; cycloexane/ethyl acetate 95:5;
1H NMR (200 MHz, DMSO-d6)
: 3.88 (s, 3H, CH3), 3.94 (s, 3H, CH3), 6.97 (dd, 1H, J = 2.5, 8.9 Hz,
H-6”), 7.47–7.58 (m, 3H, ArH), 7.93–8.05 (m, 4H, ArH), 8.08 (s, 1H, H-2”), 8.18 (s, 1H, H-5), 8.22 (dd,
1H, J = 1.6, 8.6 Hz, ArH), 8.66 (s, 1H, ArH); 13C NMR (50 MHz, DMSO-d6)
: 33.0 (q), 55.2 (q), 102.3 (d),
δ
δ
109.2 (s), 110.9 (d), 111.5 (d), 112.3(d), 124.3 (d), 124.6 (d), 125.2 (d), 126.1 (s), 126.6 (d), 127.6 (d), 128.2
(d), 128.3 (d), 130.9 (d), 131.9 (s), 132.2 (s), 132.6 (s), 133.2 (s), 153.7 (s), 155.0 (s), 162.7 (s). Anal. Calcd.
for C23H18N2OS C (74.57%) H (4.90%) N (7.56%) found C (74.25%) H (5.15%) N (7.37%).
5-Bromo-1-methyl-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6b)
Conditions: 60 ◦C for 6 h; cycloexane/ethyl acetate 9:1; orange solid; yield 98%; mp: 182–183 ◦C;
1H NMR (200 MHz, DMSO-d6)
δ:3.90 (s, 3H, CH3), 7.45 (dd, 1H, J = 1.9, 8.8 Hz, H-6”), 7.51–7.61 (m,
3H, ArH), 7.95 (d, 1H, J = 2.8 Hz, ArH), 7.98–8.06 (m, 2H, ArH), 8.12 (s, 1H, H-2”), 8.20 (dd, 1H, J = 1.4,
8.6 Hz, ArH), 8.29 (s, 1H, H-5), 8.54 (d, 1H, J = 1.7 Hz, ArH), 8.61 (s, 1H, ArH); 13C NMR (50 MHz,
DMSO-d6) δ: 33.1 (q), 109.0 (s), 111.6 (d), 112.9 (d), 113.9 (s), 122.7 (d), 124.3 (d), 124.6 (d), 125.0 (d),
126.1(s), 126.2 (d), 126.6 (d),127.7 (d), 128.1 (d), 128.4 (d), 131.7 (s), 132.1 (d), 132.6 (s), 133.1 (s), 135.8 (s),
154.0 (s), 162.0 (s). Anal. Calcd. for C22H15BrN2S C (63.01%) H (3.61%) N (6.68%) found C (63.36%) H
(3.41%) N (6.85%).
5-Fluoro-1-methyl-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6c)
Conditions: 60 ◦C for 6 h; cycloexane/ethyl acetate 8:2; orange solid; yield 75%; mp: 182–183 ◦C;
1H NMR (200 MHz, DMSO-d6)
δ
: 3.91 (s, 3H, CH3), 7.20 (dt, J = 2.6, 9.1, H-6”), 7.50–7.65 (m, 3H, ),
7.93–8.14 (m, 5H, H-2”, ArH), 8.22 (d, 1H, J = 1.7, 8.6 Hz, ArH), 8.31 (s, 1H, H-5), 8.63 (s, 1H, ArH);
13C NMR (50 MHz, DMSO-d6)
: 105.4 (d, JC4”-F = 24.7 Hz), 109.5 (s), 110.7 (d, JC6”-F = 26.0 Hz), 112.0 (d),
δ
111.0 (d), 112.1 (d), 124.3 (d), 124.3 (s), 124.6 (d), 126.1 (d), 126.5 (d), 127.6 (d), 128.3 (d, JC7”-F = 3.8 Hz),
131.7 (s), 132.6 (s), 133.2 (s), 133.4 (d), 133.8 (s), 154.0 (s), 162.3 (s) Anal. Calcd. for C22H15FN2S C
(73.72%) H (4.22%) N (7.82%) found C (73.36%) H (4.45%) N (7.95%).
1-Methyl-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6d)
◦
Conditions: reflux for 24 h; cycloexane/ethyl acetate 9:1; orange solid; yield 99%; mp: 166–167 C;
1H NMR (200 MHz, DMSO-d6)
δ
: 3.91 (s, 3H, CH3), 7.30–7.38 (m, 2H, ArH ), 7.53–7.61 (m, 3H, ArH),
7.93–8.08 (m, 3H, ArH ), 8.10 (s, 1H, H-2”), 8.20 (d, 1H, J = 1.4 Hz, ArH), 8.25 (s, 1H, H-5), 8.39–8.44 (m,
1H, ArH), 8.66 (s, 1H, ArH); 13C NMR (50 MHz, DMSO-d6)
: 32.8 (q), 109.5 (s), 110.6 (d), 110.7 (s),
111.2 (d), 120.6 (d), 121.1 (d), 122.5 (d), 124.4 (d), 124.6 (d), 126.1 (d), 126.5 (d), 127.6 (d), 128.2 (d), 128.3
δ

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(d), 130.7 (d), 131.8 (s), 132.6 (s), 133.2 (s), 137.1 (s), 153.9 (s), 162.6 (s). Anal. Calcd per C22H16N2S: C
(77.62%) H (4.74%) N (8.23%) found C (77.45%) H (4.39%) N (8.05%).
5-Methoxy-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6e)
◦
Conditions: 60 C for 6 h; cycloexane/ethyl acetate 9:1; light brown solid; yield 48%; mp:
145–146 ◦C; IR: 3019 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ: 3.94 (s, 3H, CH3), 6.92 (dd, 1H,
J = 2.5, 8.8 Hz, H-6”), 7.43 (d, 1H, J = 8.8 Hz, H-7”), 7.53–7.58 (m, 2H, ArH), 7.94 (d, 1H, J = 2.5 Hz,
H-4”), 7.98–8.05 (m, 3H, H-2”, ArH), 8.08 (s, 1H, H-5), 8.15 (d, 1H, J = 2.8 Hz, ArH), 8.22 (dd, 1H, J = 1.5,
8.6 Hz, ArH), 8.66 (s, 1H, ArH), 11.70 (s, 1H; NH); 13C NMR (50 MHz, DMSO-d6)
δ: 55.2 (q), 102.1 (d),
110.3 (s), 110.9 (d), 112.4 (d), 113.0 (d), 124.3 (d), 124.6 (d), 124.9 (s), 126.1 (d), 126.6 (d), 127.2 (d), 127.6
(d), 128.2 (d), 128.3 (d), 131.6 (s), 131.9 (s), 132.6 (s), 133.2 (s), 153.7 (s), 154.7 (s), 163.1 (s). Anal. Calcd.
for C22H16N2OS C (74.13%) H (4.52%) N (7.86%) found C (74.44%) H (4.87%) N (7.66%).
5-Bromo-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6f)
◦
Conditions: 60 C for 3 h; cycloexane/ethyl acetate 8:2; dark brown solid; yield 75%; mp:
224–225 ◦C; IR 3202 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ:7.40 (dd, 1H, J = 1.8, 8.7 Hz, H-6”),
7.49 (s, 1H, H-4”), 7.54–7.62 (m, 2H, H-7”, ArH), 7.94–8.06 (m, 3H, H-2”, ArH ), 8.12 (s, 1H, H-5), 8.21
(dd, 1H, J = 1.4, 8.6 Hz, ArH), 8.28 (d, 1H, J = 2.8 Hz, ArH), 8.55 (d, 1H, J = 1.4 Hz, ArH), 8.62 (s, 1H,
ArH), 12.02 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
δ: 110.1 (s), 111.6 (d), 113.4 (s), 114.3 (d), 122.6
(d), 124.3 (d), 124.6 (d), 125.1 (d), 126.0 (s), 126.2 (d), 126.6 (d), 127.7 (d), 128.2 (d), 128.3 (d), 128.4 (d),
131.8 (s), 132.6 (s), 133.2 (s), 135.3 (s), 153.9 (s), 162.4 (s). Anal. Calcd. for C21H13BrN2S C (62.23%) H
(3.23%) N (6.91%) found C (62.38%) H (3.11%) N (7.23%).
5-Fluoro-3-[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6g)
◦
Conditions: reflux for 24 h; cycloexane/ethyl acetate 7:3; brown solid; yield 60%; mp: 192–193 C;
1
IR 3205 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6)
δ
: H NMR (200 MHz, DMSO-d6)
δ
: 7.13 (dt, 1H,
J = 2.6, 9.2 Hz, H-6”), 7.51–7.61 (m, 3H, ArH), 7.93–8.13 (m, 5H, H-2”, H-5, ArH), 8.23 (dd, 1H, J = 1.7,
8.6 Hz, ArH), 8.28 (d, 1H, J = 2.9 Hz, ArH), 8.64 (s, 1H, ArH), 11.93 (s, 1H, NH); 13C NMR (50 MHz,
DMSO-d6) δ: 105.2 (d, JC6”-F = 24.4 Hz), 110.5 (d), 110.7 (s), 111.3 (d), 113.3 (d), 113.5 (d), 113.6 (d), 124.4
(d), 124.6 (d), 124.7 (s), 126.2 (s), 126.3 (d, JC6”-F =18.5 Hz ), 127.6 (d), 128.3 (d, JC6”-F = 3.7 Hz), 128.7 (d),
131.8 (s), 132.6 (s), 133.2 (s), 133.3 (s), 157.1 (s, JC5”-F =322.8 Hz), 162.7 (s). Anal. Calcd. for C21H13FN2S
C (73.23%) H (3.80%) N (8.13%) found C (72.98%) H (4.17%) N (8.31%).
3-[4-(Naphthalen-2-yl)-1,3-thiazol-2-yl]-1H-indole (6h)
◦
◦
Conditions: 60 C for 24 h; cycloexane/ethyl acetate 9:1; orange solid; yield 60%; mp: 172–173 C;
IR 2972 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6)
: 7.29–7.35 (m, 2H, ArH), 7.55–7.62 (m, 3H, ArH),
7.97–8.13 (m, 3H, ArH), 8.14 (s, 1H, H-2”), 8.24–8.30 (m, 2H, H-5, ArH), 8.42–8.47 (m, 1H, ArH), 8.70
(s, 1H, ArH), 11.85 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
: 110.6 (s), 111.2 (d), 112.2 (d), 120.2 (s),
δ
δ
120.4 (d), 120.9 (d), 122.4 (d), 124.3 (d), 124.6 (d), 126.1 (d), 126.5 (d), 126.8 (d), 127.6 (d), 128.2 (d), 128.3
(d), 131.8 (s), 132.6 (s), 133.2 (s), 136.6 (s), 153.9 (s), 163.0 (s). Anal. Calcd. for C21H14N2S C (77.27%) H
(4.32%) N (8.58%) found C (77.55%) H (4.47%) N (8.65%).
3.2. Biology Studies
3.2.1. Biology
HCT 116 cells (colorectal carcinoma), MDA-MB-435 cells (melanoma) and MCF-7 cells (breast
cancer) were purchased from American Type Culture Collection, Rockville, MD, USA and grown
in RPMI medium supplemented with L-glutamine (2 mM), 10% fetal bovine serum (FBS), penicillin
(100 U/mL), streptomycin (100
phase by seeding twice a week at a density of 3
µ
g/mL) and gentamicin (5
µg/mL). Cells were maintained in log
×
108 cells/L in humidified 5% CO2 atmosphere, at

Mar. Drugs 2016, 14, 226
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◦
37 C. In all experiments, cells were made quiescent through overnight incubation before the treatment
with the compounds or vehicle alone (control cells) No differences were found between cells treated
with DMSO 0.1% and untreated cells in terms of cell number and viability.
3.2.2. Viability Assay In Vitro
Cytotoxic activity of the compounds against human tumor cell lines was determined by the
MTT colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-
2H-tetrazolium to purple formazan by mitochondrial dehydrogenases of living cells. This method is
commonly used to illustrate inhibition of cellular proliferation. Monolayer cultures were treated with
various concentrations (0.1–100
in 96-well plates containing 200
and incubated with the compounds in RPMI. After 72 h incubation, cells were washed, and 50
µ
M) of the drugs. Briefly, all cell lines were seeded at 2
L RPMI. When appropriated, cells were washed with fresh medium
×
104 cells/well
µ
µL
FBS-free medium containing 5 mg/mL MTT were added. The medium was discarded after 2 h
incubation at 37 ◦C by centrifugation, and formazan blue formed in the cells was dissolved in DMSO.
The absorbance, measured at 570 nm in a microplate reader (Bio-RAD, Hercules, CA, USA), of MTT
formazan of control cells was taken as 100% of viability. The growth inhibition activity of compounds
was defined as GI50 value which represents the log of the molar concentration of the compound that
inhibits 50% cell growth. Each experiment was repeated at least three times in triplicate to obtain the
mean values.
3.2.3. Measurement of Phosphatidylserine (PS) Exposure
The apoptosis-induced PS externalization to the cell surface was measured by flow cytometry
by double staining with Annexin V-Fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide
(PI). Annexin V binding to phosphatidylserine is used to identify the earliest stage of apoptosis. PI,
which does not enter cells with intact membranes, is used to distinguish between early apoptotic
cells (Annexin V-FITC positive and PI negative), late apoptotic cells (Annexin V-FITC/PI-double
positive) or necrotic cells (Annexin VFITC negative and PI positive). MCF-7 cells were treated
with 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-1H-indoles 4a–l 3-[2-(naphthalen-2-yl)-1,3-thiazol-4-yl]-
1H-pyrrolo[2,3-b]pyridines 5a , and 3-[4-(naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 5a–h,
–
d
prepared as described above. The compounds were dissolved in dimethyl sulfoxide (DMSO) and
then diluted in culture medium to have a DMSO concentration not exceeding 0.1%. MCF-7 cells
(5.0
the cells were washed with fresh medium and incubated with the compounds or vehicle alone (control
cells) in RPMI for 24 h. Then, cells were harvested by trypsinization and adjusted at 1.0
106 cells/mL
with combining buffer according to the manufacturer’ instructions (eBioscience, San Diego, CA, USA).
One hundred L of cell suspensions were added to a new tube, and incubated with Annexin V-FITC
and PI solution at room temperature in the dark for 15 min. Then samples of at least 1.0
104 cells
were subjected to fluorescence-activated cell sorting (FACS) analysis by Epics XL flow cytometer
×
104 cells/cm2) were seeded in triplicate in 24-wells culture plates. After an overnight incubation,
×
µ
×
™
using Expo32 software (Beckman Coulter, Fullerton, CA, USA) using appropriate bidimensional
gating method.
3.2.4. Cell Cycle Analysis
Cell cycle stage was analyzed by flow cytometry. MCF-7 cells (5.0
×
104 cells/cm2) were seeded
in triplicate in 24-wells culture plates. After an overnight incubation, the cells were washed with fresh
medium and incubated with the compounds or vehicle alone (control cells) in RPMI for 24 h. Then
cells were harvested by trypsinization. Aliquots of 1
in the dark in a PBS solution containing 20 µg/mL propidium iodide (PI) and 200 µg/mL RNase, for
30 min, at room temperature. Then samples of at least 1.0
104 cells were subjected to FACS analysis.
×
106 cells were washed with PBS and incubated
×

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4. Conclusions
New thiazole nortopsentin analogs in which one of the two indole units was replaced by a
naphthalyl portion were conveniently synthesized. Among these, compounds 4a 6a and 6d showed
,
good antiproliferative activity in particular against MCF7 cell line with GI50 values in the micromolar
range. Biological studies performed to clarify their mechanism of action showed that the three
compounds act as pro-apoptotic agents inducing a clear shift of viable cells towards early apoptosis
in MCF-7 cells after 24 h treatment, while not exerting necrotic effects. They also caused cell
cycle perturbation with significant decrease in the percentage of cells in the G0/G1 and S phases,
accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a
subG1-cell population.
Acknowledgments: This work was financially supported by Ministero dell’Istruzione dell’Università e della
Ricerca (MIUR).
Author Contributions: Barbara Parrino, Virginia Spanò, Stella Cascioferro, Anna Carbone and
Alessandra Montalbano performed chemical research and analyzed the data. Alessandro Attanzio and
Luisa Tesoriere performed biological research and analyzed the data. Girolamo Cirrincione, Patrizia Diana,
Paola Barraja and Luisa Tesoriere participated in the design of the research and the writing of the manuscript.
All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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