3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines, nortopsentin Analogues with antiproliferative activity

Article abstract of DOI:10.3390/md13041901

A new series of nortopsentin analogues, in which the imidazole ring of the natural product was replaced by thiazole and the indole unit bound to position 2 of the thiazole ring was substituted by a 7-azaindole moiety, was efficiently synthesized. Two of t

Full text of DOI:10.3390/md13041901

Mar. Drugs 2015, 13, 1901-1924; doi:10.3390/md13041901
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
3
-[4-(1H-Indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines,
Nortopsentin Analogues with Antiproliferative Activity
Barbara Parrino, Anna Carbone, Gloria Di Vita, Cristina Ciancimino, Alessandro Attanzio,
Virginia Spanò, Alessandra Montalbano, Paola Barraja, Luisa Tesoriere, Maria Antonia Livrea,
Patrizia Diana and Girolamo Cirrincione *
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, STEBICEF,
via Archirafi 32, 90123 Palermo, Italy; E-Mails: barbara.parrino@unipa.it (B.P.);
anna.carbone@unipa.it (A.C.); gloria.divita@unipa.it (G.D.); cristina.ciancimino@unipa.it (C.C.);
alessandro.attanzio@unipa.it (A.A.); virginia.spano@unipa.it (V.S.);
alessandra.montalbano@unipa.it (A.M.); paola.barraja@unipa.it (P.B.);
luisa.tesoriere@unipa.it (L.T.); maria.livrea@unipa.it (M.A.L.); patrizia.diana@unipa.it (P.D.)
*
Author to whom correspondence should be addressed; E-Mail: girolamo.cirrincione@unipa.it;
Tel.: +39-091-238-96810.
Academic Editor: Anake Kijjoa
Received: 22 February 2015 / Accepted: 25 March 2015 / Published: 3 April 2015
Abstract: A new series of nortopsentin analogues, in which the imidazole ring of the
natural product was replaced by thiazole and the indole unit bound to position 2 of the
thiazole ring was substituted by a 7-azaindole moiety, was efficiently synthesized. Two of
the new nortopsentin analogues showed good antiproliferative effect against the totality of
the NCI full panel of human tumor cell lines (~60) having GI50 values ranging from low
micromolar to nanomolar level. The mechanism of the antiproliferative effect of these
derivatives, investigated on human hepatoma HepG2 cells, was pro-apoptotic, being associated
with externalization of plasma membrane phosphatidylserine and mitochondrial dysfunction.
Moreover, the compounds induced a concentration-dependent accumulation of cells in the
subG0/G1phase, while confined viable cells in G2/M phase.
Keywords: marine alkaloids; indolyl alkaloids; nortopsentin analogues; 3-[4-(1H-indol-3-
yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines; antiproliferative activity

Mar. Drugs 2015, 13
. Introduction
New natural or synthetic heterocyclic scaffolds have been recently identified and developed as possible
1902
1
anticancer agents [1–19]. Among the various natural sources, viz plants, microbes, animals, and marine
organisms, the latter are increasingly contributing a large number of biologically active alkaloids. An
important class of deep-sea sponge metabolites with remarkable biological activities, such as
anti-inflammatory, antimicrobial, antiviral, and antitumor, is constituted by bis-indolyl alkaloids,
characterized by two indole units connected, through their position 3, by a spacer [20–23].
The bis-indolyl alkaloid spacer can have several structural features such as an acyclic chain or a
carbocyclic ring. Thus, hyrtiosin B, isolated from Hyrtios erecta, 2,2-bis(6′-bromo-3′-indolyl)ethylamine,
isolated from the tunicate Didemnum candidum and Coscinamides A–C, isolated from deep marine sponge
Coscinoderma, showed antiproliferative or anti-HIV activity, as well as asterriquinone, isolated from
Aspergillus fungi, and bearing a six-membered carbocyclic symmetrical structure (Chart 1) [24–27].
O
O
OH
NH2
HO
Br
Br
N
H
N
H
N
H
N
H
Hyrtiosin B
2,2-bis(6'-bromo-3'-indolyl)ethylamine
H
N
O
N
O
R
O
N
H
N
H
HO
R1
OH
O
N
Coscinamide A R = Br; R = H
1
Coscinamide B R = R = H
1
Coscinamide C R = Br; R = OH
Asterriquinone
1
Chart 1. bis-Indolyl alkaloids with acyclic and carbocyclic spacers.
The spacer can also be constituted by differently sized heterocycles. Thus, dragmacidin and
dragmacidins A−E, isolated from a large number of deep water sponges such as Dragmacidon,
Halicortex, Spongosorites, Hexadella and the tunicate Didemnum candidum, exhibit the saturated
six-membered heterocyclic link piperazine or a pyrazinone spacer, and showed several biological properties
(Chart 2) [25,28–31].
More recently, hyrtinadine A, isolated from the marine sponge Hyrtios and bearing a pyrimidine ring as
a spacer, exhibited in vitro cytotoxicity [32].
Topsentins A, B1 and B2, which were isolated from Mediterranean sponge Topsentia genitrix, bear a
2-acyl imidazole spacer and showed antitumor and antiviral activities [33]. Later, from Spongosorites sp.
and from Hexadella sp. were obtained the same natural products, which were named deoxy-topsentin,
topsentin, and bromotopsentin, respectively [34,35]. More recently, topsentin C, isobromotopsentin,

Mar. Drugs 2015, 13
1903
bromodeoxytopsentin, and its isomer isobromodeoxytopsentin, were isolated from Hexadella sp.,
Spongosorites sp and Spongosorites genitrix, respectively [29,36–38].
H
N
NH2
H
N
+
H
R1
N
H
N
N
Br
Br
R3
N
N
R
N
H
O
Br
_
N
H
N
R2
CF CO2
3
HO
H
Dragmacidin
Dragmacidin A R = R = R = H; R = Me
R = H; R = Me; R = Br; R = OH
1 2 3
Dragmacidin D
2
3
1
Dragmacidin B R = R = Me; R = R = H
1
2
3
Dragmacidin C R = R = R = R = H
H N
H
1
2
3
2
N
N
HN
Br
H
N
N
OH
N
H
O
N
HO
N
H
N
HO
N
H
Dragmacidin E
Hyrtinadine A
O
R
R3
Y Z
X
N
X
Y N
R1
H
N
N
N
H
R
R1
R2
N
H
R2
Nortopsentin A X = N; Y = CH; Z = NH; R = R = H; R = R = Br
1
2
3
X-Y = CH=C
Topsentin A
Deoxytopsentin
Nortopsentin B X = N; Y = CH; Z = NH; R = R = R = H; R = Br
1
2
3
R = R = R = H
1 2
Nortopsentin C X = N; Y = CH; Z = NH; R = R = R = H; R = Br
1
2
3
Thiazoles X = N; Y = CH; Z = S; R or R = H, Me; R or R = H, OMe, Br
1
2
3
Topsentin B1
Topsentin
R = R = H; R = OH
Thiophenes X = S; Y = Z = CH; R = R = H, Me, OMe, Cl, Br;
3
2
1
R = R =H, Me, SO Ph
1
2
2
Pyrazoles X = CH; Y = N; Z = NH,NMe; R = R = H,Me,OMe,Cl,Br;
3
Topsentin B2
R = Br; R = OH; R = H
R = R = Me
1 2
1
2
Bromotopsentin
Furans X = O; Y = Z = CH; R = R = H, Me, OMe; R = R = Me
3
1
2
[
1,2]Oxazoles X = CH; Y = N; Z = O; R = R = H, Me, OMe, Cl, Br;
3
Isobromotops. R = H; R = Br; R = OH;
1
2
R = R = Me
1
2
Br-deoxytops.
R = Br R = R = H
1 2
Pyrroles X = NH; Y = Z = CH; R = R = Me; R = R = H, Me, OMe, Cl, Br
1
2
3
IsoBr-deoxytops. R = R = H; R = Br
2
1
1
,2,4-Thiadiazoles X = Z = N; Y = S; R or R = H, OMe, NO , Br;
3 2
R or R = H
1
2
X-Y = CH -CH
2
Topsentin C
R = R = Br; R = H
1 2
Chart 2. bis-Indolyl alkaloids and analogues with heterocyclic spacers.
Nortopsentins A−C, isolated from Spongosorites ruetzleri, have a 2,4-disubstituted imidazole ring as a
spacer and showed in vitro cytotoxicity against P388 cells (IC50, 4.5–20.7 μM). Methylation
of the two-indole nitrogen atoms produced a significant improvement in cytotoxicity (IC50
,
0.64–1.70 μM) [39].

Mar. Drugs 2015, 13
1904
Since marine organisms allow the isolation of very small amount of the biologically active substances
from the natural material, several total synthesis of nortopsentins were proposed [40–43]. Moreover, due to
the considerable activities shown, indolyl alkaloids have attracted remarkable attention by researchers
becoming an interesting field in medicinal chemistry. Thus, several dragmacidin analogues bearing six
membered heterocycles, such as pyridine, pyrazine, pyrazinone and pyrimidine, as spacer and showing
antiproliferative activity were synthesized [44–47].
Also, nortopsentins were considered lead compounds and several reports dealt with the synthesis
and the evaluation of the antiproliferative activity of nortopsentin analogues bearing five-membered
heterocycles, which replaced the imidazole ring of the natural product, such as bis-indolyl-thiophenes [48],
-pyrazoles [49], -furans [50], -isoxazoles [50], -pyrroles [51], and -1,2,4-thiadiazoles [52]. Most of these
analogues showed antiproliferative activity often reaching GI50 values at submicromolar level.
Beside the eterocyclic spacer, the structural manipulation of the natural nortopsentins, was extended to
one or both indole units and produced 3-[(2-indolyl)-5-phenyl]pyridine and phenylthiazolyl-7-azaindole
derivatives, which showed antiproliferative activity and inhibited CDK1 [53,54].
More recently, due to the good results obtained by the aza-substitution of the indole moiety,
3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-4-azaindole derivatives were synthesized and tested against
breast cancer, prostate cancer, pancreatic carcinoma and peritoneal mesothelioma and showed cytotoxic
activity, inhibited CDK1, and increased caspase-9 and caspase-3 activity (Chart 3) [55].
S
S
R2
X
R
N
N
Y
N
N
N
N
N
R
N
R1
R2
R1
4
7
-aza Series: X = N; Y = CH;
-aza Series: X = CH; Y = N
1
R
S
N
S
R2
N
N
N
N
N
N
N
Cl
R1
R2
R
R1
2
3
Chart 3. Nortopsentin aza-analogues.
Contemporaneously, 3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-7-azaindoles were synthesized and
tested against the NCI full panel of human cancer cell lines and STO and MesoII cells, derived from human
diffuse malignant peritoneal mesothelioma (DMPM). The most active compounds, which also act as CDK1
inhibitors, administrated with paclitaxel produced a synergistic cytotoxic effect. In the mouse model,
intraperitoneal (i.p.) administration of active derivatives was effective, resulting in a significant tumor
volume inhibition of DMPM xenografts (range, 58%–75%) at well-tolerated doses, and two complete
responses were observed in each treatment group [56].
Lately, two new series of nortopsentin analogues, 1 and 2, were efficiently synthesized and inhibited the
growth of HCT-116 colorectal cancer cells at low micromolar concentrations, whereas they did not affect
the viability of normal-like intestinal cells. A compound of the series 1 induced apoptosis. Instead, a

Mar. Drugs 2015, 13
1905
derivative of the series 2 at low concentrations (GI30) induced morphological changes characteristic 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 [57].
In this paper, continuing our studies on indole and related azaindole systems [58–64], we report the
synthesis of derivatives of type 3, nortopsentin analogues, in which indole and 7-azaindole units of the
preceding very active 7-aza series were switched. We also report the antiproliferative activity of these new
nortopsentin analogues and studies directed to elucidate their mode of action.
2
. Results and Discussion
2
.1. Chemistry
Substituted 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines of type 3 (Scheme 1,
Table 1) were obtained by Hantzsch reaction between pyrrolo[2,3-b]pyridine-carbothioamides 6a,b and
3-haloacetyl compounds 9a–d.
R1
S
ii
NH2
N
R
N
1
5
N
R
N
S
4
''
R2
5''
4
'
4
a,b R = CN
5'
1
3
'
3''
i
6
6
a R = H
b R = Me
N
5
a,b R = CONH
6''
1
2
6
'
3
2'
N
2''
N
7
''
N
F
vii
R1
R
3
3
3
a-d
e-h
i-k
N
R1
viii
7
7
7
a R = H
1
b R = Me
c R =Ts
iii
1
v
iv
1
X
O
O
vi
R2
N
N
R1
R1
8
8
8
a R = H
9a R = Me, R = F, X = Cl
9b R = Ts, R = F, X = Cl
1 2
9c R = Me, R = H, X = Br
1 2
1
1
2
iii
iv
b R = Me
1
c R =Ts
1
9
d R = Ts, R = H, X = Br
1 2
Scheme 1. Synthesis of substituted 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-
b] pyridines 3a–k. Reagents: (i) (a) H SO , 25 °C, 15–60 min; (b) NaOH, 95%–99%;
ii) Lawesson’s reagent, THF, reflux, 30 min, 88%–99%; (iii) (a) t-BuOK, toluene,
tris[2-(2-methoxyethoxy)ethyl]amine (TDA-1), 25 °C, 5–8 h; (b) MeI, 25 °C, 1–2 h,
0%–98%; (iv) (a) NaH, THF, 0 °C–25 °C, 1–24 h; (b) p-TsCl, 25 °C, 4–24 h, 90%–96%;
v) AlCl , CH Cl , ClCOCH Cl, 1 h, 50%–55%; (vi) Br , MeOH, reflux, 2 h, 40%, 70%;
vii) EtOH, reflux, 30 min, 30%–97%; (viii) KOH, EtOH, reflux, 1–2 h, 40%–98%.
2
4
(
8
(
(
3
2
2
2
2

Mar. Drugs 2015, 13
1906
The key intermediates 6a,b were obtained from the corresponding pyrrolo[2,3-b]pyridine-3-
carbonitriles 4a,b through the formation of carboxamides 5a,b, as previously reported [57].
N-protected 5-fluoroindoles 7b,c and 1-(1H-indol-3-yl)ethanones 8b,c were synthesized, in good
yields (80%–98%), from commercially available 5-fluoroindole 7a or 1-(1H-indol-3-yl)ethanone 8a
by methylation [54], or reaction with tosyl chloride using sodium hydride (NaH) as the base in
tetrahydrofuran (THF).
3
-Haloacetyl indoles 9a–d were prepared from the corresponding indoles 7b,c or 1-(1H-indol-3-
yl)ethanones 8b,c. In particular, 3-chloroacetyl derivatives 9a,b were obtained by reaction of the
corresponding N-methyl or N-tosyl 5-fluoroindole 7b,c with chloroacetyl chloride (ClCOCH Cl) in the
presence of aluminum chloride (AlCl ) in dichloromethane (CH Cl ) in moderate yields (50%–55%); while
-bromoacetyl compounds 9c,d were synthesized (40%–70%) from the corresponding N-methyl or N-tosyl
2
3
2
2
3
1
-(1H-indol-3-yl)ethanone 8b,c using bromine in refluxing methanol (MeOH).
Reaction between pyrrolo[2,3-b]pyridine-carbothioamides 6a,b and 3-haloacetyl compounds 9a–d in
ethanol under reflux afforded the desired 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines
3a–h in moderate to excellent yields (30%–97%) (Table 1).
The subsequent deprotection of N-tosyl derivatives 3e–h using potassium hydroxide in refluxing ethanol
gave the corresponding compounds 3i–k (40%–98%).
Table 1. Substituted 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine 3a–k.
Compound
R
H
R
1
R
2
Yield% Compound
R
H
R
1
R
2
Yield%
90
3
a
Me
Me
Me
Me
Ts
F
F
97
30
60
60
97
40
3g
3h
3i
Ts
Ts
H
H
H
F
3
b
Me
H
Me
H
50
3
c
H
H
F
98
3
d
Me
H
3l
Me
H
H
F
40
3
e
3j
H
H
H
98
3
f
Me
Ts
F
3k
Me
H
98
2
.2. Biology
All the synthesized thiazoles 3a–k were evaluated by the National Cancer Institute (Bethesda MD) for
cytotoxicity against the NCI-60 cell line panel using standard protocols [65]. Initially, the selected
−5
derivatives 3c,d,g,h,j,k were tested at a single dose (10 M) on the full panel of approximately 60 human
tumor cell lines derived from 9 human cancer cell types, that have been grouped in disease
sub-panels including leukemia, non-small-cell lung, colon, central nervous system (CNS), melanoma,
ovarian, renal, prostate and breast cancers (data not shown). Compounds 3d and 3k were further selected
−4
−8
for full evaluation at five concentration levels (10 –10 M).
The antitumor activity of compounds 3d and 3k was given by three parameters for each cell line: GI50
(the molar concentration of the compound that inhibits 50% net cell growth), TGI (the molar concentration
of the compound leading to total inhibition of net cell growth), and LC50 (the molar concentration of the
compound that induces 50% net cell death). The average values of mean graph midpoint (MG_MID) were
calculated for each of these parameters.

Mar. Drugs 2015, 13
1907
An evaluation of the data reported in Table 2 pointed out that compounds 3d and 3k exhibited
antiproliferative activity against all the human cell lines at GI50 values from micromolar to nanomolar
(13.0–0.03 and 14.2–0.04 µM, respectively).
Table 2. In vitro inhibition of cancer cell line growth by indolyl-thiazolyl-pyrrolopyridines 3d, 3k a.
Cell Lines
GI50 (µM )
Cell Lines
GI50 (µM ) Cell Lines
3d 3k
CNS Cancer
GI50 (µM )
3d 3k
3
d
3k
Leukemia
Renal Cancer
CCRF-CEM
0.40
0.29
0.07
0.67
0.45
0.06
0.43 SF-268
0.32 SF-295
0.12 SF-539
0.66 SNB-19
0.65 SNB-75
0.14 U251
2.44 0.94 786-0
0.24 0.31 A498
0.18 0.25 ACHN
0.63 0.64 CAKI-1
0.16 0.14 RXF393
0.41 0.43 SN12C
TK-10
0.97 0.72
0.50 0.37
0.72 0.51
0.38 0.44
0.33 0.52
12.1 5.48
0.60 0.99
0.90 0.69
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
UO-31
Non-Small Cell Lung Cancer
Melanoma
Prostate Cancer
0.46 0.56
0.39 0.56
A549/ATCC
EKVK
0.56
0.91
0.81
0.35
0.74
0.72
0.71 LOX IMVI
0.91 MALME-3M
0.93 M14
0.54 0.51 PC-3
1.04 0.74 DU-145
0.27 0.28
HOP-62
HOP-92
0.31 MDA-MB-435 0.03 0.04
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0.65 SK-MEL-2
0.58 0.41
1.20 0.76
0.24 0.27
13.0 14.2
0.30 0.42
0.67 SK-MEL-28
0.76 ND b SK-MEL-5
0.30
0.04
0.37 UACC-257
0.05 UACC-62
Colon Cancer
Ovarian Cancer
1.33 1.18 MCF7
0.10 0.25 MDA-MB-231/ATCC 2.82 1.29
Breast Cancer
COLO-205
0.19
1.13
0.37
0.17
0.25
0.21
0.22
0.19 IGROV1
1.18 OVCAR-3
0.39 OVCAR-4
0.29 OVCAR-5
0.19 OVCAR-8
0.33 0.37
HCC-2998
HCT-116
HCT-15
HT29
7.22 0.52 HS 578T
1.41 2.39 BT-549
0.59 0.66
0.47 0.44
0.28 0.23
0.66 0.82 MDA-MB-468
KM12
0.40 NCI/ADR-RES 0.09 0.15
0.30 SK-OV-3 0.57 0.52
SW-620
a
b
Data obtained from NCI’s in vitro disease-oriented tumor cells screen; ND = not determined.
Indolyl-thiazolyl-pyrrolopyridine derivatives 3d and 3k were selective with respect to the leukemia
cancer subpanel having all the subpanel cell lines GI50 in the range 0.06–0.67 μM and 0.12–0.66 μM,
respectively. In particular, the most sensitive cell lines were K-562 (GI50 0.07 μM and 0.12 μM,
respectively), and SR (GI50 0.06 μM and 0.14 μM, respectively). Derivatives 3d and 3k were also
particularly sensitive against NCI-H522 (GI50 0.04 μM and 0.05 μM, respectively) of non-small cell lung
cancer, NCI/ADR-RES (GI50 0.09 μM and 0.15 μM, respectively) of ovarian cancer, and
MDA-MB-435 (GI50 0.03 μM and 0.04 μM, respectively) of melanoma subpanel.
Cell growth inhibitory activity of 3d and 3k was also investigated on human HepG2 hepatocarcinoma.

Mar. Drugs 2015, 13
1908
Cell growth inhibitory activity of compounds 3d and 3k was also investigated on human HepG2
hepatocarcinoma cells, a cell line not included in the NCI panel and of interest in the drug discovery
because provided with a very active microsomal system for detoxification of xenobiotics. Monolayer
cultures treated for 72 h with 0.1–10 μM concentrations of the compounds were examined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell growth. As shown in
Figure 1, both the compounds inhibited the HepG2 cells growth in dose-dependent manner. Based on the
dose response curve, the GI50 values were 1.69 µM) and 0.21 µM for 3d and 3k, respectively. Under
identical conditions, both the nortopsentin analogues did not substantially impaired viability of normal
immortalized human liver cells (Chang), suggesting high selectivity towards tumor cells.
Figure 1. Effect 3d (A) and 3k (B) on the growth of HepG2 (black bars) or Chang cells
(
grey bars) as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT). Cell monolayers were incubated for 24 h in the absence (control) or in the presence
(
of the compounds at the indicated concentrations and cell growth was assessed by MTT
test as reported Methods. Results are indicated as the percentage of viable cells with
respect to untreated controls. Values are the mean ± SD of three separate experiments
carried out in triplicate.
The distribution of HepG2 cells in the cell cycle phases after 24 h treatment with the two norptopsentin
analogues, was assessed by flow cytometric analysis after staining of DNA with PI. Both compounds 3d
and 3k caused a significant dose-dependent 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 2).

Mar. Drugs 2015, 13
1909
Figure 2. Effect of 3d and 3k on the cell cycle distribution of human hepatoma HepG2
cells. Flow cytometric analysis of propidium iodide (PI)-stained cells, as determined by
flow cytometry after 24 h treatment with the compounds or vehicle alone (control). The
percentage of cells in the different phases of the cycle was calculated by Expo32 software.
Values are the mean ± SD of three separate experiments in triplicate.
Figure 3. Apoptosis induced by 3d an 3k nortopsentin analogues in HepG2 cells. (A)
Percentage of Annexin V/propidium iodide (PI) double-stained cells, as determined by
flow cytometry; (B) Fluorescence micrographs of ethidium bromide/acridine orange
double-stained cells. Control, cells treated with vehicle; 3d and 3k, cells treated for 24 h
with either Nortopsentin derivative at GI50 concentration. (A) Representative images of
three experiments with comparable results; (B) Representative images, in 200 × magnification,
of three experiments with comparable results.

Mar. Drugs 2015, 13
1910
Apoptosis induction by 3d and 3k in HepG2 cells was investigated by externalization of plasma
membrane phosphatidylserine and changes of mitochondrial transmembrane potential. Flow cytometry
analysis of Annexin V-FITC/PI-stained cells after 24 h treatment with the Norptopsentin analogues,
assayed at their relevant GI50 values, indicated a high percentage of cells in early apoptosis, with
externalized phosphatidylserine (Figure 3A).
To confirm apoptotic mechanism of cytotoxicity of the nortopsentin analogues, we carried out
morphological evaluation of HepG2 cells using AO and EB double staining. After 24 h of treatment with
3d or 3k at GI50 concentration, fluorescent microscopy revealed the appearance of cells containing bright
green patches in the nuclei as a consequence of chromatin condensation and nuclear fragmentation, which
are typical features of apoptosis. Moreover, fluorescing orange cells owing to increase of cell permeability
to ethidium bromide, cell shrinkage and nuclear fragmentation were also evident as cells in late apoptosis
(Figure 3B). Taken together, these findings provided strong evidence that the synthesized nortopsentin
analogues induced apoptosis in HepG2 cells.
Figure 4. Mitochondrial dysfunction induced by 3d and 3k in HepG2 cells. (A) Fluorescence
intensity of 3,30-dihexyloxacarbocyanine iodide-treated cells, as determined by flow
cytometry; (B) Fluorescence intensity of 2′,7′-dichlorofluorescin diacetate-stained cells, as
determined by flow cytometry. Control, cells treated with vehicle; 3d and 3k, cells treated
for 24 h with either Nortopsentin derivative at GI50 concentration. Representative images of
three experiments with comparable results.

Mar. Drugs 2015, 13
1911
Involvement of mitochondria in apoptosis induced in HepG2 cells by the synthesized nortopsentin
analogues, was assessed. Loss of mitochondrial trans-membrane potential was indicated by decreased
mitochondrial 3,30-dihexyloxacarbocyanine iodide-red fluorescence (Figure 4A). Mitochondrial
dysfunction in HepG2 cells following 24 h treatment with 3d and 3k, was also evident from the levels of
intracellular ROS, revealed by cytofluorimetric analysis with 2′,7′-dichlorofluorescindiacetate, significantly
higher than cell control (Figure 4B).
3
3
3
. Experimental Section
.1. Chemistry
.1.1. General
All melting points were taken on a Büchi-Tottoly capillary apparatus. IR spectra were determined in
1
13
bromoform with a Shimadzu FT/IR 8400S spectrophotometer. H and C NMR spectra were measured at
2
00 and 50.0 MHz, respectively, in dimethylsulfoxide (DMSO)-d
6
or CDCl
3
solution, using a Bruker
1
Avance II series 200 MHz spectrometer. Compounds 3a,b 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 ±0.4% of theoretical values and were performed with a VARIO EL III
elemental analyzer. Purity of all the tested compounds was greater than 98%, determined by HPLC as
described below.
3
.1.2. Synthesis of 5-Fluoro-1-methyl-1H-indole (7b)
To a cold solution of 5-fluoroindole 7a (0.8 g, 5.0 mmol) in anhydrous toluene (50 mL), potassium
t-butoxide (0.8 g, 6.8 mmol) and tris[2-(2-methoxyethoxy)ethyl]amine (TDA-1) (1–2 drops) were added.
The reaction mixture was stirred at room temperature for 5 h and then methyl iodide (0.3 mL,
5
mmol) was added. TLC analysis (dichloromethane (DCM)/petroleum ether (9/1) revealed that
methylation was complete after 1 h. The solvent was evaporated under reduced pressure and the residue,
treated with water (15 mL), was extracted with DCM (3 × 15 mL), dried (Na
2
SO ) and evaporated to afford
4
the pure methyl derivative 7b.
Yellow solid; yield: 98%; mp: 55–56 °C. Analytical and spectroscopic data were in agreement with
those previously reported [50].
3
.1.3. Synthesis of 5-Fluoro-1-[(4-methylphenyl)sulfonyl]-1H-indole (7c)
To a stirred solution of 5-fluoroindole 7a (1.0 g, 7.4 mmol) in tetrahydrofuran (THF) (5.0 mL) sodium
hydride (60% dispersion in mineral oil, 0.4 g, 9.6 mmol) was added at 0 °C and the mixture was stirred at
room temperature for 24 h. p-Toluenesulfonyl chloride (2.1 g, 11.1 mmol) was added to the reaction and
the mixture was stirred at room temperature for 4 h. The residue was evaporated under reduced pressure,
treated with water (20 mL) and extracted with ethyl acetate (EtOAc) (3 × 20 mL). The organic phase was
dried (Na
2
SO
4
), evaporated under reduced pressure and purified by column chromatography using
petroleum ether/DCM (9/1) as eluent. White solid; yield: 96%; mp: 111–112 °C; IR 1373,

Mar. Drugs 2015, 13
1912
), 6.82–6.84 (m, 1H,
−1
1
1
172 (SO
2
) cm ; H NMR (200 MHz, DMSO-d
6
) δ: 2.32 (s, 3H, CH
3
H-7), 7.19 (td, 1H, J = 2.6, 9.2, 11.0 Hz, H-6), 7.39 (d, 2H, J = 8.7 Hz, H-3′, H-5′), 7.45 (d, 1H,
J = 2.6 Hz, H-3), 7.87 (d, 2H, J = 8.7 Hz, H-2′, H-6′), 7.88–7.97 (m, 2H, H-2, H-4); 13C NMR
(
50 MHz, DMSO-d
6
) δ: 21.0 (q), 107.0 (d, JC4–F = 24.1 Hz), 109.3 (d, JC3–F = 4.2 Hz), 112.5 (d,
C6–F = 25.8 Hz), 114.4 (d, JC7–F = 9.6 Hz), 126.7 (2xd), 128.9 (d), 130.2 (2xd), 130.6 (s), 131.6 (d,
C3a–F = 10.4 Hz), 133.9 (s), 145.6 (s), 158.9 (d, JC5–F = 237.7 Hz). Anal. Calcd for: C15 S: C,
J
J
H
12FNO
2
62.27; H, 4.18; N, 4.84. Found: C, 62.57; H, 4.38; N, 4.69.
3
.1.4. Synthesis of 1-(1-Methyl-1H-indol-3-yl)ethanone (8b)
To a cold solution of 3-acetylindole 8a (1.0 g, 6.3 mmol) in anhydrous toluene (50 mL), potassium
t-butoxide (1.0 g, 8.6 mmol) and TDA-1 (1–2 drops) were added. The reaction mixture was stirred at room
temperature for 8 h and then methyl iodide (0.4 mL, 6.3 mmol) was added. TLC analysis (DCM/EtOAc
9/1) revealed that methylation was complete after 2 h. The solvent was evaporated under reduced pressure.
The residue was treated with water (50 mL), extracted with DCM (3 × 50 mL), dried (Na SO ),
2
4
evaporated under reduced pressure, and purified by column chromatography using DCM/EtOAc (9/1)
as eluent.
White solid; yield 80%; mp: 108–109 °C. Analytical and spectroscopic data were in agreement with
those previously reported [48].
3
.1.5. Synthesis of 1-{1-[(4-Methylphenyl)sulfonyl]-1H-indol-3-yl}ethanone (8c)
To a solution of 3-acetylindole 8a (1.0 g, 6.3 mmol) in THF (5.0 mL) sodium hydride (60% dispersion
in mineral oil, 0.3 g, 6.3 mmol) was added at 0 °C and the mixture was stirred at room temperature for 1 h.
p-toluenesulfonyl chloride (1.2 g, 6.3 mmol) was added and the mixture was stirred at room temperature
for 24 h. The residue was evaporated under reduced pressure, treated with water (20 mL) and extracted
with EtOAc (3 × 20 mL). The organic phase was dried (Na
2
SO ), evaporated under reduced pressure and
4
purified by column chromatography using DCM as eluent. White solid; yield: 90%; mp: 148–149 °C; IR
−
1 1
1
662 (CO), 1382, 1299 (SO
CH ), 7.32–7.39 (m, 2H, H-5, H-6), 7.43 (d, 2H, J = 7.8 Hz, H-3′, H-5′), 7.95 (d, 1H, J = 7.5 Hz, H-7), 8.04
d, 2H, J = 7.8 Hz, H-2′, H-6′), 8.19 (d, 1H, J = 7.5 Hz, H-4), 8.81 (s, 1H, H-2); C NMR (50 MHz,
DMSO-d ) δ:21.0 (q), 27.8 (q), 113.0 (d), 120.7 (s), 122.3 (d), 124.8 (d), 125.6 (d), 127.0 (s), 127.2 (2xd),
30.5 (2xd), 133.5 (s), 134.0 (s), 134.2 (d), 146.2 (s), 193.9 (s). Anal. Calcd for: C17 S: C, 65.16; H,
2
) cm ; H NMR (200 MHz, DMSO-d
6
) δ: 2.33 (s, 3H, CH
3
), 2.60 (s, 3H,
3
13
(
6
1
4
H
15NO
3
.82; N, 4.47. Found: C, 65.28, H, 5.06, N, 4.37.
3
.1.6. Synthesis of 2-Chloro-1-(5-fluoro-1H-indol-3-yl)ethanones (9a,b)
To a stirred suspension of anhydrous aluminium chloride (1.1 g, 8.5 mmol), in DCM (18.0 mL), was
added dropwise at 0 °C a solution of the suitable indole 7b,c (1.2 mmol) in DCM (5.0 mL) under nitrogen
atmosphere. Then, chloroacetyl chloride (0.3 mL, 3.6 mmol) was slowly added to the reaction mixture,
which was stirred at room temperature for 1 h and then poured in ice and water (20 mL) and extracted with
DCM (3 × 20 mL). The organic phase was dried (Na
2
SO ), evaporated under reduced pressure and purified
4
by column chromatography using DCM as eluent.

Mar. Drugs 2015, 13
.1.6.1. 2-Chloro-1-(5-fluoro-1-methyl-1H-indol-3-yl)ethanone (9a)
1913
3
−
1 1
White solid; yield: 50%; mp: 185–186 °C; IR 1653 (CO) cm ; H NMR (200 MHz, DMSO-d
s, 3H, CH ), 4.84 (s, 2H, CH
H-7), 7.84 (dd, 1H, J = 2.6, 11.0 Hz, H-4), 8.51 (bs, 1H, H-2); C NMR (50 MHz, DMSO-d
6
) δ: 3.89
(
3
2
), 7.20 (td, 1H, J = 2.6, 9.2, 11.0 Hz, H-6), 7.62 (dd, 1H, J = 4.5, 9.2 Hz,
1
3
6
) δ: 33.7 (q),
46.2 (t), 106.1 (d, JC4–F = 24.8 Hz), 111.3 (d, JC6–F = 26.1 Hz), 112.3 (s), 112.4 (d, JC7–F = 9.8 Hz), 126.4 (d,
J
C3a–F = 11.0 Hz), 133.9 (s), 139.5 (d), 159.0 (d, JC5–F = 236 Hz), 185.6 (s). Anal. Calcd for C11 ClFNO:
H
9
C, 58.55; H, 4.02; N, 6.21. Found: C, 58.85; H, 4.14; N, 6.01.
3
.1.6.2. 2-Chloro-1-{5-fluoro-1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}ethanone (9b)
−1 1
White solid; yield: 55%; mp: 155–156 °C; IR 1683 (CO), 1375, 1171 (SO
CDCl ) δ: 2.39 (s, 3H, CH ), 4.54 (s, 2H, CH ), 7.13 (td, 1H, J = 2.6, 8.9, 11.0 Hz, H-6), 7.31 (d, 2H,
J = 8.0 Hz, H-3′, H-5′), 7.82 (d, 2H, J = 8.0 Hz, H-2′, H-6′), 7.90 (dd, 1H, J = 4.9, 8.9 Hz, H-7), 7.98 (dd,
2
) cm ; H NMR (200 MHz,
3
3
2
13
2
H, J = 2.6, 11.0 Hz, H-4), 8.34 (s, 1H, H-2); C NMR (50 MHz, CDCl
C6–F = 25.5 Hz), 114.1 (s), 114.2 (d), 114.5 (d, JC4–F = 14.0 Hz), 117.9 (s, JC7a–F = 4.5 Hz), 127.2 (2xd),
30.2 (s), 130.4 (2xd), 133.5 (d), 134.1 (s), 146.5 (s), 160.1 (s, JC5–F = 241 Hz), 186.7 (s). Anal. Calcd for
13ClFNO S: C, 55.82; H, 3.58; N, 3.83. Found: C, 55.99; H, 3.36; N, 4.10.
3
) δ: 21.7 (q), 45.8 (t), 108.9 (d,
J
1
C
17
H
3
3
.1.7. Synthesis of 2-Bromo-(1H-indol-3-yl)ethanones (9c,d)
To a cold suspension of the appropriate 3-acetylindole 8b,c (1.9 mmol) in anhydrous methanol
(3.0 mL) bromine (0.1 mL, 1.9 mmol) was added dropwise. The mixture was heated at 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 (Na
2
SO ), evaporated under reduced pressure and purified by column
4
chromatography using DCM as eluent.
3
.1.7.1. 2-Bromo-1-(1-methyl-1H-indol-3-yl)ethanone (9c)
Brown solid; yield: 70%; mp: 205–206 °C. Analytical and spectroscopic data were in agreement with
those previously reported [48].
3
.1.7.2. 2-Bromo-1-{1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}ethanone (9d)
−1 1
Brown solid; yield: 40%; mp: 134–135 °C; IR 1668 (CO), 1379, 1177 (SO
CDCl ) δ: 2.38 (s, 3H, CH ), 4.36 (s, 2H, CH ), 7.29 (d, 2H, J = 8.2 Hz, H-3′, H-5′), 7.36–7.40 (m, 2H,
H-5, H-6), 7.83–7.87 (m, 2H, H-2′, H-6′), 7.91–7.96 (m, 1H, H-7), 8.28–8.34 (m, 2H, H-2, H-4); C NMR
50 MHz, CDCl ) δ: 21.7 (q), 31.4 (t), 113.1 (d), 123.1 (d), 124.3 (s), 125.1 (d), 126.1 (d), 127.2 (2xd),
27.5 (s), 130.4 (2xd), 132.8 (d), 134.2 (s), 134.8 (s), 146.2 (s), 187.0 (s). Anal. Calcd. for: C17 S:
C, 52.05; H, 3.60; N, 3.57. Found: C, 52.25; H, 3.70; N, 3.35.
2
) cm ; H NMR (200 MHz,
3
3
2
13
(
3
1
H
14BrNO
3

Mar. Drugs 2015, 13
.1.8. Synthesis of 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines (3a–h)
A suspension of the appropriate pyrrolo[2,3-b]pyridine-carbothioamide 6a,b (5.0 mmol) and
1914
3
halo-acetyl compounds 9a–d (5.0 mmol) in ethanol (3.0 mL) was heated under reflux for 30 min. The
precipitate, obtained after cooling, was filtered off, dried and recrystallized from ethanol to afford
derivatives 3a–h.
3
.1.8.1. 3-[4-(5-Fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (3a)
−1 1
Yellow solid; yield: 97%; mp: 237–238 °C; IR 3550 (NH) cm ; H NMR (200 MHz, DMSO-d
6
) δ:
3
.90 (s, 3H, CH ), 7.12 (t, 1H, J = 8.2 Hz, H-6″), 7.44 (dd, 1H, J = 5.2, 7.4 Hz, H-5′), 7.54 (dd, 1H,
3
J = 4.6, 8.2 Hz, H-7″), 7.73 (s, 1H, H-2″), 7.94 (dd, 1H, J = 9.7, 2.5 Hz, H-4″), 8.16 (bs, 1H, H-2′), 8.38 (s,
H, H-5), 8.45 (d, 1H, J = 3.7 Hz, H-6′), 8.89 (d, 1H, J = 7.4 Hz, H-4′), 12.74 (bs, 1H, NH). Anal. Calcd for
S: C, 65.50; H, 3.76; N, 16.08. Found: C, 65.76; H, 3.55; N, 16.22.
1
C
19
H
13FN
4
3.1.8.2. 3-[4-(5-Fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (3b)
1
Yellow solid; yield: 30%; mp: 286–287 °C; H NMR (200 MHz, DMSO-d
6
) δ: 2.56 (s, 3H, CH ), 4.03
3
3
(s, 3H, CH ), 6.02 (dd, 1H, J = 4.3, 10.7 Hz, H-7″), 6.76 (td, 1H, J = 2.3, 9.5, 10.7 Hz, H-6″), 6.98 (dd, 1H,
J = 5.0, 8.4 Hz, H-5′), 7.25 (dd, 1H, J = 2.3, 9.5 Hz, H-4″), 7.72 (d, 1H, J = 8.4 Hz,
H-4′), 8.19-8.81 (m, 4H, H-2’, H-2’’, H-5, H-6′). Anal. Calcd for C20
H
15FN S: C, 66.28; H, 4.17; N, 15.46.
4
Found: C, 66.53; H, 3.90; N, 15.23.
3
.1.8.3. 3-[4-(1-Methyl-1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (3c)
−1 1
Yellow solid; yield: 60%; mp: 274–275 °C, IR 3427 (NH) cm ; H NMR (200 MHz, DMSO-d
6
) δ:
3
.90 (s, 3H, CH ), 7.21–7.30 (m, 2H, H-5″, H-6″), 7.36 (dd, 1H, J = 4.9, 7.7 Hz, H-5′), 7.53 (d, 1H,
3
J = 7.1 Hz, H-7″), 7.66 (s, 1H, H-2″), 8.05 (s, 1H, H-5), 8.19 (d, 1H, J = 7.1 Hz, H-4″), 8.30 (d, 1H,
13
J = 2.0 Hz, H-2′), 8.40 (d, 1H, J = 4.9 Hz, H-6′), 8.79 (d, 1H, J = 7.7 Hz, H-4′), 12.40 (bs, 1H, NH); C
NMR (50 MHz, DMSO-d ) δ: 32.6 (q), 106.5 (d), 109.6 (s), 110.2 (d), 117.1 (d), 117.3 (s), 119.9 (d), 120.1
d), 121.6 (d), 124.9 (s), 126.9 (d), 129.2 (d), 129.7 (d), 137.1 (s), 143.2 (d), 143.3 (s), 147.9 (s), 150.1 (s),
6
(
1
61.0 (s). Anal. Calcd for C19
H
14
N S: C, 69.07; H, 4.27; N, 16.96. Found: C, 68.90; H, 4.46; N, 16.75.
4
3
.1.8.4. 1-Methyl-3-[4-(1-methyl-1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (3d)
1
Yellow solid; yield: 60%; mp: 290–291 °C; H NMR (200 MHz, DMSO-d
6
) δ: 3.90 (s, 3H, CH ), 3.94
3
3
(s, 3H, CH ), 7.17–7.30 (m, 2H, H-5″, H-6″), 7.39 (dd, 1H, J = 4.7, 7.9 Hz, H-5′), 7.53 (dd, 1H,
J = 1.4, 6.9 Hz, H-7″), 7.66 (s, 1H, H-2″), 8.05 (s, 1H, H-2′), 8.19 (dd, 1H, J = 1.4, 6.9 Hz, H-4″), 8.41 (s,
13
1
H, H-5), 8.45 (dd, 1H, J = 1.3, 4.7 Hz, H-6′), 8.78 (dd, 1H, J = 1.3, 7.9 Hz, H-4′); C NMR (50 MHz,
DMSO-d ) δ: 31.4 (q), 32.6 (q), 106.5 (d), 108.3 (s), 110.0 (s), 110.2 (d), 117.2 (d), 117.5 (s), 119.9 (d),
20.1 (d), 121.6 (d), 124.9 (s), 129.3 (d), 129.8 (d), 130.5 (d), 137.0 (s), 143.1 (d), 147.0 (s), 150.1 (s),
60.7 (s). Anal. Calcd for C20 S: C, 69.74; H, 4.68; N, 16.27. Found: C, 69.50; H, 4.84; N, 16.06.
6
1
1
H N
16 4

Mar. Drugs 2015, 13
1915
3
.1.8.5. 3-(4-{5-Fluoro-1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}-1,3-thiazol-2-yl)-1H-pyrrolo
[2,3-b]pyridine (3e)
1
Yellow solid; yield: 97%; mp: 257–258 °C; IR 3337 (NH), 1370, 1171 (SO
DMSO-d ) δ: 2.31 (s, 3H, CH ), 7.28–7.32 (m, 2H, ArH), 7.39–7.42 (m, 2H, H-3‴, H-5‴), 7.94–7.98 (m,
H, H-2‴, H-6‴), 8.03–8.15 (m, 3H, ArH), 8.42–8.49 (m, 3H, ArH), 8.74 (d, 1H, J = 7.4 Hz,
2
); H NMR (200 MHz,
6
3
2
13
H-4′), 12.67 (s, 1H, NH); C NMR (50 MHz, DMSO-d
6
) δ: 21.1 (q), 107.3 (d, JC4″–F = 24.1 Hz), 109.3 (s),
1
1
1
12.6 (d, JC6″–F = 26.8 Hz), 113.4 (d), 114.9 (d, JC7″–F = 9.9 Hz), 117.2 (d), 117.4 (s), 117.5 (s), 117.6 (s),
26.3 (d), 126.9 (2xd), 127.8 (d), 128.8 (s), 129.0 (s), 130.2 (d), 130.4 (2xd), 131.1 (s), 133.6 (s), 142.3 (d),
45.9 (s), 146.9 (s), 159.4 (d, JC5″–F = 245.5 Hz). Anal. Calcd. for C25 : C, 61.46; H, 3.51; N,
4 2 2
H17FN O S
11.47. Found: C, 61.73; H, 3.25; N, 11.31.
3
.1.8.6. 3-(4-{5-Fluoro-1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}-1,3-thiazol-2-yl)-1-methyl-1H-
pyrrolo[2,3-b]pyridine (3f)
−1 1
White solid; yield: 40%; mp: 230–231 °C; IR 1380, 1299 (SO
2
) cm ; H NMR (200 MHz, DMSO-d )
6
δ: 2.32 (s, 3H, CH
3
), 3.94 (s, 3H, CH ), 7.28–7.43 (m, 2H, ArH), 7.41 (d, 2H, J = 7.8 Hz, H-3‴,
3
H-5‴), 7.87–7.94 (m, 2H, H-2‴, H-6‴), 7.99–8.13 (m, 3H, ArH), 8.42–8.48 (m, 3H, ArH), 8.60 (d, 1H,
13
J = 7.3 Hz, H-4′); C NMR (50 MHz, DMSO-d
6
) δ: 21.0 (q), 31.2 (q), 107.4 (d, JC4″–F = 25.2 Hz), 107.8
(s), 112.0 (d), 113.1 (d, JC6″–F = 27.5 Hz), 114.8 (d, JC7″–F = 9.1 Hz), 116.9 (s), 117.4 (d), 117.5 (d,
J
C7″–a = 4.0 Hz), 126.3 (d), 126.9 (2xd), 128.6 (d), 128.8 (s), 129.0 (s), 130.4 (s), 130.8 (2xd), 131.1 (s),
33.6 (s), 143.8 (d), 145.9 (s), 146.9 (s), 147.6 (s), 159.3 (d, JC5″–F = 246.5 Hz). Anal. Calcd. for
: C, 62.13; H, 3.81; N, 11.15. Found: C, 61.98; H, 3.99; N, 10.90.
1
C
26 4 2 2
H19FN O S
3
.1.8.7. 3-(4-{1-[(4-Methylphenyl)sulfonyl]-1H-indol-3-yl}-1,3-thiazol-2-yl)-1H-pyrrolo[2,3-b]
pyridine (3g)
−1
1
Yellow solid; yield: 90%; mp: 260–261 °C; IR 3498 (NH), 1374, 1174 (SO ) cm ; H NMR
2
(
200 MHz, DMSO-d
.01–8.07 (m, 2H, ArH), 8.36–8.39 (m, 4H, ArH), 8.64 (d, 1H, J = 7.7 Hz, H-4′), 12.39 (bs, 1H, NH); C
NMR (50 MHz, DMSO-d ) δ: 21.0 (q), 109.1 (s), 111.8 (d), 113.4 (d), 116.7 (s), 117.2 (d), 117.7 (s), 121.7
d), 124.0 (d), 124.5 (d), 125.2 (d), 126.8 (2xd), 127.2 (d), 127.9 (s), 128.6 (d), 130.3 (2xd), 133.8 (s), 134.6
s), 144.0 (d), 145.7 (s), 147.3 (s), 148.7 (s), 162.1 (s). Anal. Calcd. for C25 : C, 63.81; H, 3.86;
6
) δ: 2.30 (s, 3H, CH
3
), 7.31–7.41 (m, 5H, ArH), 7.94–7.98 (m, 2H, ArH),
13
8
6
(
(
18 4 2 2
H N O S
N, 11.91. Found: C, 63.55; H, 4.02; N, 11.76.
3
.1.8.8. 1-Methyl-3-(4-{1-[(4-methylphenyl)sulfonyl]-1H-indol-3-yl}-1,3-thiazol-2-yl)-1H-pyrrolo
[2,3-b]pyridine (3h)
−1 1
Yellow solid; yield: 50%; mp: 250–251 °C; IR 1374, 1174 (SO
2
) cm ; H NMR (200 MHz, DMSO-d )
6
δ: 2.30 (s, 3H, CH
H, ArH), 8.31–8.47 (m, 4H, ArH), 8.66 (d, 1H, J = 7.5 Hz, H-4′); C NMR (50 MHz, DMSO-d
q), 31.4 (q), 108.0 (s), 112.0 (d), 113.4 (d), 117.2 (s), 117.4 (d), 117.6 (s), 121.7 (d), 124.0 (d), 124.6 (d),
25.2 (d), 126.8 (2xd), 127.8 (s), 129.3 (d), 130.3 (2xd), 130.9 (d), 133.8 (s), 134.6 (s), 145.7 (s), 147.1 (s),
3
), 3.94 (s, 3H, CH ), 7.37–7.45 (m, 5H, ArH), 7.93–7.97 (m, 2H, ArH), 8.01–8.09 (m,
3
13
2
6
) δ: 21.0
(
1

Mar. Drugs 2015, 13
1916
: C, 64.44; H, 4.16, N, 11.56. Found: C,
147.4 (s), 148.2 (d), 161.6 (s). Anal. Calcd for: C26
20 4 2 2
H N O S
64.19; H, 4.33; N, 11.25.
3
.1.9. Synthesis of 3-[4-(1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines (3i–k)
To a suspension of derivatives 3e–h (0.6 mmol) in ethanol (2 mL), potassium hydroxide (0.1 g,
1.8 mmol) was added and the mixture was heated at reflux for 1–2 h. The precipitate, obtained after
cooling, was filtered off, dried and recrystallized from ethanol to afford derivatives 3i–k.
3
.1.9.1. 3-[4-(5-Fluoro-1H-indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (3i)
Conditions: reflux for 1.5 h. Yellow solid; yield: 98%; mp: 248–249 °C; IR 3600 and 3550(2xNH),
−1
1
cm ; H NMR (200 MHz, DMSO-d
6
) δ: 7.05 (t, 1H, J = 7.8 Hz, H-6″), 7.47–7.53 (m, 2H, H-5′,
H-7″), 7.78 (bs, 1H, H-2″), 7.92 (d, 1H, J = 8.9 Hz, H-4″), 8.19 (bs, 1H, H-2′), 8.47 (m, 2H, H-5,
13
H-6′), 8.95 (d, 1H, J = 7.4 Hz, H-4′), 11.70 (bs, 1H, NH), 12.93 (bs, 1H, NH); C NMR (50 MHz,
DMSO-d ) δ: 104.7 (d, JC4″–F = 21.6 Hz), 107.3 (d), 109.7 (d, JC6″–F = 26.1 Hz), 109.8 (s), 110.7 (s), 113.0
d, JC7″–F = 10.0 Hz), 117.2 (d), 119.0 (s), 124.6 (d, JC3″–F = 10.5 Hz), 127.2 (d), 128.2 (d), 132.3 (d), 133.3
s), 140.6 (d), 144.7 (s), 149.6 (s), 157.5 (d, JC5″–F = 232 Hz), 160.5 (s). Anal. Calcd. for C18 S: C,
6
(
(
H
11FN
4
64.66; H, 3.32; N, 16.76. Found: C, 64.37; H, 3.04; N, 16.57.
3
.1.9.2. 3-[4-(5-Fluoro-1H-indol-3-yl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (3l)
−1 1
Conditions: reflux for 1 h. Yellow solid; yield 40%; mp: 243–244 °C; IR 3557 (NH) cm ; H NMR
200 MHz, DMSO-d ) δ: 3.93 (s, 3H, CH ), 7.04 (td, 1H, J = 2.1, 9.1, 10.9 Hz, H-6″), 7.35 (dd, 1H,
(
6
3
J = 4.6, 7.8 Hz, H-5′), 7.49 (dd, 1H, J = 4.6, 9.1 Hz, H-7″), 7.70 (s, 1H, H-2″), 7.96 (dd, 1H, J = 2.1, 10.9
Hz, H-4″), 8.11 (bs, 1H, H-2′), 8.38 (s, 1H, H-5), 8.42 (d, 1H, J = 4.6 Hz, H-6′), 8.69 (d, 1H,
13
J = 7.8 Hz, H-4′), 11.64 (bs, 1H, NH); C NMR (50 MHz, DMSO-d
6
) δ: 31.2 (q), 104.9 (d,
C4″–F = 24 Hz), 106.5 (d), 108.3 (s), 109.7 (d, JC6″–F = 26 Hz), 111.3 (d, JC7″–F = 4.5 Hz), 112.9 (d,
C7″–F = 9.5 Hz), 117.0 (s), 117.2 (d), 124.8 (d, JC4″–F = 10.1 Hz), 126.8 (d), 129.0 (d), 130.2 (d), 133.3 (s),
J
J
1
43.7 (d), 147.6 (s), 150.2 (s), 157.4 (d, JC5″–F = 231.9 Hz), 160.8 (s). Anal. Calcd. for C19
H
13FN S: C,
4
65.50; H, 3.76; N, 16.08. Found: C, 65.36; H, 3.47; N, 15.82.
3
.1.9.3. 3-[4-(1H-Indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (3j)
−1
Conditions: reflux for 2 h. Yellow solid; yield: 98%; mp: 297–298 °C; IR 3676 and 3557 (2xNH) cm ;
1H NMR (200 MHz, DMSO-d
) δ: 7.13–7.23 (m, 2H, H-5″, H-6″), 7.39 (dd, 1H, J = 4.9, 7.9 Hz,
6
H-5′), 7.47–7.51 (m, 1H, H-7″), 7.68 (bs, 1H, H-2″), 8.05 (d, 1H, J = 2.6 Hz, H-2′), 8.18 (dd, 1H,
J = 2.7, 7.1 Hz, H-4″), 8.34 (s, 1H, H-5), 8.42 (dd, 1H, J = 1.5, 4.9 Hz, H-6′), 8.82 (dd, 1H, J = 1.5, 7.9 Hz,
13
H-4′), 11.46 (bs, 1H, NH), 12.50 (bs, 1H, NH); C NMR (50 MHz, DMSO-d
6
) δ: 106.6 (d), 109.7 (s),
1
1
10.9 (s), 111.9 (d), 117.1 (d), 117.6 (s), 119.8 (d), 120.0 (d), 121.5 (d), 124.6 (s), 125.1 (d), 127.1 (d),
30.3 (d), 136.6 (s), 142.7 (d), 147.3 (s), 150.5 (s), 160.8 (s). Anal. Calcd for C18
H
12
N S: C, 68.33; H, 3.82;
4
N, 17.71. Found: C, 68.06; H, 4.07; N, 17.45.

Mar. Drugs 2015, 13
.1.9.4. 3-[4-(1H-Indol-3-yl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (3k)
1917
3
−1 1
Conditions: reflux for 1.5 h. Yellow solid; yield: 98%; mp: 276–277 °C; IR 3500 (NH) cm ; H NMR
200 MHz, DMSO-d ) δ: 3.95 (s, 3H, CH ), 7.14–7.25 (m, 2H, H-5″, H-6″), 7.41 (dd, 1H,
(
6
3
J = 5.0, 7.7 Hz, H-5′), 7.50 (dd, 1H, J = 2.1, 7.4 Hz, H-7″), 7.69 (bs, 1H, H-2″), 8.05 (d, 1H, J = 5.0 Hz,
H-6′), 8.16–8.21 (m, 1H, H-4″), 8.45–8.48 (m, 2H, H-5, H-2′), 8.78 (dd, 1H, J = 2.0, 7.7 Hz, H-4′), 11.49
13
(
s, 1H, NH); C NMR (50 MHz, DMSO-d
6
) δ: 31.6 (q), 106.8 (d), 108.4 (s), 110.7 (s), 111.9 (d), 117.3 (d),
1
1
4
19.8 (d), 119.9 (d), 121.6 (d), 124.6 (s), 125.1 (d), 125.9 (s), 130.3 (d), 130.8 (d), 135.7 (s), 136.6 (s),
42.6 (d), 150.3 (s), 160.6 (s). Anal. Calcd for C19
H
14
N S: C, 69.07; H, 4.27; N, 16.96. Found: C, 69.31; H,
4
.50; N, 16.70.
3
.2. HPLC Analysis
Analysis of nortopsentin analogues was carried out using a Gilson modular liquid chromatography
system (Gilson Inc., Middleton, WI, USA) equipped with M 302 and 305 pumps, and injector model 77–25
Rheodyne, Berkeley, CA, USA) with a 20 μL injector loop and a M 802 manometric module. The
(
chromatographic column was a µPorasil column (300 × 3.9 mm; Waters, Milford, MA, USA) provided
with relevant guard cartridge (5 × 3.9 mm, Waters). Detection at 283 nm was by an M 118 UV-vis detector,
used along with the Gilson 712 HPLC system controller software. Sensitivity was 0.05% absorbance unit
(AUFS). Elution was with a 20 min linear gradient elution from solvent A (dichlomethane:ethyl acetate,
1:1) to 100% solvent B (ethyl acetate), at a flow rate of 1 mL/min. Retention times of 3d and 3k were
8.10 min and 8.33 min, respectively.
3
.3. Biology
3
.3.1. Viability Assay in Vitro
The tested compounds 3d and 3k were dissolved in DMSO and then diluted in culture medium so that
the effective DMSO concentration did not exceed 0.1%. Human cell lines of hepatoma HepG2 and Chang
liver were purchased from American Type Culture Collection, Rockville, MD, USA. Cells were grown in
RPMI supplemented with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin
and 5 μg/mL gentamicin. HepG2 culture medium also contained 1.0 mM sodium pyruvate. Cells were
8
maintained in log phase by seeding twice a week at a density of 3 × 10 cells/L in humidified 5% CO
2
atmosphere, at 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. Cytotoxic activity of the
compounds was determined by the MTT quantitative colorimetric assay based on the reduction of the
3
-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into purple formazan by
4
mitochondrial dehydrogenases of living cells. Briefly, HepG2 and Chang cells were plated at 5 × 10 cells/
well in 96-well plates containing 200 μL RPMI. After an overnight incubation, cells were washed with
fresh medium and incubated with the compounds in RPMI. After a 72 h incubation, cells were washed, and
50 µL FBS-free medium containing 5 mg/mL MTT were added. The medium was discarded after 4 h
incubation at 37 °C, and formazan blue formed in the cells was dissolved in DMSO. The absorbance at

Mar. Drugs 2015, 13
1918
540 nm was measured in a microplate reader (Bio-RAD, Hercules, CA, USA). 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.
3
.3.2. Cell Cycle Analysis
Cell cycle stage was analyzed by flow cytometry. Aliquots of 1 × 10 cells were harvested by
6
centrifugation, washed with PBS and incubated 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 were
subjected to fluorescence-activated cell sorting (FACS) analysis by Epics XL™ flow cytometer
4
using Expo32 software (Beckman Coulter, Fullerton, CA, USA). At least 1 × 10 cells were analyzed for
each sample.
3
.3.3. Measurement of Phosphatidylserine 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
V-FITC negative and PI positive). HepG2 cells were seeded in triplicate in 24-wells culture plates at a
5
2
density of 2.0 × 10 cells/cm . After an overnight incubation, cells were washed with fresh medium and
incubated with the compounds in RPMI. After 24 h, cells were harvested by trypsinization and adjusted at
5
2
.0 × 10 cells/mL with combining buffer. One hundred μL of cell suspended solution was added to a new
tube, and incubated with 5 µL annexin V and 10 μL of a 20 μg/mL PI solution at room temperature in the
4
dark for 15 min. Then at least 1.0 × 10 cells were immediately subjected to FACS analysis using
appropriate 2-bidimensional gating method.
3
.3.4. Acridine Orange and Ethidium Bromide Morphological Fluorescence Dye Staining
Acridine orange (AO) stains DNA bright green, allowing visualization of the nuclear chromatin pattern
and stains both live and dead cells. Ethidium bromide (EB) stains DNA orange but is excluded by viable
cells. Dual staining allows separate enumeration of populations of viable non-apoptotic, viable (early)
apoptotic, nonviable (late) apoptotic, and necrotic cells. Live cells appear uniformly green. Early apoptotic
cells stain green and contain bright green dots in the nuclei as a consequence of chromatin condensation
and nuclear fragmentation. Late apoptotic cells incorporate EB and therefore stain orange, but, in contrast
to necrotic cells, the late apoptotic cells show condensed and often fragmented nuclei. Necrotic cells stain
orange, but have a nuclear morphology resembling that of viable cells, with no condensed chromatin.
Briefly, after HepG2 cells were treated with 3d or 3k compounds for 24 h, the medium was discarded.
Cells were washed with saline 5 mM phosphate buffer, pH 7.4 (PBS) and then incubated with 100 μL
PBS containing 100 μg/mL of EB plus 100 μg/mL of AO. After 20 s, EB/AO solution was discarded and
cells immediately visualized by means of fluorescent microscope equipped with an automatic

Mar. Drugs 2015, 13
1919
photomicrograph system (Leica, Wetzlar, Germany). Multiple photos were taken at randomly selected
areas of the well to ensure that the data obtained are representative.
3
.3.5. Measurement of Mitochondrial Transmembrane Potential
Mitochondrial transmembrane potential was assayed by flow cytofluorometry, using the cationic
lipophilic dye 3,30-dihexyloxacarbocyanine iodide (Molecular Probes, Inc., Eugene, OR, USA), which
accumulates in the mitochondrial matrix. Changes in mitochondrial membrane potential are indicated by a
reduction in the 3,30-dihexyloxacarbocyanine iodide-induced fluorescence intensity. Cells were incubated
with 3,30-dihexyloxacarbocyanine iodide at a 40 nmol/L final concentration, for 15 min at 37 °C. After
centrifugation, cells were washed with PBS and suspended in 500 µL PBS. Fluorescent intensities were
4
analyzed in at least 1 × 10 cells for each sample.
3
.3.6. Measurement of Intracellular Reactive Oxygen Species
ROS level was monitored by measuring fluorescence changes that resulted from intracellular oxidation
of 2′,7′-dichlorofluorescindiacetate (DCFH). DCFH, at 10 mM final concentration, was added to the cell
medium 30 min before the end of the treatment. The cells were collected by centrifugation for 5 min at
2
000 rpm at 4 °C, washed, suspended in PBS and immediately subjected to fluorescence-activated cell
4
sorting analysis. At least 1 × 10 cells were analyzed for each sample.
4
. Conclusions
A new series of nortopsentin analogues in which the imidazole ring of the natural product was replaced
by thiazole and the indole unit bound to the position 2 of thiazole was substituted by a 7-azaindole moiety
was efficiently synthesized. Two of the new nortopsentin derivatives, 3d and 3k, showed good
antiproliferative activity against the totality of the about 60 human tumor cell lines of NCI full panel with
GI50 values ranging from low micromolar to nanomolar level (13.0–0.03 and 14.2–0.04 μM, respectively.
Moreover, they have shown potent cytotoxic activity on HepG2 hepatocarcinoma cells, while under
identical conditions, they did not affect normal immortalized human liver cells (Chang). Both the
compounds induced a concentration-dependent accumulation of cells in the subG0/G1phase while confined
viable cells in G2/M phase. The mechanism of the anti-proliferative effect of the nortopsentin derivatives
was pro-apoptotic, being associated with externalization of plasma membrane phosphatidylserine and
mitochondrial dysfunction.
Acknowledgments
This work was financially supported by Ministero dell’Istruzione dell’Università e della
Ricerca (MIUR).
Author Contributions
Anna Carbone, Barbara Parrino, Gloria Di Vita Virginia Spanò, Alessandra Montalbano performed
chemical research and analyzed the data. Alessandro Attanzio, Luisa Tesoriere, and Maria Antonia Livrea,
performed biological research and analysed the data. Girolamo Cirrincione, Patrizia Diana, Paola Barraja,

Mar. Drugs 2015, 13
1920
Luisa Tesoriere, Maria Antonia Livrea, 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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