Synthesis and antiproliferative activity of thiazolyl-bis-pyrrolo[2,3-b]pyridines and indolyl-thiazolyl-pyrrolo[2,3-c]pyridines, nortopsentin analogues

Article abstract of DOI:10.3390/md13010460

Two new series of nortopsentin analogues, in which the imidazole ring of the natural product was replaced by thiazole and indole units were both substituted by 7-azaindole moieties or one indole unit was replaced by a 6-azaindole portion, were efficiently synthesized. Compounds belonging to both series 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 former series induced apoptosis, evident as externalization of plasma membrane phosphatidylserine (PS), and changes of mitochondrial trans-membrane potential, while blocking the cell cycle in G2/M phase. In contrast, a derivative of the latter series elicited distinct responses in accordance with the dose. Thus, 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.

Full text of DOI:10.3390/md13010460

Mar. Drugs 2015, 13, 460-492; doi:10.3390/md13010460
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Synthesis and Antiproliferative Activity of
Thiazolyl-bis-pyrrolo[2,3-b]pyridines and
Indolyl-thiazolyl-pyrrolo[2,3-c]pyridines, Nortopsentin Analogues
Anna Carbone, Barbara Parrino, Gloria Di Vita, Alessandro Attanzio, Virginia Spanò,
Alessandra Montalbano, Paola Barraja, Luisa Tesoriere, Maria Antonia Livrea,
Patrizia Diana and Girolamo Cirrincione *
Biological, Chemical and Pharmaceutical Sciences and Technologies Department, STEBICEF,
Università degli Studi di Palermo, via Archirafi 32, 90123 Palermo, Italy;
E-Mails: anna.carbone@unipa.it (A.C.); barbara.parrino@unipa.it (B.P.);
gloria.divita@unipa.it (G.D.V.); 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.: +39-91-238-96810.
Academic Editor: Yoshihide Usami
Received: 14 November 2014 / Accepted: 4 January 2015 / Published: 16 January 2015
Abstract: Two new series of nortopsentin analogues, in which the imidazole ring of the
natural product was replaced by thiazole and indole units were both substituted by
7-azaindole moieties or one indole unit was replaced by a 6-azaindole portion, were efficiently
synthesized. Compounds belonging to both series 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 former series induced apoptosis, evident as
externalization of plasma membrane phosphatidylserine (PS), and changes of mitochondrial
trans-membrane potential, while blocking the cell cycle in G2/M phase. In contrast, a
derivative of the latter series elicited distinct responses in accordance with the dose. Thus, 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.

Mar. Drugs 2015, 13
Keywords: marine alkaloids; bis-indolyl alkaloids; nortopsentins; thiazolyl-bis-pyrrolo
461
[2,3-b]pyridines; indolyl-thiazolyl-pyrrolo[2,3-c]pyridines; apoptosis; autophagic death;
antiproliferative activity
1. Introduction
In recent years, a great deal of attention has been paid to the identification and development of new
molecules based on natural or synthetic heterocyclic scaffolds as possible novel cancer therapeutic
agents [1–12]. In particular, bis-indolyl alkaloids, characterized by two indole units bound to a spacer
through their position 3, constitute a class of deep-sea sponge metabolites with potent biological
activities such as antiinflammatory, antimicrobial, antiviral, and antitumor [13–16].
The spacer structural profile can vary from a linear chain to carbocycles or heterocycles differently
sized. Thus, coscinamides A–C, isolated from deep marine sponge Coscinoderma sp. bearing a linear
chain as a spacer, showed HIV inhibitory activity (Chart 1) [17]. Asterriquinone, isolated from
Aspergillus fungi, bears a quinone symmetrical structure and showed in vivo activity against Ehrlich
carcinoma, ascites hepatoma AH13, and mouse P388 leukemia [18]. Dragmacidins have been isolated
from a large number of deep water sponges such as Dragmacidon, Halicortex, Spongosorites,
Hexadella and the tunicate Didemnum candidum, and present different spacers and diverse related
activities. Dragmacidin and dragmacidins A–C, which exhibit the saturated six-membered heterocyclic
link piperazine, showed modest cytotoxic activity [19–21]. Instead, a more complex member of this
family, dragmacidin D, bearing a pyrazinone moiety as central core, exhibited several biological
properties such as inhibition of serine-threonine protein phosphatases, antiviral, antimicrobial, and
anticancer activities [22,23].
Topsentins A, B1 and B2, isolated from Mediterranean sponge Topsentia genitrix, bearing the
five-membered ring spacer imidazole, showed antitumor and antiviral activities [24,25].
Nortopsentins A–C also bearing the imidazole ring as a spacer, were isolated from Spongosorites
ruetzleri, and showed in vitro cytotoxicity against P388 cells (GI50, 4.5–20.7 μM). Replacement of the
indole nitrogen with a methyl group led to derivatives that showed a significant improvement in
cytotoxicity against P388 cells (GI50, 0.8–2.1 μM) [26,27].
Due to a great limitation in the use of the reservoir of marine organism that allow the isolation of
very small amount of the biologically active substances from the natural material, several total
synthesis of nortopsentins were proposed [28–31].
Moreover, due to the considerable activities shown, indolyl alkaloids have become an attractive
field in medicinal chemistry and several dragmacidin analogues bearing six membered rings such as
pyridine, pyrazine, pyrazinone and pyrimidine as spacer between the two indole units were
synthesized. These analogues showed good antiproliferative activity against a wide range of
human tumor cell lines [32–35]. Many papers reported 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 [36], -pyrazoles [37], -furans [38],
-isoxazoles [38], -pyrroles [39], and -1,2,4-thiadiazoles [40]. Most of these analogues exhibited good

Mar. Drugs 2015, 13
462
antiproliferative activity against wide range of human tumor cell lines often reaching GI50 values at
submicromolar level.
H
N
O
N
O
O
R
O
HO
N
N
H
N
H
R1
OH
Coscinamide A R=Br; R1=H
Coscinamide B R=R1=H
Coscinamide C R=Br; R1=OH
Asterriquinone
H
N
R1
Br
N
NH2
R3
+
H
NH
H
N
N
N
R
Br
Br
N
N
H
R2
N
H
O
_
Dragmacidin
R=H; R1=Me; R2=Br; R3=OH
N
H
CF3CO2
OH
Dragmacidin A R=R2=R3=H; R1=Me
Dragmacidin B R=R1=Me; R2=R3=H
Dragmacidin C R=R1=R2=R3=H
Dragmacidin D
R
R3
Y
Z
O
N
X
R1
N
N
N
H
N
H
R1
R2
R
N
H
Nortopsentin A X=N; Y=CH; Z=NH; R1=R2=H; R=R3=Br
Nortopsentin B X=N; Y=CH; Z=NH; R=R1=R2=H; R3=Br
Nortopsentin C X=N; Y=CH; Z=NH; R1=R2=R3=H; R=Br
Topsentin A R=R1=H
Topsentin B1 R=H; R1=OH;
Topsentin B2 R=Br; R1=OH;
Thiazoles X=N; Y=CH; Z=S; R1 or R2=H, Me; R or R3=H, OMe, Br
Thiophenes X=S; Y=Z=CH; R=R3=H, Me, OMe, Cl, Br;
R1=R2=H, Me, SO2Ph
Pyrazoles X=CH; Y=N; Z=NH,NMe; R=R3=H,Me,OMe,Cl,Br;
R1=R2= Me
Furans X=O; Y=Z=CH; R=R3=H,Me,OMe; R1=R2=Me
[1,2]Oxazoles X=CH; Y=N; Z=O; R=R3=H, Me, OMe, Cl, Br;
R1=R2=Me
Pyrroles X=NH; Y=Z=CH; R1=R2=Me; R=R3=H, Me, OMe, Cl, Br
1,2,4-Thiadiazoles X=Z=N; Y=S; R or R3=H, OMe, NO2, Br;
R1or R2=H
Chart 1. Bis-indolyl alkaloids and analogues.
Moreover, the structural manipulation of the natural nortopsentins, beside the heterocyclic spacer,
was extended to one or both indole units and led to 3-[(2-indolyl)-5-phenyl]pyridines and
phenylthiazolyl-7-azaindole derivatives. Both these series of compounds showed antiproliferative
activity against a wide range of human tumor cell lines in the micromolar-submicromolar range and
were able to inhibit the activity of the cyclin-dependent kinase 1 (CDK1) with GI50 values lower than
1 μM [41,42].
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, in which the 4-azaindole ring
substituted one indole system and the thiazole moiety replaced the imidazole nucleus of nortopsentin

Mar. Drugs 2015, 13
463
were synthesized and tested against a panel of cell lines with different histologic origin, including
breast cancer, androgen-independent prostate cancer, pancreatic carcinoma and peritoneal
mesothelioma (Chart 2). Four compounds showed GI50 values ranging from 2.20 to 19.36 μM, and
were also able to inhibit CDK1 with GI50 in the range 0.64–0.87 μM. Moreover, the most active
compound also reduced the cyclin B1-associated CDK1 kinase activity in a peritoneal mesothelioma
cell line and increased by 4-fold and 3-fold caspase-9 and caspase-3 activity respectively [43].
S
S
R
R2
S
X
R
N
N
N
N
Y
N
N
N
N
N
R
N
N
N
Cl
R1
R2
R1
R1
R2
4-aza Series: X=N; Y=CH;
7-aza Series: X=CH; Y=NH
1
2-4
Chart 2. Nortopsentin aza-analogues.
Contemporaneously, 3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl)-1H-7-azaindoles, in which the spacer is
constituted by the thiazole ring and one of the indole units is replaced by a 7-azaindole moiety
were synthesized.
All compounds of this series were 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, that also act as CDK1 inhibitors, consistently reduced DMPM cell
proliferation and induced a caspase-dependent apoptotic response, with a concomitant reduction
of the expression of the active Thr34-phosphorylated form of the anti-apoptotic protein survivin.
Moreover, the combined treatment of DMPM cells with the most active derivative and paclitaxel
produced a synergistic cytotoxic effect, which was parallel by an enhanced apoptotic response. In the
mouse model, 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 [44].
In this paper, continuing our studies on indole and related azaindole systems [45–49], we report the
synthesis of derivatives of type 1, nortopsentin analogues in which both indole units are replaced by a
7-azaindole moiety, and of derivatives of type 2–4 in which one indole unit was replaced by a
6-azaindole moiety. 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
The synthesis of thiazolyl-bis-pyrrolo[2,3-b]pyridines of type 1 involved the construction of the thiazole
ring by Hantzsch reaction between the two key intermediates pyrrolo[2,3-b]pyridine-carbothioamides
7 and 3-bromoacetyl-pyrrolo[2,3-b]pyridines 9 (Scheme 1). The Hantzsch reaction was also used for the
synthesis of indolyl-thiazolyl-pyrrolo[2,3-c]pyridines of type 2–4, but, in this case the key intermediates
were indolocarbothioamides 16–18 and 3-bromoacetyl-pyrrolo[2,3-c]pyridines 23, 24 (Scheme 2).

Mar. Drugs 2015, 13
464
The starting material for the preparation of pyrrolo[2,3-b]pyridine-carbothioamides 7a,b
(Scheme 1) were the corresponding carboxamides 6a,b conveniently prepared in excellent yields
(95%–99%) by reaction of the corresponding pyrrolo[2,3-b]pyridine-3-carbonitrile 5a,b with
sulfuric acid and subsequent alkalinization. Treatment of carboxamides 6a,b with Lawesson’s reagent
in refluxing tetrahydrofuran (THF) gave the desired pyrrolo[2,3-b]pyridine-carbothioamides 7a,b
(88%–99%).
Pyrrolo[2,3-b]pyridines 8a,c,e were converted into the corresponding N-methyl derivatives 8b,d,f
(60%–96%) using potassium tert-butoxide (t-BuOK), tris[2-(2-methoxyethoxy)ethyl]amine (TDA-1)
as a catalyst and methyl iodide (MeI) in toluene [42].
3-Bromoacetyl-pyrrolo[2,3-b]pyridines 9a–f were efficiently synthesized (70%–92%) by
acylation of the corresponding pyrrolo-pyridines 8a–f with bromoacetyl bromide (BrCOCH2Br) in the
presence of aluminium chloride (AlCl3) in dichloromethane (DCM) (Scheme 1) [42]. Reaction of
pyrrolo[2,3-b]pyridine-carbothioamides 7a,b and 3-bromoacetyl-pyrrolo[2,3-b]pyridines 9a–f in
ethanol (EtOH) under reflux gave the desired thiazolyl-bis-pyrrolo[2,3-b]pyridines 1a–l in good to
excellent yields (60%–94%) (Scheme 1).
S
O
CN
NH2
NH2
ii
iii
N
R
N
R
N
R
N
N
N
5a R = H
5b R1 = Me
6a R = H
6b R= Me
7a R = H
7b R = Me
i
R2
S
vi
N
N
N
N
R
N
R1
1a-l
O
Br
R2
R2
v
N
R1
N
N
R1
N
8a R1 = R2 = H
9a R1 = R2 = H
iv
iv
iv
8b R1 = Me, R2 = H
8c R1 = H, R2 = Br
8d R1 = Me, R2 = Br
8e R1 = H, R2 = F
8f R1 = Me, R2 = F
9b R1 = Me, R2 = H
9c R1 = H, R2 = Br
9d R1 = Me, R2 = Br
9e R1 = H, R2 = F
9f R1 = Me, R2 = F
Scheme 1. Synthesis of thiazolyl-bis-pyrrolo[2,3-b]pyridines 1a–l.

Mar. Drugs 2015, 13
465
S
O
R
NH2
R
NH2
R
iii
iv
N
N
N
R1
R1
R1
16d
R1 = H
13d R1 = H
14a-d R1 = Me
15a-c R1 = Boc
10a-d R1 = H
11a-d R1 = Me
12a-c R1 = Boc
i
17a-d R1 = Me
18a-c R1 = Boc
ii
R
S
viii
N
N
a R = H, b R = OMe, c R = Br, d R = F
N
N
Cl
R1
R2
2a-j
3a-f
4a-f
ix
O
NO2
Br
v
vii
Cl
N
19
N
N
N
N
R2
Cl
R2
Cl
Br
21 R2 = H
22 R2 = Me
23 R2 = H
24 R2 = Me
Mg
vi
20
Scheme 2. Synthesis of indolyl-thiazolyl-pyrrolo[2,3-c]pyridines 2a–j, 3a–f, 4a–f.
Reagents: (i) (a) t-BuOK, toluene, TDA-1, rt, 6 h; (b) MeI, rt, 1 h, 85%; (ii) (a) H2SO4, rt, 15–60 min;
(b) NaOH, 95%–99%; (iii) Lawesson’s reagent, THF, reflux, 30 min, 88%–99%; (iv) (a) t-BuOK,
toluene, TDA-1, rt, 3 h; (b) MeI, rt, 1 h, 60%–96%; (v) AlCl3, DCM, BrCOCH2Br, reflux, 40 min,
70%–92%; (vi) EtOH, reflux, 30 min, 60%–94%.
Indole-3-carbothioamides 16d, 17a–d and 18a–c (Scheme 2) were obtained from the corresponding
indoles 10d, 11a–d, and 12a–c through the formation of amides 13d, 14a–d and 15a–c as previously
reported [43,44].
7-Chloro-1H-pyrrolo[2,3-c]pyridine 21 was obtained by reaction of 2-chloro-3-nitropyridine 19
with vinylmagnesium bromide 20 in THF under nitrogen atmosphere as previously reported [50].
Compound 21 was converted into the corresponding N-methylated derivative 22, and both
pyrrolo[2,3-c]pyridines were transformed into the corresponding 3-bromoacetyl-pyrrolo[2,3-c]pyridines
23, 24 in excellent yields (88%–90%) by usual acylating procedure [42].
The reaction of indolocarbothioamides 16d, 17a–d and 18a–c with 3-bromoacetyl-pyrrolo
[2,3-c]pyridines 23, 24 provided the indolyl-thiazolyl-pyrrolo[2,3-c]pyridines 2a–j and 3a–f in good to
excellent yields (65%–98%) (Scheme 2). The subsequent deprotection of N-tert-butylcarboxylate
derivatives 3a–f using trifluoacetic acid (TFA) in DCM under reflux afforded, after neutralization
with hydrogen carbonate (NaHCO3), the corresponding thiazoles 4a–f in good to excellent yields
(62%–99%) (Table 1).

Mar. Drugs 2015, 13
466
Table 1. Substituted thiazolyl-bis-pyrrolo[2,3-b]pyridines 1a–l and indolyl-thiazolyl-
pyrrolo[2,3-c]pyridines 2a–j, 3a–f, 4a–f.
Compd
1a
1b
1c
R
H
R1
H
R2
H
Yield% Compd
R
OMe
Br
R1
Me
Me
Me
H
R2
Me
Me
Me
Me
H
Yield%
79
63
60
75
90
94
90
60
90
60
85
80
72
98
65
93
97
96
2f
2g
2h
2i
H
H
Br
F
98
H
H
F
95
1d
1e
Me
Me
Me
Me
Me
Me
H
H
H
F
82
H
Br
F
2j
F
H
66
1f
H
3a
3b
3c
3d
3e
3f
H
Boc
Boc
Boc
Boc
Boc
Boc
H
H
84
1g
1h
1i
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
OMe
Br
H
77
Br
F
H
77
H
Me
Me
Me
H
91
1j
H
OMe
Br
92
1k
1l
H
Br
F
91
H
4a
4b
4c
4d
4e
4f
H
93
2a
2b
2c
H
H
OMe
Br
H
H
62
OMe
Br
F
H
H
H
98
H
H
H
Me
Me
Me
75
2d
2e
H
OMe
Br
H
73
H
Me
H
99
Reagents: (i) (a) t-BuOK, toluene, TDA-1, rt, 1–24 h; (b) MeI, rt, 0.5–1 h, 96%–98%; (ii) (Boc)2O,
TEA, THF, reflux, 24–48 h, 90%–100%; (iii) (a) CSI, MeCN, 0 °C -rt, then rt 0.5–2 h or reflux 15 min;
(b) 10% KOH, aq. acetone, 40%–60%; (iv) Lawesson’s reagent, toluene or benzene, reflux, 0.5–24 h,
90%–98%; (v) THF, −78 °C, 8 h, 33%; (vi) (a) t-BuOK, toluene, TDA-1, rt, 5 h; (b) MeI, rt, 2 h, 70%;
(vii) AlCl3, DCM, BrCOCH2Br, reflux, 40 min, 88%–90%; (viii) EtOH, reflux, 1–3 h, 65%–98%;
(ix) (a) TFA, DCM, reflux, 24 h; (b) aqueous NaHCO3, 62%–99%.
2.2. Biology
All the synthesized thiazoles 1a–l, 2a–j, 3a–f and 4a–f were evaluated by the National Cancer
Institute (Bethesda MD) for cytotoxicity against the NCI-60 cell line panel using standard
protocols [51]. Initially, all derivatives were tested at a single dose (10−5 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 1k and 4c
were further selected for full evaluation at five concentration levels (10−4–10−8 M).
The antitumor activity of compounds 1k and 4c was given by three parameters for each cell line:
GI50 (GI50 is the molar concentration of the compound that inhibits 50% net cell growth), TGI (TGI is
the molar concentration of the compound leading to total inhibition of net cell growth), and LC50
(LC50 is 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. An evaluation of the
data reported in Table 2 pointed out that compounds 1k and 4c exhibited antiproliferative activity

Mar. Drugs 2015, 13
467
against most of the human cell lines at GI50 values from micromolar to submicromolar (0.81–27.7 and
0.93–4.70 µM respectively).
Table 2. In vitro inhibition of cancer cell line growth by compounds 1k, 4c a.
Cell Lines
GI50 (µM )
Cell Lines
GI50 (µM )
Cell Lines
GI50 (µM )
1k
4c
1k
4c
1k
4c
Leukemia
CCRF-CEM
HL-60(TB)
K-562
CNS Cancer
SF-268
Renal Cancer
6.81
3.16
6.01
3.01
27.7
6.74
2.18
2.70
4.06
2.48
1.87
3.25
2.37
2.05
786-0
A498
9.65
10.9
2.35
1.56
2.05
ND
1.37
1.56
2.02
1.96
1.48
3.35
4.16
0.93
>100 2.64
8.76 2.73
SF-295
SF-539
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
MOLT-4
RPMI-8226
SR
>100 3.02
>100 4.03
ND b 1.27
SNB-19
SNB-75
U251
3.77
ND
Non-Small Cell Lung Cancer
Melanoma
A549/ATCC
EKVK
2.59
1.27
2.39
5.03
1.97
2.80
3.83
3.11
2.11
LOX IMVI
MALME-3M
M14
4.26
3.01
4.06
1.63
ND
Prostate Cancer
HOP-62
2.22
3.43
4.09
1.85
2.61
2.68
2.19
PC-3
4.35
3.51
3.86
1.76
HOP-92
2.43 MDA-MB-435 3.17
DU-145
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
2.40
2.42
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
19.8
ND
Breast Cancer
>100 3.54
2.05
ND
MCF7
MDA-MB-231/ATCC
HS 578T
6.77
3.02
2.43
20.5
1.80
0.81
2.20
1.68
3.70
4.70
3.12
1.18
2.98
4.86
2.16
2.28
3.47
BT-549
T-47D
Colon Cancer
COLO-205
HCC-2998
HCT-116
HCT-15
Ovarian Cancer
MDA-MB-468
ND
1.80
IGROV1
2.21
2.91
2.03
2.51
3.45
3.25
>100 2.22
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
2.91
13.7
6.75
5.70
4.46
2.35
1.40
2.66
>100 3.14
HT29
3.72
3.65
2.71
2.78
KM12
2.15 NCI/ADR-RES 3.88
SW-620
1.92 SK-OV-3 3.25
a Data obtained from NCI’s in vitro disease-oriented tumor cells screen; b ND = not determined.
The indolyl-thiazolyl-pyrrolo[2,3-c]pyridine derivative 4c resulted more active than thiazolyl-bis-
pyrrolo[2,3-b]pyridines derivative 1k in terms either of GI50 (mean value 2.59 and 5.26 µM
respectively) and percentage of sensitive cell lines out of the total number of cell lines investigated
(100% and 89%, respectively).
Moreover for compound 4c positive TGI and LC50 values were observed with respect to a good
number of cell lines (98% and 81%, respectively; data not shown).
Derivative 4c was shown to be selective with respect to the renal cancer subpanel having
all the subpanel cell lines GI50 in the range 0.93–4.16 µM . The most sensitive cell lines are UO-31
(GI50 0.93 µM), 786-0 (GI50 1.37 µM ), RXF393 (GI50 1.48 µM), and A498 (GI50 1.56 µM). Cell lines

Mar. Drugs 2015, 13
468
sensitive to derivative 4c were also SR (GI50 1.27 µM ) of leukemia, HCT-15 (GI50 1.40 µM ) of colon
cancer, and MDA-MB-468 (GI50 1.18 µM ) of breast cancer subpanel.
Derivative 1k showed selectivity with respect to MDA-MB-468 (GI50 0.81 µM ) of breast cancer,
EKVK (GI50 1.27 µM ) of non small cell lung, and CAKI-1 (GI50 1.56 µM ) of renal cancer subpanel.
Selective toxicity of 1k and 4c nortopsentin analogues towards tumor cells was investigated. To this
aim human HTC-116 colorectal carcinoma cells, against which both compounds exhibited a
comparable antiproliferative effects (GI50 2.91 and 2.35 μM, Table 2), and intestinal normal-like
differentiated Caco-2 cells were exposed to the compounds for 24 h and viability compared.
While both derivatives, in the range 5 to 100 μM, dose-dependently inhibited the intestinal HCT-116
cell proliferation, they did not affect the differentiated Caco-2 cell viability, suggesting tumor cells as
the main target of their cytocidal action (Figure 1). GI50 values of 1k or 4c calculated after 24 h
treatment of HCT-116 cells, were 33.55 ± 2.31 μM and 13.15 ± 0.95 μM, respectively. Taking in
account the GI50 values measured by NCI on the same cell line after 72 h treatment, the cytotoxic
effects of the nortopsentin analogues appeared time-dependent.
100
75
50
25
0
0
25
50
75
100
Concentration (mM)
Figure 1. Effect of 1k (triangle) and 4c (circle) on the viability of human intestinal cell
lines either tumoral (HCT-116; full line) or normal-like (differentiated Caco-2; dashed line).
Cells were treated with the compounds 1k and 4c and cell survival was measured after 24 h
by MTT assay in comparison to cells treated with vehicle alone (control). Values are the
mean of three separate experiments in triplicate.
2.2.1. Cell Cycle Alterations
We next determined alterations in the cell cycle caused by derivatives 1k and 4c in colorectal
cancer cells. Flow cytometry analysis of nuclear DNA content after 24 h treatment of HCT-116 cells is
reported in Figure 2A. Drug concentrations were chosen on the basis that they represent values above
and below the respective GI50 values. Compared to control cells, 1k caused a dose-dependent
accumulation of cells in G2/M phase, paralleled by a reduction in the percentage of cells in the G1
phase and by a significant increase of cells in the sub-G1 phase, which is representative of cells with
fragmented DNA. On the other hand, 4c induced a dose-dependent accumulation of cells in G1 phase
accompanied by a decrement in the percentage of cells in G2/M phases. Moreover, accumulation of
sub-G1 population was significantly higher than control (p < 0.05) only at high concentration of the

Mar. Drugs 2015, 13
469
drug. These results indicated that the two nortopsentin analogues caused arrest of the HCT-116 cancer
cell growth involving different check points of the cell cycle.
2.2.2. Cell Death
To determine whether HCT-116 cells undergo apoptosis upon treatment with the nortopsentin
analogues, cells were treated with 1k or 4c for 24 h, stained with both propidium iodide (PI) and
Annexin V-fluorescein isothiocyanate (FITC), and analyzed by flow cytometry.
Neither compound caused cell necrosis (Figure 2B). Rather, while the percentage of cells in late
apoptosis increased at the increase of the 1k doses, apoptotic effects of 4c were evident only at high
concentrations (GI70), when cells in early apoptosis appeared significantly increased with respect to
control (p < 0.05).
Mitochondria play a critical role in regulating the apoptotic machinery. We then examined
mitochondrial membrane potential (Δψm) loss using DiOC6, a fluorescent mitochondria-specific and
voltage-dependent dye.
Figure 2. Effect of 1k and 4c on the cell cycle distribution and apoptosis of HCT-116
cells. Flow cytometric analysis of propidium iodide-stained cells (A) and percentage of
Annexin V/propidium iodide (PI) double-stained cells (B), 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 or of apoptosis was calculated
by Expo32 software. Values are the mean ± SD of three separate experiments in triplicate.
As indicated by the decrement in DiOC6 green-associated fluorescence, treatment of HCT-116 cells
with 1k, for 24 h, induced a remarkable dose-dependent dissipation of Δψm (Figure 3).
On the other hand, incubation with 4c did not cause mitochondrial dysfunction at GI30, whereas
induced a significant increase in Δψm loss at higher concentrations.

Mar. Drugs 2015, 13
470
Figure 3. Effects of nortopsentin analogues 1k and 4c on mitochondrial transmembrane
potential in HCT-116 cells. The Δψm was detected by fluorescence intensity of
3,30-dihexyloxacarbocyanine iodide-treated cells, as determined by flow cytometry.
Control, cells treated with vehicle. Representative images of three experiments with
comparable results.
Overall our findings indicated that, although the nortopsentin derivatives inhibited the HCT-116
tumor cell growth, they may elicit different molecular pathways of programmed cell death.
Morphology of HTC-116 cells treated for 24 h with nortopsentin analogues 1k and 4c was assessed by
microscopy analysis after Giemsa staining. Both compounds caused an evident dose-dependent
reduction of the cell population with respect to control (Figure 4A). However, whereas 1k caused
highly condensate cells, as a sign of their apoptotic fate, more complex alterations were observed after
treatment with 4c. A low concentration of drug (GI30) caused evident expansion of whole cytoplasm,
with a massive accumulation of multiple-membrane bounded vacuoles without apparent loss of nuclear
material, a morphology characteristic of autophagic cell death. On the other hand, when HCT-116 cells
were treated with higher concentration of 4c (GI70), vacuolization was less evident and cells with
condensed morphology prevailed, indicating evolution of the cell fate towards apoptosis. Then, we
performed FACS analysis of acridine-orange stained acid vacuoles (AVO) using the red to green ratio
as an indicator of acid vacuolar organelle accumulation [52]. As shown in Figure 4B, treatment of the
cells with 4c (GI30), for 24 h, led to a 10-fold increase of the bright red to green fluorescence intensity
ratio of acrydine orange, indicating an elevated induction of cell vacuolization associated to autophagy.
Instead, the treatment of cells with the highest drug concentration did not significantly modify the
percent of cells with AVO with respect to control, supporting the concept of a different cell death.

Mar. Drugs 2015, 13
471
Figure 4. Nortopsentin analogue 4c induces accumulation of acid vacuoles in HCT-116
cells. (A) Micrographs of Giemsa-stained cells in 40× magnification. Control, cells
treated with vehicle. Representative images of three experiments with comparable results;
(B) The percentage of bright red (FL3) fluorescence-positive cells after Acridine Orange
(AO) staining and fluorescence-activated cell sorting (FACS) analysis. Results are the
mean ± SD of two triplicate experiments. Mean values with unlike superscript letters were
significantly different (p < 0.05; Bonferroni’s test).
A comparison of the antiproliferative activity of the natural products nortopsentins A–C with 1k
and 4c, the most active derivatives of the two new series of analogues, cannot be performed since
nortopsentins were tested against only one cell line (murine leukemia P388), whereas, compounds 1k
and 4c showed to be active against the 89% and 100% respectively, of the tested cell lines belonging to
the NCI full panel of human cancer cell lines (ca. 60) with GI50 values in the low micromolar range.

Mar. Drugs 2015, 13
472
A comparison of the antiproliferative activity of 1k and 4c with the series of nortopsentin analogues
previously reported reveals that these compounds showed to be active against a very large range of
human cell lines at the same level of both the analogues in which the central heterocycle was replaced
by thiophene, pyrazole, furan, isoxazole, and pyrrole [33–39]; and those series in which also the indole
units were substituted by azaindole systems [42–44].
The feature that makes these series of analogues interesting was revealed by the investigations into
the cell cycle alterations and cell death modes which suggest that these derivatives inhibiting the
HCT-116 tumor cell growth may elicit different molecular pathways of programmed cell death. In fact,
1k induced apoptosis, and 4c exhibited different responses depending on the dose: low concentrations
induced morphological changes typical of autophagic death with extensive formation of cytoplasmic
acid vacuoles without apparent loss of nuclear material, and with arrest of cell cycle at the G1 phase,
instead, higher concentrations induced apoptosis with arrest of cell cycle at the G1 phase.
3. Experimental Section
3.1. Chemistry
3.1.1. General
All melting point were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR spectra
1
13
were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. H and C NMR
spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 or CDCl3 solution, using a
Bruker Avance II series 200 MHz spectrometer. Compounds 2d–f,h,j, and 3e,f were characterized
1
only by H NMR spectra, because of their poor solubility the 13C spectra were not performed.
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 (Agilent 1100 Series).
3.1.2. Synthesis of 1-Methyl-1H-pyrrolo[2,3-b]pyridine-3-carbonitrile (5b)
To a cold solution of 5a (1.0 g, 7.0 mmol) in anhydrous toluene (20 mL), t-BuOK (1.1 g, 9.5 mmol)
and TDA-1 (1 or 2 drops) were added at 0 °C. The reaction mixture was stirred at room temperature
for 6 h and then MeI (7.0 mmol, 0.44 mL) was added at 0 °C. TLC analysis (DCM/ethyl acetate 9/1)
revealed that methylation was complete after 1 h. The solvent was evaporated under reduced pressure.
The residue was treated with water, extracted with DCM (3 × 20 mL), dried (Na2SO4), evaporated and
purified by column chromatography using DCM/ethyl acetate (9/1) as eluent. White solid; yield 85%;
1
mp: 106–107 °C; IR 2224 (CN) cm−1; H NMR (200 MHz, DMSO-d6) δ: 3.90 (s, 3H, CH3), 7.35
(dd, 1H, J = 4.7, 8.0 Hz, H-5), 8.15 (dd, 1H, J = 1.5, 8.0 Hz, H-4), 8.47 (dd, 1H, J = 1.5, 4.7 Hz, H.6),
13
8.48 (s, 1H, H-2); C NMR (50 MHz, DMSO-d6) δ: 31.6 (q), 81.9 (s), 115.3 (s), 118.1 (d), 119.2 (s),
127.6 (d), 138.4 (d), 144.8 (d), 146.4 (s). Anal. Calcd for C9H7N3: C, 68.78; H, 4.49; N, 26.74.
Found: C, 68.61; H, 4.21; N, 26.92.

Mar. Drugs 2015, 13
473
3.1.3. General Procedure for the Synthesis of 1H-Pyrrolo[2,3-b]pyridine-3-carboxamides (6a,b)
A solution of appropriate carbonitriles 5a,b (6.3 mmol) in concentrate sulphuric acid (3.32 mL) was
stirred at room temperature for 15–60 min. The solution was slowly poured into ice and basified with
concentrated NaOH. The residue was extracted with ethyl acetate (3 × 20 mL), dried (Na2SO4) and
concentrated under reduced pressure to afford the carboxamides 6a,b.
1H-Pyrrolo[2,3-b]pyridine-3-carboxamide (6a)
Conditions: room temperature for 60 min. Brown solid; yield: 95%; mp: 273–274 °C; IR 3389,
1
3335 (NH2), 3021 (NH), 1636 (CO) cm−1; H NMR (200 MHz, DMSO-d6) δ: 7.16 (dd, 1H, J = 4.7,
7.9 Hz, H-5), 7.52 (bs, 2H, NH2), 8.16 (d, 1H, J = 2.5 Hz, H-2), 8.27 (dd, 1H, J = 1.7, 4.7 Hz, H-6),
13
8.46 (dd, 1H, J = 1.7, 7.9 Hz, H-4), 12.10 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 109.3 (s),
116.8 (d), 118.5 (s), 128.6 (d), 129.2 (d), 143.3 (d), 148.4 (s), 165.8 (s). Anal. Calcd for C8H7N3O: C,
59.62; H, 4.38; N, 26.07. Found: C, 59.46; H, 4.59; N, 26.25.
1-Methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (6b)
Conditions: room temperature for 15 min. White solid; yield: 99%; mp: 218–219 °C; IR 3347,
1
3326 (NH2), 1614 (CO) cm−1; H NMR (200 MHz, DMSO-d6) δ: 3.85 (s, 3H, CH3), 7.20 (dd, 1H,
J = 4.7, 7.9 Hz, H-5), 7.48 (bs, 2H, NH2), 8.17 (s, 1H, H-2), 8.33 (dd, 1H, J = 1.7, 4.7 Hz, H-6),
13
8.45 (dd, 1H, J = 1.7, 7.9 Hz, H-4); C NMR (50 MHz, DMSO-d6) δ: 31.2 (q), 108.2 (s), 117.0 (d),
118.7 (s), 129.4 (d), 132.3 (d), 143.2 (d), 147.4 (s), 165.5 (s). Anal. Calcd for C9H9N3O: C, 61.70;
H, 5.18; N, 23.99. Found: C, 61.55; H, 5.44; N, 24.19.
3.1.4. General Procedure for the Synthesis of 1H-Pyrrolo[2,3-b]pyridine-3-carbothioamides (7a,b)
A mixture of Lawesson’s reagent (0.33 g, 0.8 mmol) and suitable carboxamides 6a,b (1.3 mmol) in
THF (10 mL) was heated under reflux for 30 min. The solution was cooled to room temperature and
the solvent was removed under reduced pressure. The residue was purified by column chromatography
using ethyl acetate as eluent to give the carbothioamides 7a,b.
1H-Pyrrolo[2,3-b]pyridine-3-carbothioamide (7a)
Yellow solid; yield: 88%; mp: 220–222 °C; IR 3306, 3180 (NH2), 3020 (NH), 1590 (CS); 1H NMR
(200 MHz, DMSO-d6) δ: 7.22 (dd, 1H, J = 4.7, 8.0 Hz, H-5), 8.23 (d, 1H, J = 2.7 Hz, H-2), 8.29
(dd, 1H, J = 1.5, 4.7 Hz, H-6), 8.99 (dd, 1H, J = 1.5, 8.0 Hz, H-4), 9.00 (s, 1H, SH), 9.10 (s, 1H, NH),
13
12.30 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 114.7 (s), 117.1 (d), 118.8 (s), 127.6 (d),
130.3 (d), 143.5 (d), 148.9 (s), 193.0 (s). Anal. Calcd for C8H7N3S: C, 54.22; H, 3.98; N, 23.71.
Found: C, 54.02; H, 4.08; N, 23.91.
1-Methyl-1H-Pyrrolo[2,3-b]pyridine-3-carbothioamide (7b)
1
Yellow solid; yield: 99%; mp: 232–233 °C; IR 3336, 3182 (NH2), 1540 (CS) cm−1; H NMR
(200 MHz, DMSO-d6) δ: 3.86 (s, 3H, CH3), 7.26 (dd, 1H, J = 4.7, 8.0 Hz, H-5), 8.28 (s, 1H, H-2), 8.33

Mar. Drugs 2015, 13
474
(dd, 1H, J = 1.6, 4.7 Hz, H-6), 8.93 (dd, 1H, J = 1.6, 8.0 Hz, H-4), 8.97 (s, 1H, SH), 9.16 (bs, 1H, NH);
13C NMR (50 MHz, DMSO-d6) δ: 31.3 (q), 113.7 (s), 117.4 (d), 118.7 (s), 130.3 (d), 131.8 (d),
143.3 (d), 147.8 (s), 192.6 (s). Anal. Calcd for C9H9N3S: C, 56.52; H, 4.74; N, 21.97. Found: C, 56.32;
H, 4.59; N, 22.27.
3.1.5. General Procedure for the Synthesis of 1-Methyl-1H-pyrrolo[2,3-b]pyridines (8b,d,f)
To a cold solution of appropriate pyrrolo-pyridines 8a,c,e (2.5 mmol) in anhydrous toluene (25 mL),
t-BuOK (0.38 g, 3.4 mmol) and TDA-1 (1 or 2 drops) were added at 0 °C. The reaction mixture was
stirred at room temperature for 3 h and then MeI (2.5 mmol, 0.2 mL) was added at 0 °C. TLC analysis
(ethyl acetate) revealed that methylation was complete after 1 h. The solvent was evaporated under
reduced pressure. The residue was treated with water, extracted with DCM (3 × 20 mL), dried
(Na2SO4), evaporated and purified by column chromatography using DCM/ethyl acetate (9/1) as eluent
to give derivatives 8b,d,f [42].
1-Methyl-1H-pyrrolo[2,3-b]pyridine (8b)
Oil; yield: 96%; Analytical and spectroscopic data were previously reported [42].
5-Bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine (8d)
1
Brown solid; yield: 85%; mp: 62–63 °C; H NMR (200 MHz, DMSO-d6) δ: 3.83 (s, 3H, CH3),
6.44 (d, 1H, J = 3.4 Hz, H-3), 7.60 (d, 1H, J = 3.4 Hz, H-2), 8.21 (d, 1H, J = 2.1 Hz, H-4), 8.33 (d, 1H,
13
J = 2.1 Hz, H-6); C NMR (50 MHz, DMSO-d6) δ: 31.0 (q), 98.5 (d), 110.7 (s), 121.7 (s), 130.3 (d),
131.8 (d), 142.2 (d), 145.7 (s). Anal. Calcd for C8H7BrN2: C, 45.53; H, 3.34; N, 13.27. Found: C,
45.38; H, 3.24; N, 13.45.
5-Fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridine (8f)
1
Brown solid; yield: 60%; mp: 72–74 °C; H NMR (200 MHz, DMSO-d6) δ: 3.83 (s, 3H, CH3),
6.47 (1H, d, J = 3.4 Hz, H-3) 7.63 (d, 1H, J = 3.4 Hz, H-2), 7.87 (dd, 1H, J = 2.7, 9.5 Hz, H-4),
13
8.24–8.27 (m, 1H, H-6); C NMR (50 MHz, DMSO-d6) δ: 31.1 (q), 98.8 (d, JC3-F = 4.5 Hz), 113.9
(d, JC4-F = 20.5 Hz), 119.9 (s, JC3a–f = 7.5 Hz), 130.4 (d, JC6-F = 29.2 Hz), 132.3 (d), 144.3 (s), 155.0
(s, JC5-F = 238.0 Hz). Anal. Calcd for C8H7FN2: C, 63.99; H, 4.70; N, 18.66. Found: C, 63.69; H, 4.91;
N, 18.48.
3.1.6. General Procedure for the Synthesis of 2-Bromo-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethanones
(9a–f)
To a solution of appropriate pyrrolo-pyridines 8a–f (2.5 mmol) in 10 mL of anhydrous DCM,
anhydrous AlCl3 (1.2 g, 8.8 mmol) was slowly added. The reaction mixture was heated under reflux
and BrCOCH2Br (2.5 mmol, 0.2 mL) in 2 mL of anhydrous DCM was added dropwise. The resulting
solution was allowed to stir under reflux for 40 min. After cooling, water and ice were slowly added
and the obtained precipitate (for derivative 9a) was filtered off or the oil residue (for derivatives 9b–f)

Mar. Drugs 2015, 13
475
was extracted with DCM (3 × 20 mL) and purified by column chromatography using DCM/ethyl
acetate (9/1) as eluent [42].
2-Bromo-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)pthanone (9a)
White solid; yield: 92%; mp: 280–282 °C; Analytical and spectroscopic data were previously
reported [42].
2-Bromo-1-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9b)
White solid; yield: 80%; mp: 116–117 °C; Analytical and spectroscopic data were previously
reported [42].
2-Bromo-1-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9c)
1
Brown solid; yield: 75%; mp: 244–245 °C; IR 3019 (NH), 1655 (CO) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 4.72 (s, 2H, CH2), 8.46 (d, 1H, J = 2.2 Hz, H-4), 8.57 (d, 1H, J = 2.2 Hz, H-6), 8.71
13
(d, 1H, J = 3.2 Hz, H-2), 13.00 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 33.1 (t), 111.7 (s),
113.8 (s), 119.4 (s), 131.2 (d), 136.9 (d), 144.8 (d), 147.4 (s), 186.6 (s). Anal. Calcd for C9H6Br2N2O:
C, 34.00; H, 1.90; N, 8.81. Found: C, 33.87; H, 2.11; N, 8.99.
2-Bromo-1-(5-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9d)
Brown solid; yield: 70%; mp: 166–167 °C; IR 1653 (CO) cm−1; 1H NMR (200 MHz, DMSO-d6) δ:
3.89 (s, 3H, CH3), 4.66 (s, 2H, CH2), 8.49 (d, 1H, J = 2.2 Hz, H-4), 8.54 (d, 1H, J = 2.2 Hz, H-6),
13
8.74 (s, 1H, H-2); C NMR (50 MHz, DMSO-d6) δ: 31.9 (q), 32.9 (t), 110.2 (s), 114.2 (s), 119.6 (s),
131.4 (d), 139.9 (d), 144.6 (d), 146.5 (s), 186.1 (s). Anal. Calcd for C10H8Br2N2O: C, 36.18; H, 2.43;
N, 8.44. Found: C, 36.02; H, 2.67; N, 8.64.
2-Bromo-1-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9e)
1
Brown solid; yield: 74%; mp: 206–207 °C; IR 3555 (NH), 1654 (CO) cm−1; H NMR (200 MHz,
DMSO- d6) δ: 4.71 (s, 2H, CH2), 8.20 (dd, 1H, J = 2.8, 9.1 Hz, H-4), 8.37–8.39 (m, 1H, H-6), 8.73
13
(d, 1H, J = 3.3 Hz, H-2), 12.88 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 33.7 (t), 112.2
(s, JC7a–f = 3.9 Hz), 115.0 (d, JC4-F = 21.5 Hz), 118.1 (s, JC3a–f = 7.9 Hz), 133.0 (d, JC6-F = 26.5 Hz),
137.4 (d), 145.7 (s), 156.4 (s, JC5-F = 243.0 Hz), 186.5 (s). Anal. Calcd for C9H6BrFN2O: C, 42.05;
H, 2.35; N, 10.90. Found: C, 41.85; H, 2.56; N, 10.76.
2-Bromo-1-(5-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (9f)
Brown solid; yield: 74%; mp: 149–150 °C; IR 1668 (CO) cm−1; 1H NMR (200 MHz, DMSO- d6) δ:
3.90 (s, 3H, CH3), 4.65 (s, 2H, CH2), 8.20 (dd, 1H, J = 2.8, 9.0 Hz, H-4), 8.42–8.44 (m, 1H, H-6), 8.78
13
(s, 1H, H-2); C NMR (50 MHz, DMSO-d6) δ: 32.0 (q), 32.8 (t), 110.6 (s, JC7a–f = 3.9 Hz), 115.3
(d, JC4-F = 21.9 Hz), 118.3 (s, JC3a–f = 7.9 Hz), 132.8 (d, JC6-F = 29.3 Hz), 140.4 (d), 144.8 (s), 156.7

Mar. Drugs 2015, 13
476
(s, JC5-F = 243.5 Hz), 186.0 (s). Anal. Calcd for C10H8BrFN2O: C, 44.31; H, 2.97; N, 10.33. Found: C,
44.14; H, 2.73; N, 10.53.
3.1.7. General Procedure for the Synthesis of Substituted-(1,3-thiazole-2,4-diyl)-bis(1H-pyrrolo
[2,3-b]Pyridines (1a–l)
A suspension of the appropriate pyrrolo[2,3-b]pyridine-carbothioamides 7a,b (5 mmol) and the
proper 3-bromo-acetyl-pyrrolo[2,3-b]pyridines 9a–f (5 mmol) in anhydrous EtOH (20 mL) was heated
under reflux for 30 min. The precipitate, obtained after cooling, was filtered off, dried and crystallized
with EtOH to afford derivatives 1a–l.
3,3′-(1,3-Thiazole-2,4-diyl)bis(1H-pyrrolo[2,3-b]pyridine) (1a)
1
Yellow solid; yield: 63%; mp: 346–347 °C; IR 3410, 3166 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 7.48 (dd, 1H, J = 5.0, 7.9 Hz, ArH), 7.60 (dd, 1H, J = 5.5, 7.9 Hz, ArH), 8.00 (s, 1H,
Ar), 8.39 (s, 1H, ArH), 8.46 (s, 1H, ArH), 8.48 (d, 1H, J = 5.5 Hz, ArH), 8.55 (d, 1H, J = 5.0 Hz,
ArH), 8.87 (d, 1H, J = 7.9 Hz, ArH), 9.11 (d, 1H, J = 7.9 Hz, ArH), 12.75 (bs, 1H, NH), 12.90 (bs, 1H,
NH). 13C NMR (50 MHz, DMSO-d6) δ: 109.6 (d), 110.5 (s), 116.3 (d), 117.3 (d), 118.7 (s), 125.8 (s),
126.9 (d), 127.9 (d), 129.6 (s), 131.4 (d), 134.8 (d), 137.2 (d), 141.5 (d), 143.2 (s), 145.9 (s), 147.9 (s),
149.2 (s). Anal. Calcd for C17H11N5S: C, 64.34; H, 3.49; N, 22.07. Found: C, 64.54; H, 3.70; N, 22.23.
5-Bromo-3-[2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (1b)
1
Yellow solid; yield: 60%; mp: 390–391 °C; IR 3334, 3350 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 7.35 (dd, 1H, J = 4.8, 8.0 Hz, H-5′), 7.87 (s, 1H, H-5), 8.24 (d, 1H, J = 2.6 Hz, H-2″),
8.35 (d, 1H, J = 2.1 Hz, H-6″), 8.38–8.42 (m, 2H, H-2′, H-6′), 8.80 (dd, 1H, J = 1.2, 8.0 Hz, H-4′), 8.81
(d, 1H, J = 2.1 Hz, H-4″), 12.26 (bs, 1H, NH), 12.46 (bs, 1H, NH); 13C NMR (50 MHz,
DMSO-d6) δ: 108.2 (d), 109.5 (s), 109.8 (s), 111.5 (s), 117.1 (d), 118.3 (s), 118.7 (s), 126.8 (d),
127.5 (d), 130.3 (d), 130.9 (d), 131.6 (s), 142.0 (d), 143.0 (d), 147.0 (s), 149.0 (s), 161.0 (s).
Anal. Calcd for C17H10BrN5S: C, 51.53; H, 2.54; N, 17.67. Found: C, 51.30; H, 2.26; N, 17.86.
5-Fluoro-3-[2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (1c)
1
Yellow solid; yield: 75%; mp: 373–374 °C; IR 3247, 3156 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 7.37 (dd, 1H, J = 4.8, 7.9 Hz, H-5′), 7.83 (s, 1H, H-5), 8.27 (d, 1H, J = 2.6 Hz, H-2″),
8.31 (t, 1H, J = 1.7 Hz, H-6″), 8.36 (d, 1H, J = 2.2 Hz, H-2′), 8.41 (dd, 1H, J = 1.3, 4.8 Hz, H-6′),
8.48 (d, 1H, J = 1.7 Hz, H-4″), 8.80 (dd, 1H, J = 1.3, 7.9 Hz, H-4′), 12.19 (bs, 1H, NH), 12.49 (bs, 1H,
13
NH); C NMR (50 MHz, DMSO-d6) δ: 108.1 (d), 110.0 (s), 114.0 (d, JC-4” = 21.2 Hz), 116.9 (s),
117.1 (s), 117.2 (d), 127.7 (d), 128.1 (d), 131.2 (d, JC-6” = 28.6 Hz), 132.7 (d), 140.4 (d), 144.8 (s),
145.5 (s), 147.4 (s), 149.2 (s), 153.0 (s), 159.2 (s, JC-5” = 149 Hz). Anal. Calcd for C17H10FN5S: C,
60.88; H, 3.01; N, 20.88. Found: C, 60.58; H, 2.80; N, 21.04.

Mar. Drugs 2015, 13
477
1-Methyl-3-[4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridine (1d)
Yellow solid; yield: 90%; mp: 278–279 °C; IR 3450 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ:
3.95 (s, 3H, CH3), 7.39 (dd, 1H, J = 4.8, 7.9 Hz, ArH), 7.55 (dd, 1H, J = 5.4, 7.8 Hz, ArH), 7.93
(s, 1H, ArH), 8.33 (s, 1H, ArH), 8.44–8.53 (m, 3H, 3xArH), 8.69 (d, 1H, J = 7.9 Hz, ArH), 9.05
13
(d, 1H, J = 7.8 Hz, ArH), 12.80 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 31.4 (q), 107.7 (s),
109.0 (d), 110.7 (s), 115.8 (s), 116.2 (d), 117.3 (d), 120.0 (s), 126.6 (d), 129.4 (d), 130.7 (d),
134.18 (d), 137.7 (d), 142.4 (s), 143.2 (d), 146.7 (s), 148.1 (s), 151.0 (s). Anal. Calcd for C18H13N5S:
C, 65.24; H, 3.95; N, 21.13. Found: C, 65.06; H, 4.19; N, 21.43.
3-[4-(5-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1e)
Yellow solid; yield: 94%; mp: 311 °C; IR 3347 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ: 3.94
(s, 3H, CH3), 7.36 (dd, 1H, J = 4.7, 7.9 Hz, H-5′), 7.86 (s, 1H, H-5), 8.23 (d, 1H, J = 2.6 Hz, H-2″),
8.39 (d, 1H, J = 2.2 Hz, H-6″), 8.41 (s, 1H, H-2′), 8.44 (dd, 1H, J = 1.6, 4.7 Hz, H-6′), 8.72 (dd, 1H,
13
J = 1.6, 7.9 Hz, H-4′), 8.81 (d, 1H, J = 2.2 Hz, H-4″), 12.27 (bs, 1H, NH); C NMR (50 MHz,
DMSO-d6) δ: 31.6 (q), 108.1 (d), 108.3 (s), 109.4 (s), 111.5 (s), 117.2 (d), 117.6 (s), 118.8 (s),
126.8 (d), 130.2 (d), 130.4 (d), 130.8 (d), 142.6 (d), 142.9 (d), 146.5 (s), 147.0 (s), 149.0 (s), 161.0 (s).
Anal. Calcd for C18H12BrN5S: C, 52.69; H, 2.95; N, 17.07. Found: C, 52.84; H, 3.19; N, 16.93.
3-[4-(5-Fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine (1f)
1
Yellow solid; yield: 90%; mp: 316 °C; IR 3377 (NH) cm−1; H NMR (200 MHz, DMSO-d6) δ:
3.94 (s, 3H, CH3), 7.38 (dd, 1H, J = 4.8, 7.9 Hz, H-5′), 7.83 (s, 1H, H-5), 8.27 (d, 1H, J = 2.7 Hz,
H-2″), 8.32 (t, 1H, J = 1.7 Hz, H-6″), 8.43–8.48 (m, 3H, H-2′, H-4″, H-6′), 8.71 (dd, 1H, J = 1.5,
13
7.9 Hz, H-4′), 12.18 (bs, 1H, NH). C NMR (50 MHz, DMSO-d6) δ: 31.5 (q), 107.6 (d), 108.2 (s),
109.9 (s, JC7″a–f = 4.2 Hz), 114.1 (d, JC4″-F = 21.3 Hz), 117.0 (s, JC3″a–f = 7.27 Hz), 117.3 (d), 117.6 (s),
127.5 (d), 127.6 (d), 130.4 (d, JC6″-F = 27.2 Hz), 130.9 (d), 131.5 (d), 142.7 (d), 145.5 (s), 146.6 (s),
149.2 (s), 155.3 (s, JC5″-F = 239 Hz), 160.9 (s). Anal. Calcd for C18H12FN5S: C, 61.88; H, 3.46; N,
20.04. Found: C, 61.68; H, 3.72; N, 20.23.
3,3′-(1,3-Thiazole-2,4-diyl)bis(1-methyl-1H-pyrrolo[2,3-b]pyridine) (1g)
1
Yellow solid; yield: 60%; mp: 310–311 °C; H NMR (200 MHz, DMSO-d6) δ: 3.94 (s, 3H, CH3),
3.98 (s, 3H, CH3), 7.36–7.45 (m, 2H, 2xArH), 7.82 (s, 1H, ArH), 8.32 (s, 1H, ArH), 8.42 (s, 1H, ArH),
8.45–8.52 (m, 2H, 2xArH), 7.75 (dd, 1H, J = 1.0, 7.9 Hz, ArH), 8.81 (d, 1H, J = 7.8 Hz, ArH);
13C NMR (50 MHz, DMSO-d6) δ: 31.4 (q), 31.7 (q), 108.2 (d), 109.5 (s), 116.1 (d), 117.3 (d),
117.4 (s), 129.9 (d), 130.8 (d), 131.5 (d), 140.0 (d), 140.1 (d), 143.0 (d), 144.9 (s), 145.1 (s), 145.2 (s),
146.9 (s), 148.7 (s), 161.2 (s). Anal. Calcd for C19H15N5S: C, 66.07; H, 4.38; N, 20.27. Found: C,
65.92; H, 4.20; N, 20.40.

Mar. Drugs 2015, 13
478
5-Bromo-1-methyl-3-[2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H pyrrolo
[2,3-b]pyridine (1h)
1
Yellow solid; yield: 90%; mp: 308 °C; H NMR (200 MHz, DMSO-d6) δ: 3.92 (s, 3H, CH3),
3.93 (s, 3H, CH3), 7.36 (dd, 1H, J = 4.7, 7.9 Hz, H-5′), 7.85 (s, 1H, H-5), 8.31 (s, 1H, H-2″), 8.40
(s, 1H, H-2′), 8.42 (dd, 1H, J = 1.6, 4.7 Hz, H-6′), 8.45 (d, 1H, J = 2.2 Hz, H-6″), 8.73 (dd, 1H,
13
J = 1.6, 7.9 Hz, H-4′), 8.79 (d, 1H, J = 2.2 Hz, H-4″); C NMR (50 MHz, DMSO-d6) δ: 31.2 (q),
31.5 (q), 107.9 (d), 108.2 (s), 108.3 (s), 111.7 (s), 117.2 (d), 117.5 (s), 118.7 (s), 130.0 (d), 130.4 (d),
130.5 (d), 130.6 (d), 130.7 (d), 142.9 (d), 146.0 (s), 146.7 (s), 148.6 (s), 161.0 (s). Anal Calcd for
C19H14BrN5S: C, 53.78; H, 3.33; N, 16.50. Found: C, 53.65; H, 3.08; N, 16.73.
5-Fluoro-1-methyl-3-[2-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo
[2,3-b]pyridine (1i)
1
Yellow solid; yield: 60%; mp: 301–302 °C; H NMR (200 MHz, DMSO-d6) δ: 3.92 (s, 3H, CH3),
3.93 (s, 3H, CH3), 7.37 (dd, 1H, J = 4.7, 7.9 Hz, H-5′), 7.81 (s, 1H, H-5), 8.35–8.49 (m, 5H, H-2′,
13
H-2″, H4″, H-6′, H-6″), 8.72 (dd, 1H, J = 1.5, 7.9 Hz, H-4′); C NMR (50 MHz, DMSO-d6) δ:
31.3 (q), 31.4 (q), 107.4 (d), 108.2 (s), 108.7 (s, JC7″a–f = 4.2 Hz), 114.2 (d, JC4″-F = 21.7 Hz), 117.0
(s, JC3″a–f = 7.3 Hz), 117.2 (d), 117.4 (s), 130.0 (d), 130.8 (d), 131.0 (d, JC6″-F = 27.1 Hz), 131.4 (d),
143.0 (d), 144.5 (s), 146.9 (s), 148.9 (s), 153.2 (s), 159.5 (s, JC5″-F = 155.0 Hz). Anal. Calcd for
C19H14FN5S: C, 62.79; H, 3.88; N, 19.27. Found: C, 62.94; H, 4.15; N, 19.38.
1-Methyl-3-[4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (1j)
Yellow solid; yield: 85%; mp: 303–304 °C; IR 3410 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ:
3.96 (s, 3H, CH3), 7.36 (dd, 1H, J = 4.9, 7.9 Hz, ArH), 7.42 (dd, 1H, J = 4.9, 7.9 Hz, ArH), 7.82
(s, 1H, ArH), 8.31 (s, 1H, ArH), 8.39 (s, 1H, ArH), 8.42–8.46 (m, 2H, 2xArH), 8.73 (dd, 1H, J = 1.5,
7.9 Hz, ArH), 8.87 (dd, 1H, J = 1.5, 7.9 Hz, ArH), 12.60 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
δ: 31.44 (q), 108.0 (d), 109.3 (s), 109.8 (s), 116.2 (d), 117.2 (d), 118.2 (s), 118.5 (s), 127.5 (d),
129.6 (d), 130.2 (d), 130.8 (d), 131.5 (s), 140.7 (s), 141.4 (d), 141.6 (s), 142.3 (d), 148.9 (s).
Anal. Calcd for C18H13N5S: C, 65.24; H, 3.95; N, 21.13. Found: C, 65.09; H, 3.69; N, 21.36.
5-Bromo-1-methyl-3-[2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (1k)
Yellow solid; yield: 80%; mp: 301 °C; IR 3174 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ: 3.92
(s, 3H, CH3), 7.35 (dd, 1H, J = 4.8, 7.9 Hz, H-5′), 7.86 (s, 1H, H-5), 8.32 (s, 1H, H-2″), 8.33 (d, 1H,
J = 3.1 Hz, H-2′), 8.41 (dd, 1H, J = 1.4, 4.8 Hz, H-6′), 8.43 (d, 1H, J = 2.2 Hz, H-6″), 8.79 (dd, 1H,
J = 1.4, 7.9 Hz, H-4′), 8.80 (d, 1H, J = 2.2 Hz, H-4″), 12.44 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
δ: 31.2 (q), 108.4 (d), 109.9 (s), 111.7 (s), 117.1 (d), 118.6 (s), 118.7 (s), 127.9 (d),
130.4 (d), 130.7 (d), 132.1 (d), 141.0 (d), 141.1 (s), 142.9 (d), 145.4 (s), 146.0 (s), 148.6 (s), 160.9 (s).
Anal. Calcd for C18H12BrN5S: C, 52.69; H, 2.95; N, 17.07. Found: C, 52.93; H, 3.09; N, 17.23.

Mar. Drugs 2015, 13
479
5-Fluoro-1-methyl-3-[2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine (1l)
Yellow solid; yield: 72%; mp: 295–296 °C; IR 3434 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ:
3.93 (s, 3H, CH3), 7.38 (dd, 1H, J = 4.8, 7.9 Hz, H-5′), 7.83 (s, 1H, H-5), 8.36–8.50 (m, 5H, H-2′,
H-2″, H-4″, H-6′, H-6″), 8.81 (dd, 1H, J = 1.4, 7.9 Hz, H-4′), 12.50 (bs, 1H, NH); 13C NMR (50 MHz,
DMSO-d6) δ: 31.3 (q), 107.8 (d), 108.7 (s, JC3a″-F = 4.1 Hz), 109.8 (s), 114.2 (d, JC6″-F = 21.8 Hz),
116.9 (s), 117.1 (d), 117.2 (d), 118.3 (s), 127.7 (d), 130.8 (d), 131.5 (d, JC4-F″ = 14.1 Hz), 141.5 (d),
144.5 (s), 145.9 (s), 148.9 (s), 153.2 (s), 159.4 (s, JC5′-F′ = 152.0 Hz). Anal. Calcd for C18H12FN5S: C,
61.88; H, 3.46; N, 20.04. Found: C, 62.04; H, 3.70; N, 20.19.
3.1.8. Synthesis of 7-Chloro-1-methyl-1H-pyrrolo[2,3-c]pyridine (22)
To a cold solution of 7-chloro-1H-pyrrolo[2,3-c]pyridine 21 (0.12 g, 0.8 mmol) in anhydrous
toluene (4 mL), t-BuOK (0.12 g, 1.1 mmol) and TDA-1 (1 or 2 drops) were added at 0 °C.
The reaction mixture was stirred at room temperature for 5 h and then MeI (0.8 mmol, 0.05 mL) was
added at 0 °C and then stirred room temperature. TLC analysis (DCM/ethyl acetate 9/1) revealed that
methylation was complete after 2 h. The solvent was evaporated under reduced pressure. The residue
was treated with water, extracted with DCM (3 × 20 mL), dried (Na2SO4), evaporated and purified
by column chromatography using DCM/ethyl acetate (95/5) as eluent. White solid; yield: 70%;
1
mp: 60–61 °C; H NMR (200 MHz, CDCl3) δ: 4.16 (s, 3H, CH3), 6.49 (d, 1H, J = 3.1 Hz, H-3),
13
7.16 (d, 1H, J = 3.1 Hz, H-2), 7.41 (d, 1H, J = 5.4 Hz, H-4), 7.95 (d, 1H, J = 5.4 Hz, H-5); C NMR
(50 MHz, CDCl3) δ: 36.7 (q), 101.0 (d), 115.2 (d), 129.2 (s), 134.1 (s), 134.7 (d), 136.5 (s), 137.5 (d).
Anal. Calcd for: C8H7ClN2: C, 57.67; H, 4.23; N, 16.81. Found: C, 57.90; H, 3.97; N, 16.63.
3.1.9. General Procedure for the Synthesis of 1-(7-Chloro-1H-pyrrolo[2,3-c]pyridin-3-yl)
ethanones (23, 24)
To a solution of appropriate pyrrolo[2,3-c]pyridines 21, 22 (2.5 mmol) in 10 mL of anhydrous
DCM, anhydrous AlCl3 (1.2 g, 8.8 mmol) was slowly added. The reaction mixture was heated
under reflux and BrCOCH2Br (2.5 mmol, 0.2 mL) in 2 mL of anhydrous DCM was added dropwise.
The resulting solution was allowed to stir under reflux for 40 min. After cooling, water and ice were
slowly added and the obtained precipitate (for derivative 23) was filtered off or the oil residue
(for derivative 24) was extracted with DCM (3 × 20 mL) and purified by column chromatography
using DCM/ethyl acetate 9/1 as eluent.
2-Bromo-1-(7-Chloro-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone (23)
1
White solid; yield: 88%; mp: 324–325 °C; IR 3557 (NH), 1676 (CO) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 4.76 (s, 2H, CH2), 8.05 (d, 1H, J = 5.2 Hz, H-4), 8.15 (d, 1H, J = 5.2 Hz, H-5), 8.74
13
(d, 1H, J = 3.0 Hz, H-2), 13.03 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 33.8 (t), 114.2 (s),
115.6 (d), 130.2 (s), 132.7 (s), 134.3 (s), 138.5 (d), 140.6 (d), 186.6 (s). Anal. Calcd for:
C9H6BrClN2O: C, 39.52; H, 2.21; N, 10.24. Found: C, 39.28; H, 2.36; N, 10.13.

Mar. Drugs 2015, 13
480
2-Bromo-1-(7-chloro-1-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone (24)
1
White solid; yield: 90%; mp: 249–250 °C; IR 1647 (CO) cm−1; H NMR (200 MHz, DMSO-d6) δ:
4.18 (s, 3H, CH3), 4.90 (s, 2H, CH2), 8.07 (d, 1H, J = 5.3 Hz, H-4), 8.12 (d, 1H, J = 5.3 Hz, H-5),
13
8.72 (s, 1H, H-2); C NMR (50 MHz, DMSO-d6) δ: 33.3 (t), 37.4 (q), 111.8 (s), 115.7 (d), 129.6 (s),
133.9 (s), 134.3 (s), 140.7 (d), 143.2 (d), 185.9 (s). Anal. Calcd for: C10H8BrClN2O: C, 41.77; H, 2.80;
N, 9.74. Found: C, 41.51; H, 3.07; N, 9.56.
3.1.10. General Procedure for the Synthesis of Indolyl-thiazolyl-pyrrolo[2,3-c]pyridines (2a–j), (3a–f)
A suspension of the appropriate indole-3-carbothioamides 16d, 17a–d, 18a–c (5.0 mmol) and the
proper 3-bromoacetyl-pyrrolo[2,3-c]pyridines 23, 24 (5.0 mmol) in anhydrous EtOH (20 mL) was
heated under reflux for 1–3 h. The precipitate, obtained after cooling, was filtered off, dried and
crystallized from EtOH to afford derivatives 2a–j and 3a–f.
7-Chloro-3-[2-(1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (2a)
Conditions: reflux for 1 h. Yellow solid; yield: 98%; mp: 326–327 °C; IR 3393 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 3.93 (s, 3H, CH3), 7.29–7.35 (m, 2H, H-5′, H-6′), 7.56–7.61
(m, 1H, H-7′), 7.85 (s, 1H, H-2′), 8.12 (d, 1H, J = 5.6 Hz, H-4″), 8.26 (s, 1H, H-5), 8.28–8.31 (m, 1H,
H-4′), 8.33 (d, 1H, J = 5.6 Hz, H-5″), 8.40 (d, 1H, J = 2.7 Hz, H-2″), 12.62 (bs, 1H, NH); 13C NMR (50
MHz, DMSO-d6) δ: 32.9 (q), 108.3 (d), 109.2 (s), 110.7 (d), 112.4 (s), 115.3 (d), 120.2 (d),
121.1 (d), 122.5 (d), 124.5 (s), 129.8 (s), 130.5 (d), 130.8 (d), 131.8 (s), 133.4 (s), 136.7 (d), 137.0 (s),
148.1 (s), 162.2 (s). Anal. Calcd for: C19H13ClN4S: C, 62.55; H, 3.59; N, 15.36. Found: C, 62.28; H,
3.34; N, 15.53.
7-Chloro-3-[2-(5-methoxy-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (2b)
Conditions: reflux for 1 h. Yellow solid; yield: 65%; mp: 216–217 °C; IR 3396 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 3.87 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 6.95 (dd, 1H, J = 2.4,
8.9 Hz, H-6′), 7.49 (d, 1H, J = 8.9 Hz, H-7′), 7.79 (s, 1H, H-2′), 7.90 (d, 1H, J = 2.4 Hz, H-4′), 8.05
(d, 1H, J = 5.5 Hz, H-4″), 8.17 (s, 1H, H-5), 8.32 (d, 1H, J = 2.7 Hz, H-2″), 8.37 (d, 1H, J = 5.5 Hz,
13
H-5″), 12.46 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 33.0 (q), 55.1 (q), 96.3 (s), 101.8 (d),
107.7 (d), 108.6 (s), 110.1 (s), 111.6 (d), 112.6 (d), 115.4 (d), 124.8 (s), 129.9 (d), 131.0 (d), 132.1 (s),
132.8 (s), 134.2 (s), 136.8 (d), 146.6 (s), 155.1 (s), 162.8 (s). Anal. Calcd for: C20H15ClN4OS: C, 60.83;
H, 3.83; N, 14.19. Found: C, 60.62; H, 3.99; N, 13.99.
3-[2-(5-Bromo-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-7-chloro-1H-pyrrolo[2,3-c]pyridine (2c)
Conditions: reflux for 1 h. Yellow solid; yield: 93%; mp: 374–375 °C; IR 3410 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 3.89 (s, 3H, CH3), 7.42 (dd, 1H, J = 1.8, 8.7 Hz, H-6′), 7.57 (d, 1H,
J = 8.7 Hz, H-7′), 7.84 (s, 1H, H-2′), 8.09 (d, 1H, J = 5.6 Hz, H-4″), 8.28 (s, 1H, H-5), 8.30 (d, 1H,
J = 5.6 Hz, H-5″), 8.34 (d, 1H, J = 2.8 Hz, H-2″), 8.49 (d, 1H, J = 1.8 Hz, H-4′), 12.56 (bs, 1H, NH);
13C NMR (50 MHz, DMSO-d6) δ: 33.1 (q), 108.4 (d), 108.9 (s), 112.5 (s), 112.8 (d), 113.8 (s),

Mar. Drugs 2015, 13
481
115.2 (d), 122.7 (d), 124.9 (d), 126.1 (s), 129.8 (s), 130.1 (d), 131.7 (s), 131.9 (d), 133.5 (s), 135.8 (s),
136.7 (d), 148.5 (s), 161.5 (s). Anal. Calcd for: C19H12BrClN4S: C, 51.43; H, 2.73; N, 12.63. Found: C,
51.17; H, 2.62; N, 12.88.
7-Chloro-3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (2d)
Conditions: reflux for 1 h. Yellow solid; yield: 97%; mp: 337–338 °C; IR 3509 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 3.91 (s, 3H, CH3), 7.18 (td, 1H, J = 2.5, 9.2, 11.7 Hz, H-6′), 7.61
(dd, 1H, J = 4.5, 9.2 Hz, H-7′), 7.82 (s, 1H, H-2′), 8.02 (dd, 1H, J = 2.5, 11.7 Hz, H-4′), 8.09 (d, 1H,
J = 5.6 Hz, H-4″), 8.25 (d, 1H, J = 5.6 Hz, H-5″), 8.30 (s, 1H, H-5), 8.34 (d, 1H, J = 2.8 Hz, H-2″),
12.50 (bs, 1H, NH). Anal. Calcd for: C19H12ClFN4S: C, 59.61; H, 3.16; N, 14.63. Found: C, 59.34; H,
3.21; N, 14.47.
7-Chloro-1-methyl-3-[2-(1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (2e)
1
Conditions: reflux for 1 h. Yellow solid; yield: 96%; mp: 265–266 °C; H NMR (200 MHz,
DMSO-d6) δ: 3.91 (s, 3H, CH3), 4.24 (s, 3H, CH3), 7.27–7.36 (m, 2H, H-5′, H-6′), 7.57–7.61 (m, 1H,
H-7′), 7.75 (s, 1H, H-2′), 8.06 (d, 1H, J = 5.5 Hz, H-4″), 8.22 (s, 1H, H-2″), 8.25 (d, 1H, J = 5.5 Hz,
H-5″), 8.33 (s, 1H, H-5), 8.35–8.38 (m, 1H, H-4′). Anal. Calcd for: C20H15ClN4S: C, 63.40; H, 3.99; N,
14.79. Found: C, 63.14; H, 3.75; N, 14.59.
7-Chloro-3-[2-(5-methoxy-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-c]
pyridine (2f)
1
Conditions: reflux for 1 h. Yellow solid; yield: 79%; mp: 231–232 °C; H NMR (200 MHz,
DMSO-d6) δ: 3.87 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 4.22 (s, 3H, CH3), 6.94 (d, 1H, J = 8.8 Hz, H-6′),
7.48 (d, 1H, J = 8.8 Hz, H-7′), 7.69 (s, 1H, H-2′), 7.87–7.91 (m, 1H, H-4′), 8.02 (d, 1H, J = 5.4 Hz,
H-4″), 8.15 (s, 1H, H-2″), 8.28 (s, 1H, H-5), 8.35 (d, 1H, J = 5.4 Hz, H-5″). Anal. Calcd for:
C21H17ClN4OS: C, 61.68; H, 4.19; N, 13.70. Found: C, 61.42; H, 4.08; N, 13.47.
3-[2-(5-Bromo-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-7-chloro-1-methyl-1H-pyrrolo[2,3-c]
pyridine (2g)
1
Conditions: reflux for 2 h. Yellow solid; yield: 98%; mp: 249–250 °C; H NMR (200 MHz,
DMSO-d6) δ: 3.90 (s, 3H, CH3), 4.23 (s, 3H, CH3), 7.44 (dd, 1H, J = 1.9, 8.7 Hz, H-6′), 7.58 (d, 1H,
J = 8.7 Hz, H-7′), 7.74 (s, 1H, H-2′), 8.04 (d, 1H, J = 5.5 Hz, H-4″), 8.24–8.28 (m, 3H, H-2″,H-5,
13
H-5″), 8.48 (d, 1H, J = 1.9 Hz, H-4′); C NMR (50 MHz, DMSO-d6) δ: 33.1 (q), 36.0 (q), 108.4 (d),
109.0 (s), 110.4 (s), 112.8 (d), 113.8 (s), 115.3 (d), 122.8 (d), 124.9 (d), 126.0 (s), 128.8 (s), 131.9 (d),
133.0 (s), 133.3 (s), 135.3 (d), 135.8 (s), 137.0 (d), 148.1 (s), 161.5 (s). Anal. Calcd for:
C20H14BrClN4S: C, 52.47; H, 3.08; N, 12.24. Found: C, 52.23; H, 2.94; N, 12.11.

Mar. Drugs 2015, 13
482
7-Chloro-3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo
[2,3-c]pyridine (2h)
1
Conditions: reflux for 1 h. Yellow solid; yield: 95%; mp: 284–285 °C; H NMR (200 MHz,
DMSO-d6) δ: 3.91 (s, 3H, CH3), 4.24 (s, 3H, CH3), 7.18 (td, 1H, J = 2.5, 9.3, 11.7 Hz, H-6′), 7.61
(dd, 1H, J = 4.4, 9.3 Hz, H-7′), 7.75 (s, 1H, H-2′), 8.02–8.07 (m, 2H, H-4′, H-4″), 8.22 (d, 1H,
J = 5.6 Hz, H-5″), 8.28 (s, 1H, H-2″), 8.35 (s, 1H, H-5). Anal. Calcd for: C20H14ClFN4S: C, 60.53; H,
3.56; N, 14.12. Found: C, 60.29; H, 3.30; N, 14.27.
7-Chloro-3-[2-(5-fluoro-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-c]pyridine (2i)
Conditions: reflux for 3 h. Yellow solid; yield: 82%; mp: 284–285 °C; IR 3399 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 4.25 (s, 3H, CH3), 7.11 (td, 1H, J = 2.5, 9.2, 11.7 Hz, H-6′), 7.53
(dd, 1H, J = 4.6, 9.2 Hz, H-7′), 7.75 (s, 1H, H-2″), 8.02 (dd, 1H, J = 2.5, 11.7 Hz, H-4′), 8.06 (d, 1H,
J = 5.5 Hz, H-4″), 8.22 (d, 1H, J = 5.5 Hz, H-5″), 8.25 (d, 1H, J = 2.9 Hz, H-2′), 8.35 (s, 1H, H-5),
11.93 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 36.6 (q), 105.2 (d, JC4′-F = 24.3 Hz), 108.2 (d),
110.5 (s), 110.7 (d, JC6′-F = 26.0 Hz), 113.4 (d, JC7′-F = 9.6 Hz), 115.1 (s), 115.2 (d), 124.5
(s, JC3a–f = 10.8 Hz), 128.7 (d), 128.9 (s), 132.9 (s), 133.2 (s), 133.4 (s), 135.8 (d), 137.0 (d), 137.5 (s),
148.1 (s), 159.7 (s, JC5′-F = 254 Hz). Anal. Calcd for: C19H12ClFN4S: C, 59.61; H, 3.16; N, 14.63.
Found: C, 59.37; H, 2.90; N, 14.83.
7-Chloro-3-[2-(5-fluoro-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (2j)
Conditions: reflux for 2 h. Yellow solid; yield: 66%; mp: 281–282 °C; IR 3418, 3557 (NH) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 7.12 (td, 1H, J = 2.5, 9.2, 11.7 Hz, H-6′), 7.53 (dd, 1H, J = 4.6,
9.2 Hz, H-7′), 7.83 (s, 1H, H-2′), 8.00 (dd, 1H, J = 2.5, 11.7 Hz, H-4′), 8.08 (d, 1H, J = 5.6 Hz, H-4″),
8.24–8.29 (m, 2H, H-5, H-5″), 8.32 (d, 1H, J = 2.8 Hz, H-2″), 11.95 (bs, 1H, NH), 12.45 (bs, 1H, NH).
Anal. Calcd for: C18H10ClFN4S: C, 58.62; H, 2.73; N, 15.19. Found: C, 58.51; H, 2.47; N, 14.94.
Tert-Butyl 3-[4-(7-Chloro-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole-1-carboxylate (3a)
Conditions: reflux for 2 h. Yellow solid; yield: 84%, mp: 225–226 °C; IR 3417 (NH), 1736 (CO)
1
cm−1; H NMR (200 MHz, DMSO-d6) δ: 1.69 (s, 9H, 3xCH3), 7.44–7.53 (m, 2H, H-5′, H-6′), 8.02
(s, 1H, H-2′), 8.09 (d, 1H, J = 5.5 Hz, H-4″), 8.16–8.21 (m, 1H, H-7′), 8.23 (d, 1H, J = 5.5 Hz, H-5″),
13
8.35–8.36 (m, 2H, H-2″, H-5), 8.47–8.51 (m, 1H, H-4′), 12.48 (bs, 1H, NH); C NMR (50 MHz,
DMSO-d6) δ: 27.6 (3xq), 84.9 (s), 110.4 (d), 112.2 (s), 114.9 (s), 115.0 (d), 121.3 (d), 123.8 (d),
123.9 (d), 125.4 (d), 125.6 (d), 126.6 (s), 129.8 (s), 129.9 (d), 131.4 (s), 133.8 (s), 135.0 (s), 137.5 (d),
148.6 (s), 149.3 (s), 159.9 (s). Anal. Calcd for: C23H19ClN4O2S: C, 61.26; H, 4.25; N, 12.42. Found: C,
61.02; H, 4.06; N, 12.13.

Mar. Drugs 2015, 13
483
Tert-Butyl 3-[4-(7-chloro-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-5-methoxy-1H-indole-1-
carboxylate (3b)
Conditions: reflux for 1 h. Yellow solid; yield: 77%; mp: 312–313 °C; IR 3418 (NH), 1650 (CO)
1
cm−1; H NMR (200 MHz, DMSO-d6) δ: 1.68 (s, 9H, 3xCH3), 3.92 (s, 3H, OCH3), 7.09 (dd, 1H,
J = 2.4, 9.1 Hz, H-6′), 7.99–8.07 (m, 4H, ArH), 8.30–8.35 (m, 3H, ArH), 12.47 (bs, 1H, NH);
13C NMR (50 MHz, DMSO-d6) δ: 27.6 (3xq), 55.1 (q), 84.7 (s), 103.2 (d), 110.2 (d), 112.2 (s),
114.5 (d), 114.8 (s), 115.2 (d), 115.8 (d), 125.9 (d), 127.5 (s), 129.5 (s), 130.0 (d), 131.6 (s), 133.7 (s),
136.9 (d), 137.2 (s), 148.5 (s), 149.2 (s), 156.1 (s), 160.2 (s). Anal. Calcd for: C24H21ClN4O3S: C,
59.93; H, 4.40; N, 11.65. Found: C, 59.66; H, 4.61; N, 11.50.
Tert-Butyl 5-bromo-3-[4-(7-chloro-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole-1-
carboxylate (3c)
Conditions: reflux for 3 h. Yellow solid; yield: 77%, mp: 241–242 °C; IR 3559 (NH),
1
1748 (CO) cm−1; H NMR (200 MHz, DMSO-d6) δ: 1.67 (s, 9H, 3xCH3), 7.60 (d, 1H, J = 8.6 Hz,
H-6′), 7.98 (s, 1H, H-2′), 8.04–8.07 (m, 2H, H-2″, H-7′ ), 8.19 (d, 1H, J = 5.3 Hz, H-4″), 8.29–8.33 (m,
2H, H-4′, H-5″), 8.65 (s, 1H, H-5), 12.45 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ:
27.6 (3xq), 85.3 (s), 110.5 (d), 112.0 (s), 114.1 (s), 114.9 (d), 116.8 (d), 123.8 (d), 126.8 (d), 127.9 (d),
128.4 (s), 129.5 (d), 129.8 (s), 129.9 (s), 131.4 (s), 133.8 (s), 134.0 (s), 137.4 (d), 148.3 (s), 149.3 (s),
159.4 (s). Anal. Calcd for: C23H18BrClN4O2S: C, 52.14; H, 3.42; N, 10.57. Found: C, 52.03; H, 3.55;
N, 10.32.
Tert-Butyl 3-[4-(7-chloro-1-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-1H-indole-1-
carboxylate (3d)
Conditions: reflux for 2 h. Yellow solid; yield: 91%; mp: 279–280 °C; IR 1736 (CO) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 1.69 (s, 9H, 3xCH3), 4.24 (s, 3H, CH3), 7.44–7.54 (m, 2H, H-5′,
H-6′), 7.96 (s, 1H, H-2′), 8.07 (d, 1H, J = 5.5 Hz, H-4″), 8.16–8.20 (m, 1H, H-7′), 8.20 (d, 1H,
J = 5.5 Hz, H-5″), 8.34 (s, 1H, H-2″), 8.37 (s, 1H, H-5), 8.48–8.52 (m, 1H, H-4′); 13C NMR (50 MHz,
DMSO-d6) δ: 27.6 (3xq), 36.6 (q), 84.9 (s), 110.2 (s), 110.4 (d), 114.9 (s), 115.0 (d), 115.1 (d),
121.4 (d), 123.8 (d), 125.5 (d), 125.6 (d), 126.6 (s), 128.9 (s), 132.8 (s), 133.3 (s), 135.0 (s), 135.7 (d),
137.4 (d), 148.6 (s), 149.6 (s), 159.9 (s). Anal. Calcd for: C24H21ClN4O2S: C, 62.00; H, 4.55; N, 12.05.
Found: C, 61.73; H, 4.41; N, 11.94.
Tert-Butyl 3-[4-(7-chloro-1-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-5-methoxy-1H-
indozle-1-carboxylate (3e)
Conditions: reflux for 1 h. Yellow solid; yield: 92%; mp: 212–213 °C; IR 1723 (CO) cm−1;
1H NMR (200 MHz, DMSO-d6) δ: 1.68 (s, 9H, 3xCH3), 3.91 (s, 3H, OCH3), 4.21 (s, 3H, CH3),
7.08 (d, 1H, J = 9.0 Hz, H-6′), 7.89–8.06 (m, 4H, ArH), 8.27–8.29 (m, 3H, ArH). Anal. Calcd for:
C25H23ClN4O3S: C, 60.66; H, 4.68; N, 11.32. Found: C, 60.45; H, 4.89; N, 11.56.

Mar. Drugs 2015, 13
484
Tert-Butyl 5-bromo-3-[4-(7-chloro-1-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)-1,3-thiazol-2-yl]-1H-
indole-1-carboxylate (3f)
1
Conditions: reflux for 3 h. Yellow solid; yield: 91%; mp: 257 °C; IR 1727 (CO) cm−1; H NMR
(200 MHz, DMSO-d6) δ: 1.69 (s, 9H, 3xCH3), 4.22 (s, 3H, CH3), 7.63 (dd, 1H, J = 1.9, 8.9 Hz, H-6′),
7.91 (s, 1H, H-2′), 8.03 (d, 1H, J = 5.5 Hz, H-4″), 8.09 (d, 1H, J = 8.9 Hz, H-7′), 8.18 (d, 1H,
J = 5.5 Hz, H-5″), 8.27 (s, 1H, H-2″), 8.35 (s, 1H, H-5), 8.61 (d, 1H, J = 1.9 Hz, H-4′).
Anal. Calcd for: C24H20BrClN4O2S: C, 53.00; H, 3.71; N, 10.30. Found: C, 53.27; H, 3.97; N, 10.16.
3.1.11. General Procedure for the Synthesis of Indolyl-thiazolyl-pyrrolo[2,3-c]pyridines (4a–f)
To a suspension of appropriate N-tert-butylcarboxylate derivatives 3a–f (0.78 mmol) in DCM
(10 mL) TFA (1.1 mL) was added. The reaction was heated under reflux for 24 h. The mixture
was neutralized with saturated aqueous NaHCO3 solution. The solvent was dried (Na2SO4),
evaporated under reduced pressure and the residue recrystallized from EtOH to afford
indolyl-thiazolyl-pyrrolo[2,3-c]pyridines 4a–f.
7-Chloro-3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (4a)
1
Green solid; yield: 93%; mp: 271–272 °C; IR 3556, 3394 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 7.21–7.31 (m, 2H, H-5′, H-6′), 7.48–7.55 (m, 1H, H-7′), 7.81 (s, 1H, H-2′), 8.08 (d, 1H,
J = 5.5 Hz, H-4″), 8.20 (d, 1H, J = 2.8 Hz, H-2″), 8.27–8.35 (m, 3H, H-5, H-4′, H-5″), 11.82 (bs, 1H,
13
NH), 12.41 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 108.0 (d), 110.4 (s), 112.2 (d), 112.5 (s),
115.3 (d), 120.2 (d), 120.8 (d), 122.4 (d), 124.2 (s), 126.8 (d), 129.4 (d), 129.8 (s), 131.4 (s), 133.9 (s),
136.6 (s), 137.5 (d), 148.6 (s), 162.5 (s). Anal. Calcd for: C18H11ClN4S: C, 61.62; H, 3.16; N, 15.97.
Found: C, 61.47; H, 3.40; N, 15.82.
7-Chloro-3-[2-(5-methoxy-1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[2,3-c]pyridine (4b)
1
Yellow solid; yield: 62%; mp: 223–224 °C; IR 3555, 3379 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 3.90 (s, 3H, OCH3), 6.89 (dd, 1H, J = 2.5, 8.8 Hz, H-6′), 7.42 (d, 1H, J = 8.8 Hz, H-7′),
7.80 (s, 1H, H-5), 7.89 (d, 1H, J = 2.5 Hz, H-4′), 8.07 (d, 1H, J = 5.6 Hz, H-4″), 8.15 (d, 1H,
J = 2.9 Hz, H-2′), 8.35 (d, 1H, J = 2.8 Hz, H-2″), 8.39 (d, 1H, J = 5.6 Hz, H-5″), 11.72 (bs, 1H, NH),
12.51 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 55.1 (q), 101.6 (d), 107.7 (d), 110.1 (s),
112.4 (s), 112.7 (d), 113.0 (d), 115.4 (d), 124.7 (s), 127.2 (d), 129.6 (d), 129.8 (s), 131.5 (s), 131.7 (s),
133.7 (s), 137.0 (d), 148.3 (s), 154.7 (s), 162.9 (s). Anal. Calcd for: C19H13ClN4OS: C, 59.92; H, 3.44;
N, 14.71. Found: C, 59.68; H, 3.33; N, 14.56.
3-[2-(5-Bromo-1H-indol-3-yl)-1,3-thiazol-4-yl]-7-chloro-1H-pyrrolo[2,3-c]pyridine (4c)
1
Yellow solid; yield: 98%; mp: 265–266 °C; IR 3395, 3124 (NH) cm−1; H NMR (200 MHz,
DMSO-d6) δ: 7.37 (dd, 1H, J = 1.9, 8.6 Hz, H-6′), 7.51 (d, 1H, J = 8.6 Hz, H-7′), 7.84 (bs, 1H, H-2″),
8.08 (d, 1H, J = 5.6 Hz, H-4″), 8.24–8.28 (m, 2H, H-5, H-5″), 8.32 (d, 1H, J = 2.9 Hz, H-2′),
8.53 (d, 1H, J = 1.9 Hz, H-4′), 12.02 (bs, 1H, NH), 12.48 (bs, 1H, NH); 13C NMR (50 MHz,

Mar. Drugs 2015, 13
485
DMSO-d6) δ: 108.3 (d), 110.0 (s), 112.5 (s), 113.4 (s), 114.3 (d), 115.2 (d), 112.6 (d), 124.9 (d),
126.0 (s), 128.2 (d), 129.6 (d), 129.8 (s), 131.6 (s), 133.8 (s), 135.3 (s), 137.1 (d), 148.6 (s), 161.9 (s).
Anal. Calcd for: C18H10BrClN4S: C, 50.31; H, 2.35; N, 13.04. Found: C, 50.55; H, 2.09; N, 12.88.
7-Chloro-3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-c]pyridine (4d)
1
Green solid; yield: 75%; mp: 210–211 °C; IR 3397 (NH) cm−1; H NMR (200 MHz, DMSO-d6) δ:
4.25 (s, 3H, CH3), 7.22–7.30 (m, 2H, H-6′, H-5′), 7.48–7.54 (m, 1H, H-7′), 7.75 (s, 1H, H-2″),
8.06 (d, 1H, J = 5.5 Hz, H-4″), 8.18 (d, 1H, J = 2.8 Hz, H-2′), 8.26 (d, 1H, J = 5.5 Hz, H-5″), 8.33
(s, 1H, H-5), 8.36–8.39 (m, 1H, H-4′), 11.82 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 36.6 (q),
108.0 (d), 110.4 (s), 110.6 (s), 112.2 (d), 115.3 (d), 120.3 (d), 120.8 (d), 122.4 (d), 124.2 (s), 126.7 (d),
128.9 (s), 132.9 (s), 133.4 (s), 136.6 (s), 135.2 (d), 137.6 (d), 148.0 (s), 162.5 (s). Anal. Calcd for:
C19H13ClN4S: C, 62.55; H, 3.59; N, 15.36. Found: C, 62.31; H, 3.45; N, 15.21.
7-Chloro-3-[2-(5-methoxy-1H-indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-c]pyridine (4e)
Orange solid; yield: 73%; mp: 184–185 °C; IR 3383 (NH) cm−1; ; 1H NMR (200 MHz, DMSO-d6)
δ: 3.89 (s, 3H, OCH3), 4.22 (s, 3H, CH3), 6.88 (dd, 1H, J = 2.5, 8.8 Hz, H-6′), 7.41 (d, 1H, J = 8.8 Hz,
H-7′), 7.69 (s, 1H, H-2″), 7.87 (d, 1H, J = 2.5 Hz, H-4′), 8.02 (d, 1H, J = 5.5 Hz, H-4″), 8.11 (d, 1H,
13
J = 2.9 Hz, H-2′), 8.28 (s, 1H, H-5), 8.36 (d, 1H, J = 5.5 Hz, H-5″), 11.67 (bs, 1H, NH); C NMR
(50 MHz, DMSO-d6) δ: 36.6 (q), 55.1 (q), 101.7 (d), 107.6 (d), 110.2 (s), 110.5 (s), 112.6 (d),
113.0 (d), 115.5 (d), 124.7 (s), 127.1 (d), 128.9 (s), 131.5 (s), 133.1 (s), 133.4 (s), 134.9 (d), 137.3 (d),
148.0 (s), 154.6 (s), 162.8 (s). Anal. Calcd for: C20H15ClN4OS: C, 60.83; H, 3.83; N, 14.19. Found: C,
60.59; H, 3.72; N, 13.94.
3-[2-(5-Bromo-1H-indol-3-yl)-1,3-thiazol-4-yl]-7-chloro-1-methyl-1H-pyrrolo[2,3-c]pyridine (4f)
Green solid; yield: 99%; mp: 273–274 °C; IR 3378 (NH) cm−1; 1H NMR (200 MHz, DMSO-d6) δ:
4.24 (s, 3H, CH3), 7.37 (dd, 1H, J = 1.9, 8.6 Hz, H-6′), 7.50 (d, 1H, J = 8.6 Hz, H-7′), 7.75 (s, 1H,
H-2″), 8.04 (d, 1H, J = 5.5 Hz, H-4″), 8.24–8.29 (m, 3H, H-2′, H-5, H-5″), 8.50 (d, 1H, J = 1.9 Hz,
13
H-4′), 12.02 (bs, 1H, NH); C NMR (50 MHz, DMSO-d6) δ: 36.6 (q), 108.4 (d), 110.0 (s), 110.5 (s),
113.4 (s), 114.3 (d), 115.3 (d), 117.8 (s), 122.5 (d), 125.0 (d), 125.9 (s), 128.2 (d), 128.9 (s), 133.1 (s),
133.3 (s), 135.2 (d), 137.2 (d), 148.1 (s), 162.0 (s). Anal. Calcd for: C19H12BrClN4S: C, 51.43; H, 2.73;
N, 12.63. Found: C, 51.19; H, 2.60; N, 12.47.
3.2. Biology
All reagents and chemicals were from Sigma Chemical Co (St. Louis, MO, USA), unless indicated.
3.2.1. Viability Assay in Vitro
Nortopsentin analogues, prepared as described above, were dissolved in dimethyl sulfoxide
(DMSO) and then diluted in culture medium to have a DMSO concentration not exceeding 0.1%.
HCT-116 and Caco-2 cell lines were purchased from American Type Culture Collection, Rockville,
MD, USA and DMEM supplemented with 10% fetal, 10% fetal bovine serum (FBS), penicillin

Mar. Drugs 2015, 13
486
(100 U/mL), streptomycin (100 μg/mL) and gentamicin (5 μg/mL). Cells were maintained in log phase
by seeding twice a week at a density of 3 × 108 cells/L in humidified 5% CO2 atmosphere, at 37 °C.
In all experiments, HCT-116 cells were made quiescent through overnight incubation before treatment
with tested compounds or vehicle alone (control cells), while Caco-2 cells were treated 15 days after
confluence, at which time the cells are differentiated in normal intestinal-like cells [53]. 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 nortopsentin derivatives 1k and 4c was determined by the colorimetric
assay based on the reduction of 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2H-tetrazolium
(MTT) to purple formazan by mitochondrial dehydrogenases. Briefly, HCT-116 and Caco-2 lines cells
were seeded at 2 × 104 cells/well in 96-well plates containing 200 μL RPMI. When appropriated,
monolayer cultures were treated for 24 h with various concentrations (5–100 μM) of the drugs.
Then cells were washed with fresh medium and 50 μL FBS-free medium containing 5 mg/mL MTT
added. Cells were incubated 2 h at 37 °C, then medium was discarded by centrifugation, formazan blue
formed in the cells dissolved in DMSO, and absorbance measured at 570 nm in a microplate reader
(Bio-RAD, Hercules, CA, USA). Formazan of control cells was taken as 100% viability. GI50 was
calculated by the curve of percent viability vs. concentration. Each experiment was repeated at least
three times in triplicate.
3.2.2. Cell Cycle Analysis
Cell cycle stage was analyzed by flow cytometry. HCT-116 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 compounds 1k and 4c in RPMI for 24 h. Then cells were harvested by
trypsinization. Aliquots of 1 × 106 cells were 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 of at least 1.0 × 104 cells were subjected to fluorescence-activated cell
sorting (FACS) analysis by Epics XL™ flow cytometer using Expo32 software (Beckman Coulter,
Fullerton, CA, USA).
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 V-FITC negative and PI positive). After 24 h treatment, HCT-116 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 μL 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 FACS analysis using
appropriate 2-bidimensional gating method.

Mar. Drugs 2015, 13
487
3.2.4. Measurement of Mitochondrial Transmembrane Potential
Changes of mitochondrial transmembrane potential (Δψm) were assessed by flow cytofluorometry,
using the cationic lipophilic dye 3,3′-dihexyloxacarbocyanine iodide(3) (DiOC6) (Molecular Probes,
Inc., Life Technologies Italia, Monza, Italy) which accumulates in the mitochondrial matrix. Changes
in mitochondrial membrane potential are indicated by a reduction in the DiOC6-induced fluorescence
intensity. After 24 h treatment, HCT116 cells were incubated with DiOC6 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 analysed in at least 1 × 104 cells for each sample.
3.2.5. Morphology
To analyse cell morphology, cells were fixed with methanol for 10 min and then stained with
Giemsa (10% in PBS) for 15 min followed by washing with distilled water. Cell images were captured
using Zeiss, AxioCam, AxioSkop microscope, (West Germany) with 40× lenses.
3.2.6. Quantification of Acidic Vesicular Organelles (AVO) by Acridine Orange (AO) Staining
AO is a fluorescent molecule used either to identify apoptotic cell death or autophagy. It can
interact with DNA emitting green fluorescence or accumulate in acidic organelles where it is
protonated forming aggregates that emit bright red fluorescence [52]. Briefly, cells were stained with a
AO solution (5 μg/mL) for 15 min. Then they were washed, re-suspended in PBS and subjected to
FACS analysis. The green (510–530 nm, FL-1) and red (650 nm, FL-3) fluorescence of AO with blue
(488 nm) excitation was determined over 10,000 events.
3.2.7. Statistics
Results are given as means and their standard deviations. Three independent observations were
carried out for each experiment, replicated three times. Statistical comparisons were made using a
one-way ANOVA, followed by Bonferroni’s test. p < 0.05 was considered statistically significant.
4. Conclusions
Two new series of nortopsentin analogues were conveniently prepared. In these series the imidazole
ring of the natural product was replaced by a thiazole, and, in one case, both indole units were
substituted by 7-azaindole moieties; in the other series, one indole moiety was replaced by a
6-azaindole system.
The newly synthesized nortopsentin analogues 1k and 4c showed cytotoxicity towards a broad
spectrum of human cancer cell lines included in the NCI panel. Importantly, the compounds did not
appreciably impair vitality of intestinal normal like cells. Investigating mechanisms underlying the
antiproliferative effect in HCT-116 colon cancer cells, we showed that 1k caused a dose-dependent
increase of the apoptotic cell population, activating the mitochondria-mediated pathway and inducing
cell cycle arrest at the G2/M phase. Comparably, previous work showed that other similar analogues
caused apoptosis and arrest of cell cycle with inhibition of cyclin-dependent kinase 1 activity

Mar. Drugs 2015, 13
488
kinase [42–44]. On the other hand 4c at a concentration lower than its GI50, induced antiproliferative
effects with massive accumulation of autophagic vacuoles without apparent signs of apoptosis.
The observed arrest of cell cycle at G1 phase, supports the autophagic fate of the cells [54].
Evolution of the cells toward apoptotic death following treatment with higher drug concentrations,
suggests that 4c may orchestrate a potential axis of autophagy and apoptosis in HCT-116 cells which
can facilitate cellular destruction. The autophagy-signaling cascade induced by 4c in HCT-116
cells is currently under investigation. Modulating authophagy appears of great interest in cancer.
Indeed, current evidence suggests that authophagic cell death can be induced as an alternative to
apoptosis with therapeutic purpose in cancer cells that are resistant to apoptosis. In this context,
4c may candidate as a lead compound for nortopsentin derivatives with autophagic activity.
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 analysed the data. Alessandro Attanzio, Luisa Tesoriere, and Maria Antonia
Livrea, performed biological research and analysed the data. Girolamo Cirrincione, Patrizia Diana,
Paola Barraja, 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.
References
1. Barraja, P.; Spanꢀ, V.; Diana, P.; Carbone, A.; Cirrincione, G.; Vedaldi, D.; Salvador, A.;
Viola, G.; Dall’Acqua, G. Pyrano[2,3-e]isoindol-2-ones, new angelicin heteroanalogue.
Bioorg. Med. Chem. Lett. 2009, 19, 1711–1714.
2. Diana, P.; Martorana, A.; Barraja, P.; Montalbano, A.; Dattolo, G.; Cirrincione, G.; Dall’Acqua, F.;
Salvador, A.; Vedaldi, D.; Basso, G.; et al. Isoindolo[2,1-a]quinoxaline derivatives, novel
potent antitumor agents with dual inhibition of tubulin polymerization and topoisomerase I.
J. Med. Chem. 2008, 51, 2387–2399.
3. Barraja, P.; Spanꢀ, V.; Diana, P.; Carbone, A.; Cirrincione, G. Synthesis of the new ring system
6,8-dihydro-5H-pyrrolo[3,4-h]-quinazoline. Tetrahedron Lett. 2009, 50, 5389–5391.
4. Barraja, P.; Caracausi, L.; Diana, P.; Carbone, A.; Montalbano, A.; Cirrincione, G.; Brun, P.;
Palꢁ, G.; Castagliuolo, I.; Dall’Acqua, F.; et al. Synthesis of pyrrolo[3,2-h]quinolinones with good
photochemotherapeutic activity and no DNA damage. Bioorg. Med. Chem. 2010, 18, 4830–4843.

Mar. Drugs 2015, 13
489
5. Diana, P.; Stagno, A.; Barraja, P.; Montalbano, A.; Carbone, A.; Parrino, B.; Cirrincione, G.
Synthesis of the new ring system pyrrolizino[2,3-b]indol-4(5H)-one. Tetrahedron 2011, 67,
3374–3379.
6. Barraja, P.; Spanò, V.; Giallombardo, D.; Diana, P.; Montalbano, A.; Carbone, A.; Parrino, B.;
Cirrincione G. Synthesis of [1,2]oxazolo[5,4-e]indazoles as antitumour agents. Tetrahedron 2013,
69, 6474–6477.
7. Diana, P.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A.M.; Dattolo, G.; Cirrincione, G.
Pyrrolo[2,1-c][1,2,4,]triazines from 2-diazopyrroles: Synthesis and antiproliferative activity.
Eur. J. Med. Chem. 2002, 37, 267–272.
8. Barraja, P.; Diana, P.; Spanꢀ, V.; Montalbano, A.; Carbone, A.; Parrino, B.; Cirrincione, G.
An efficient synthesis of pyrrolo[3′,2′:4,5]thiopyrano[3,2-b]pyridin-2-one: A new ring system of
pharmaceutical interest. Tetrahedron 2012, 68, 5087–5094.
9. Barraja, P.; Caracausi, L.; Diana, P.; Montalbano, A.; Carbone, A.; Salvador, A.; Brun, P.;
Castagliuolo, I.; Tisi, S.; Dall’Acqua, F.; et al. Pyrrolo[3,2-h]quinazolines as photochemotherapeutic
agents. ChemMedChem 2011, 6, 1238–1248.
10. Diana, P.; Martorana, A.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A.M.; Dattolo, G.;
Cirrincione, G. Isoindolo[2,1-c]benzo[1,2,4]triazines: A new ring system with antiproliferative
activity. Bioorg. Med. Chem. 2007, 15, 343–349.
11. Barraja, P.; Diana, P.; Montalbano, A.; Carbone, A.; Viola, G.; Basso, G.; Salvador, A.;
Vedaldi, D.; Dall’Acqua, F.; Cirrincione, G. Pyrrolo[3,4-h]quinolinones a new class of
photochemotherapeutic agents. Bioorg. Med. Chem. 2011, 19, 2326–2341.
12. Barraja, P.; Caracausi, L.; Diana, P.; Spanꢀ, V.; Montalbano, A.; Carbone, A.; Parrino, B.;
Cirrincione, G. Synthesis and antiproliferative activity of the ring system [1,2]oxazolo
[4,5-g]indole. ChemMedChem 2012, 7, 1901–1904.
13. Shin, J.; Seo, Y.; Cho, K.W.; Rho, J.R.; Sim, C.J. New bis(indole)alkaloids of the topsentin class
from the sponge Spongosorites genitrix. J. Nat. Prod. 1999, 62, 647–649.
14. Casapullo, A.; Bifulco, G.; Bruno, I.; Riccio, R. New bisindole alkaloids of the topsentin and
hamacanthin classes from the Mediterranean marina sponge Rhaphisia lacazei. J. Nat. Prod.
2000, 63, 447–451.
15. Bao, B.; Sun, Q.; Yao, X.; Hong, J.; Lee, C.; Sim, C.J.; Im, K.S.; Jung, J.H. Cytotoxic bisindole
alkaloids from a marine sponge Spongosorites sp. J. Nat. Prod. 2005, 68, 711–715.
16. Gul, W.; Hamann, M.T. Indole alkaloid marine natural products: An established source of cancer
drug leads with considerable promise for the control of parasitic, neurological and other diseases.
Life Sci. 2005, 78, 442–453.
17. Bokesch, H.R.; Pannell, L.K.; McKee, T.C.; Boyd, M.R. Coscinamides A, B, and C, three
new bis indole alkaloids from the marine sponge Coscinoderma sp. Tetrahedron Lett. 2000, 41,
6305–6308.
18. Shimizu, S.; Yamamoto, Y.; Inagaki, L.; Koshimura, S. Antitumor effect and structure-activity
relationship of asterriquinone analogs. Gann 1982, 73, 642–648.
19. Kohmoto, S.; Kashman, Y.; McConnel, O.J.; Rinehart, K.L., Jr.; Wrigh, A.; Koehn, F.
Dragmacidin, a new cytotoxic bis(indole)alkaloid from a deep water marine sponge, dragmacidon
sp. J. Org. Chem. 1988, 53, 3116–3118.