Synthesis and antiproliferative activity of 2,5-bis(3′-Indolyl) pyrroles, analogues of the marine alkaloid nortopsentin

Article abstract of DOI:10.3390/md11030643

2,5-bis(3′-Indolyl)pyrroles, analogues of the marine alkaloid nortopsentin, were conveniently prepared through a three step procedure in good overall yields. Derivatives 1a and 1b exhibited concentration-dependent antitumor activity towards a panel of 42 human tumor cell lines with mean IC₅₀ values of 1.54 μM and 0.67 μM, respectively. Investigating human tumor xenografts in an ex-vivo clonogenic assay revealed selective antitumor activity, whereas sensitive tumor models were scattered among various tumor histotypes.

Full text of DOI:10.3390/md11030643

Mar. Drugs 2013, 11, 643-654; doi:10.3390/md11030643
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Synthesis and Antiproliferative Activity of
2
,5-bis(3′-Indolyl)pyrroles, Analogues of the
Marine Alkaloid Nortopsentin
1
1
1
1
1
Anna Carbone , Barbara Parrino , Paola Barraja , Virginia Spanò , Girolamo Cirrincione ,
1
,
2
2
2
Patrizia Diana *, Armin Maier , Gerhard Kelter and Heinz-Herbert Fiebig
1
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, STEBICEF, via
Archirafi 32, 90123-Palermo, Italy; E-Mails: anna.carbone@unipa.it (A.C.);
barbara.parrino@unipa.it (B.P.); paola.barraja@unipa.it (P.B.); virginia.spano@unipa.it (V.S.);
girolamo.cirrincione@unipa.it (G.C.)
2
Oncotest GmbH, Institute for Experimental Oncology, Am Flughafen 12-14, 79108 Freiburg,
Germany; E-Mails: armin.maier@oncotest.de (A.M.); gerhard.kelter@oncotest.de (G.K.);
heiner.fiebig@oncotest.de (H.-H.F.)
*
Author to whom correspondence should be addressed; E-Mail: patrizia.diana@unipa.it;
Tel.: +39-091-23896815; Fax: +39-091-23860854.
Received: 21 December 2012; in revised form: 22 January 2013 / Accepted: 6 February 2013 /
Published: 1 March 2013
Abstract: 2,5-bis(3′-Indolyl)pyrroles, analogues of the marine alkaloid nortopsentin, were
conveniently prepared through a three step procedure in good overall yields. Derivatives 1a
and 1b exhibited concentration-dependent antitumor activity towards a panel of 42 human
tumor cell lines with mean IC values of 1.54 μM and 0.67 μM, respectively. Investigating
5
0
human tumor xenografts in an ex-vivo clonogenic assay revealed selective antitumor
activity, whereas sensitive tumor models were scattered among various tumor histotypes.
Keywords: bis-indolyl-pyrroles; nortopsentin analogues; marine alkaloids; antitumor;
ex-vivo xenografts
1
. Introduction
Marine organisms constitute a very important source of biologically active natural products including
some of the most potent antineoplastic agents yet discovered [1,2]. In particular, bis-indole alkaloids
Figure 1), characterized by two indole units bound to a spacer through their 3 position, constitute a class
(

Mar. Drugs 2013, 11
644
of deep-sea sponge metabolites with potent biological activity such as anti-inflammatory, antimicrobial,
antiviral and antitumor [3–6]. Bis-indole alkaloids can bear either an acyclic chain or a six membered
carbocyclic or heterocyclic ring or a five membered heterocycle to connect the two indole units.
Coscinamides A–C 2, isolated from deep marine sponge Coscinoderma sp. bearing a linear chain as a
spacer, showed HIV inhibitory activity [7]. Asterriquinone 3, isolated from Aspergillus fungi and
having, as a spacer, a six membered carbocyclic ring showed in vivo activity against Ehrlich carcinoma,
ascites hepatoma AH13 and mouse P388 leukemia [8]. The first isolated four dragmacidins 4, containing
the six membered heterocyclic link piperazine, were isolated from a large number of deep water sponges
including Dragmacidon, Halicortex, Spongosorites, Hexadella and the tunicate Didemnum candidum
and showed, among other biological properties, modest cytotoxic activity [9–11]. Successively, more
complex components of this family such as dragmacidin D 5, having a pyrazinone moiety as a spacer,
exhibited several biological properties such as inhibition of serine-threonine protein phosphatases,
antiviral, antimicrobial and anticancer activities [12,13].
Figure 1. bis-Indolyl alkaloids.
R
R
N
H
N
N
Me
Me
1
a-e
a R=H; b R=Me; c R=OMe, d R=Cl; e R=Br
N
O
HO
N
H
N
O
OH
R
O
O
2
N
H
N
H
R1
3
Asterriquinone
1
Coscinamide A R=Br; R =H
1
Coscinamide B R=R =H
1
Coscinamide C R=Br; R =OH
H2N
N
H
+
H
N
H
N
H
N
R1
Br
Br
R3
N
N
N
R
N
H
O
Br
_
N
N
CF3CO2
R2
H
4
HO
H
5
1
2
3
Dragmacidin
R=H; R =Me; R =Br; R =OH
Dragmacidin D
2
3
1
Dragmacidin A R=R =R =H; R =Me
1
2
3
Dragmacidin B R=R =Me; R =R =H
1
2
3
Dragmacidin C R=R =R =R =H
R
Y
Z
R3
O
N
N
2
X
R1
N
N
N
H
N
H
R1 7-15 R2
R
6
N
H
1
3
Nortopsentin A 7 X=N; Y=CH; Z=NH; R =R =H; R=R =Br
1
2
3
Nortopsentin B 8 X=N; Y=CH; Z=NH; R=R =R =H; R =Br
1
Topsentin A R=R =H
1
2
3
Nortopsentin C 9 X=N; Y=CH; Z=NH; R =R =R H; R=Br
Thiazoles 10 X=N; Y=CH; Z=S; R1 or R =H,Me; R or R =H,OMe,Br
1
Topsentin B1 R=OH, R =H
2
3
1
Topsentin B2 R=Br; R =OH
3
1
Thiophenes 11 X=S; Y=Z=CH; R or R =H,Me,OMe,Cl,Br; R =H,Me;
2
R =H,Me,SO2Ph
3
Pyrazoles 12 X=CH; Y=N; Z=NH,NMe; R or R =H,Me,OMe,Cl,Br;
1
2
R =R =Me
3
1
2
Furans 13 X=O; Y=Z=CH; R or R =H,Me,OMe; R =R =Me
3
Isoxazoles 14 X=CH; Y=N; Z=O; R or R =H,Me,OMe,Cl,Br;
1
2
R =R =Me
,2,4-Thiadiazoles 15 X=Z=N; Y=S; R or R =H,OMe,NO2,Br;
3
1
1
2
R =R =H

Mar. Drugs 2013, 11
645
Topsentins 6 isolated from Mediterranean sponge Topsentia genitrix exhibited antitumor and
antiviral activities [14,15].
Nortopsentins A–C 7–9, bis-indolyl alkaloids having imidazole as a five membered ring spacer,
showed in vitro cytotoxicity against P388 cells (IC 4.5–20.7 μM) and their N-methylated derivatives
50
showed significant improvement in P388 activity compared to that of the parent compounds
IC 0.8–2.1 μM) [16,17].
(
5
0
Due to the small amounts of biologically active substances extracted from natural material, several
total syntheses of Nortopsentins were proposed [18–21].
Moreover, due to their interesting biological activities, marine alkaloids are considered to be
important lead compounds for the discovery of new biologically active compounds. Thus, dragmacidin
analogues, bearing the six membered rings pyridine, pyrimidine, pyrazine and pyrazinone as spacer were
synthesized. These analogues showed strong inhibitory activity against a wide range of human tumor
cell lines [22–25].
Nortopsentin analogues bearing five membered heterocycles which replaced the imidazole ring of the
natural product were synthesized and exhibited remarkable antiproliferative activity, often reaching
IC50 values at sub-micromolar level. Thus bis-indolyl-thiazoles 10 [22,26], thiophenes 11 [27],
pyrazoles 12 [28], furans 13 [29], isoxazoles 14 [29], and 1,2,4-thiadiazoles 15, were reported [30].
Moreover, in other bis-indolyl analogues, which—apart from the heterocyclic spacer—were also
modified, one or both indole units have been described. In particular, 3-indolyl-5-phenylpyridine
showed antiproliferative activity in the range 5–15 μM and inhibited CDK1 at 0.3–0.7 μM level [31];
phenylthiazolyl-7-azaindoles showed antiproliferative activity against a wide range of human tumor cell
lines at micromolar to nanomolar concentrations and inhibited CDK1 with IC values in the range
5
0
0
.41–0.85 μM [32].
In this paper we report the synthesis of substituted 2,5-bis(3′-indolyl)pyrroles of type 1, nortopsentin
analogues in which the imidazole ring spacer of the natural product is replaced by the pyrrole ring.
Furthermore, antitumor activity of the new nortopsentin analogues was investigated by an in vitro
cytotoxicity assay using human tumor cell lines and an ex-vivo clonogenic assay using human
tumor xenograft.
2
. Results and Discussion
A general synthesis of 2,5-bis(3′-indolyl)pyrroles 1a–e is shown in Scheme 1. N-methyl indoles
6a–e were transformed into 1,4-butanediones 17a–e in good yields by a Vilsmeier-Haack reaction with
1
phosphorus oxychloride and tetramethylsuccinamide [29]. 1,4-Diketones 17a–c were purified by flash
chromatography whereas 1,4-diketones 17d,e resulted unstable and were used for the next step
as crude products. All symmetrical 1,4-diketones 17 were converted into the corresponding
2
,5-bis(3′-indolyl)pyrroles 1a–e using ammonium acetate, acetic anhydride in acetic acid under
reflux [33].
By using a monolayer cell survival and proliferation assay the five bis-indolyl-pyrroles 1a–e were
screened for in vitro antitumor activity in a panel of 12 human tumor cell lines. All compounds showed
cytotoxic activity in at least the highest test concentration of 100 μg/mL, exhibiting mean IC values in
5
0

Mar. Drugs 2013, 11
646
the range from 4.4 μg/mL to 0.37 μg/mL (Table 1). Adriamycin tested in parallel was used as cytotoxic
positive control and showed concentration-dependent anti-cancer activity towards all cell lines.
Scheme 1. Synthesis of substituted 2,5-bis(3′-indolyl)pyrroles 1a–e.
O
O
R
R
R
R
N
R
ii
i
H
N
N
N
N
N
16
17
Me
Me
Me
Me
Me
1
a R = H, b R = Me, c R = OMe, d R = Cl, e R = Br
Reagents and conditions: (i) POCl , N,N,N′,N′-tetramethylsuccinamide, then 8 h, 55–60 °C
3
(
derivative 17a) or 20 h, rt (derivatives 17b–e); (ii) NH OAc, (CH CO) O, CH COOH, reflux, 4 h.
4 3 2 3
Table 1. In vitro activity of derivatives 1a–e towards 12 human cell lines.
a
b,c
IC50
μg/mL)
0.37
0.37
3.4
Active/Total
Tumor Selectivity
Compound
(
1 (μg/mL)
10 (μg/mL)
100 (μg/mL)
A
B
+
1a
8/12 (67%) 12/12 (100%) 12/12 (100%)
8/12 (67%) 12/12 (100%) 12/12 (100%)
1/12
1/12
0/12
1712
0/12
2/12
1b
+
1c
0/12 (0%)
1/12 (8%)
0/12 (0%)
4/12 (33%)
10/12 (83%) 12/12 (100%)
−
1d
3.4
9/12 (75%)
9/12 (75%)
10/12 (83%)
12/12 (100%)
12/12 (100%)
11/12 (92%)
+
1
e
4.4
−
d
Adr
0.007
++
a
b
c
d
Responsive (T/C < 30%)/total cell lines.
A = selective (individual IC70 < 1/3 mean IC70)/total cell lines.
B = rating, − (0/10 selective), + (1/10 selective), ++ (2/10 selective), +++ (≥3/10 selective).
Adr = Adriamycin, active/total is given at 0.03, 0.3, and 3 μg/mL.
The most active candidates 1a and 1b were further profiled in monolayer cultures of 42 human tumor
cell lines, reflecting 15 different solid tumor types (Table 2A).
Compounds 1a and 1b effected concentration-dependent inhibition of tumor cell growth with mean
IC values of 1.54 μM and 0.67 μM, indicating pronounced cytotoxic potency.
5
0
Regarding compound 1a, selective activity (as defined by individual IC value of a distinct cell
5
0
line <1/2 mean IC value over the 42 cell lines) was detected in two out of three cell lines of bladder
5
0
cancer (BXF 1218L, BXF 1352L), two out of three melanoma cell lines (MEXF 1341L; MEXF 276L),
as well as in the cell lines LXFL 1121L (lung cancer), PAXF PANC-1 (pancreatic cancer), SXF SAOS-2
(sarcoma) and UXF 1138L (cancer of the uterine body). Particular less sensitive cell lines were found
among colon (HCT-116, HT-29), lung (LXFA 289L), ovarian (OVXF 899L), prostate (DU145) and
renal cancer (RXF 393NL, RXF 486L).
Compound 1b exhibited pronounced activity towards cell lines derived from bladder cancer
(
BXF 1218L), melanoma (MEXF 1341L, MEXF 276L), prostate cancer (PRXF PC3M) and sarcoma
SXF SAOS-2).
(

Mar. Drugs 2013, 11
Table 2. In vitro and ex vivo anti-tumor activity judged by IC values (μM). (A) In vitro
647
5
0
tumor cell lines (monolayer assay); (B) Ex-vivo human xenografts (clonogenic assay).
Cell line
Tumor
histotype
Bladder
histotype
Bladder
name
1218L
1a
1b
name
1a
1b
BXF
BXF
0.72 0.32
0.68 0.41
1.72 0.58
1.39 0.56
3.24 1.64
5.20 2.65
1.50 0.63
0.84 0.53
1.56 0.65
0.81 0.50
1.84 0.61
6.34 2.39
1.57 0.67
2.28 1.36
0.67 0.38
1.85 0.67
2.08 0.89
1.24 0.64
2.44 0.98
BXF 1218
BXF 1228
CXF 1103
CXF 1729
CXF 1783
CXF 280
2.45
2.89
2.35
2.86
1352L
T24
BXF
Colon
4.47 18.36
3.49 7.77
Colon
CXF
269L
CXF
HCT116
HT29
RKO
37.57 40.70
26.18 35.70
CXF
CXF
CXF 975
6.93
6.27
6.03
Gastric
GXA
MKN45
251L
Gastric
GXF 1172
GXF 251
>100
GXF
7.10 25.50
Head&Neck
Liver
HNXF
LIXF
LXFA
LXFA
LXFA
LXFL
LXFL
LXFL
MAXF
MAXF
MAXF
MEXF
MEXF
MEXF
OVXF
OVXF
PAXF
PAXF
PAXF
PRXF
PRXF
PRXF
PRXF
PXF
CAL27
575L
GXF 97
2.72
2.45
2.57
3.74
2.67
4.00
Head&Neck
Lung
HNXF 536
HNXF 908
LXFA 1012
LXFA 1584
LXFA 297
LXFA 526
LXFA 629
LXFA 677
LXFA 923
LXFE 1422
LXFL 1072
LXFL 529
LXFL 625
MAXF 1322
MAXF 1384
MAXF 401
MEXF 1539
MEXF 276
MEXF 462
MEXF 989
OVXF 1353
OVXF 899
PAXF 546
PAXF 736
PRXF DU145
PRXF PC3M
PXF 1752L
PXF 541
Lung
289L
526L
4.76 27.34
2.07 2.73
54.90 >100
629L
1121L
529L
2.96
2.23
3.15
3.63
H460
Mammary
Melanoma
401NL
MCF7
23.19 27.02
19.50 25.68
MDA231 1.18 0.49
5.90
3.44
5.07
1.72
3.42
4.46
1341L
276L
0.52 0.19
0.22 0.11
1.31 0.55
462NL
24.04 38.21
16.80 9.49
34.48 33.28
5.90 11.32
19.90 15.31
Ovarian
OVCAR3 1.46 0.58
Mammary
Melanoma
899L
PANC1
1657L
546L
5.40 2.03
0.74 0.41
2.68 1.02
2.70 1.16
1.46 0.63
4.45 1.96
2.10 0.68
0.85 0.32
2.50 0.88
0.89 0.43
1.86 0.86
1.13 0.58
1.77 0.66
3.14 1.34
3.86 1.60
0.72 0.33
1.60 0.53
Pancreatic
1.44
3.24
1.18
1.75
4.10
0.58
Prostate
22RV1
DU145
LNCAP
PC3M
1118L
1752L
698L
Ovarian
Pancreatic
Prostate
20.96 22.90
3.54 3.93
3.52 14.63
2.99 2.10
28.67 25.98
Plerameso-
thelioma
PXF
PXF
Renal
RXF
1183L
1781L
393NL
486L
2.89
3.71
2.20
6.34
2.90
2.98
2.80
4.05
0.37
3.16
3.90
2.81
RXF
Pleurameso-
thelioma
Renal
RXF
RXF
RXF 1220
RXF 486
Sarcoma
SXF
SAOS2
TE671
SXF
RXF 631

Mar. Drugs 2013, 11
648
Table 2. Cont.
Uterus
UXF
1138L
0.72
0.35
Sarcoma
SXF 1186
SXF 1301
5.71
3.54
6.17
23.95
4.06
SXF 627
3.40
geometric mean IC50
1.54
8/42
0.67
5/42
geometric mean IC50
5.69
7.25
1
)
1)
Tumor selectivity
selective/total)
Tumor selectivity
9/44
14/44
(32%)
(
(19%)
(12%)
(selective/total)
(20%)
1/32 1/16
1/8
1/4
1/2
1
2
4
8
16
32 -fold mean IC50
sensitive cell lines
resistant cell lines
1
)
Number of cell lines/xenografts with IC50 < 1/2 (mean IC )/total.
50
Inhibition of clonogenicity of tumor cells was evaluated in additional tumor models using an ex vivo
clonogenic assay (Table 2B). The anti-proliferative activity of 1a and 1b was evaluated in cell
suspensions prepared from 44 human tumor xenografts of 13 different tumor types, which were cultured
as solid tumors in serial passage on immune deficient nude mice. The results confirmed the
concentration-dependent activity of 1a and 1b on cell lines with mean IC values of 5.69 μM (1a) and
5
0
7
.25 μM (1b), respectively. With regard to 1a, IC50 values ranged from 1.18 μM to 54.9 μM,
corresponding to a 46-fold difference. Selective activity was found against 9 out of the 44 tumors tested,
while these sensitive tumors were scattered among various tumor histotypes, like bladder, gastric, head
and neck, and lung cancer, as well as melanoma and pleuramesothelioma.
Pronounced tumor selectivity was found for compound 1b, with 14 out of 44 tumors (32%) showing
IC values <3.6 μM (=1/2 mean IC value). IC values for 1b ranged from 0.37 μM (PXF 541) to
5
0
50
50
>100 μM (GXF 1172, LXFA 297), equivalent to more than 270-fold difference between resistant and
sensitive tumor models. Sensitive tumors were found among bladder, head and neck, lung, pancreatic,
prostate and renal cancer as well as melanoma and pleuromesothelioma.
Cells that show anchorage independent growth in semi-solid medium contain, to a certain extent,
tumor stem cells which are considered to be responsible for the metastatic and infiltrative potential of a
tumor [34–37]. Thus, the clonogenic assay may inter alia be used to identify candidate tumors for
subsequent in vivo studies [34,35,38–40]. First in vivo efficacy studies, using the patient-derived
melanoma explants MEXF 276 and MEXF 989, did not result in tumor growth inhibition (data not
shown). Further in vivo efficacy studies of candidate tumors as selected by the results of the clonogenic
assay may be warranted.
3
3
3
. Experimental Section
.1. Chemistry
.1.1. General Procedure
All the commercially available reagents and solvents were used without further purification.
1,2-Diaza-1,3-diene (DD) 13 was synthesized as a mixture of E/Z isomers as previously reported [15,16].
Column chromatography was performed with Merck silica gel 230–400 Mesh ASTM or with Büchi
Sepacor chromatography module (prepacked cartridge system). TLC analysis was performed on

Mar. Drugs 2013, 11
649
pre-loaded (0.25 mm) glass supported silica gel plates (Kieselgel 60); compounds were visualized by
exposure to UV light and by dipping the plates in 1% Ce(SO )·4H O, 2.5% (NH ) Mo O ·4H O in
4
2
4 6
7
24
2
1
13
1
0% sulfuric acid followed by heating on a hot plate. H NMR and C NMR spectra were recorded in
DMSO-d solution on 200 (Bruker AC) MHz instrument. Proton and carbon spectra were referenced
6
internally to solvent signals, using values of δ = 2.49 ppm for proton (middle peak) and δ = 39.50 ppm
for carbon (middle peak) in DMSO-d . The following abbreviations are used to describe peak patterns
6
where appropriate: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. All coupling
constants (J) are given in Hz. All melting points were taken on a Büchi-Tottoli capillary apparatus and
are uncorrected; IR spectra were determined in bromoform or nujol with a Jasco FT/IR 5300
spectrophotometer. Mass spectra were recorded in the EI mode (70 eV) on a Shimadzu GC-MS
QP5050A spectrometer. Elemental analyses (C, H, N) were within ±0.4% of the theoretical values.
3
.1.2. Synthesis of N,N,N′,N′-Tetramethylsuccinamide
5
.3 mL (0.05 mol) of succinyl chloride at 0 °C was added dropwise to a solution of dimethylamine
(40% in water, 2 mmol). The mixture was stirred for 30 min and then extracted with DCM, dried and
evaporated to afford the pure N,N,N′,N′-tetramethylsuccinamide. Analytical and spectroscopic data are
reported elsewhere [21].
3
.1.3. General Procedure for the Preparation of 1,4-bis(Indol-3-yl)butane-1,4-diones (17a–e)
Phosphorus oxychloride (5.3 mL, 57 mmol) was slowly added to N,N,N′,N′-tetramethylsuccinamide
(2.58 g, 15 mmol) at 10–20 °C and the mixture was stirred for 24 h. Then N-methylindoles 16a–e
(30 mmol) were slowly added keeping the temperature below 45 °C. After the addition was complete the
mixture was heated for 8 h to 55–60 °C (for derivative 17a) or stirred at rt for 20 h (for derivatives
7b–e). The solution was poured onto crushed ice, made basic with sodium hydroxide 10 M and filtered.
1
The solid was washed with water, dried and purified by chromatography using (DCM/ethyl acetate 9/1)
as eluent to afford the pure derivatives 17a–c; whereas derivatives 17d,e were used for next step without
purification. Analytical and spectroscopic data are reported elsewhere [29].
3
.1.4. General Procedure for the Preparation of Pyrroles (1a–e)
Butanediones 17a–e (2 mmol) was refluxed under N for 4 h with ammonium acetate (3.93 g,
2
5
1 mmol) and acetic anhydride (1.6 mL, 16.9 mmol) in acetic acid (20 mL). The solution was poured
into ice water, and the solid obtained was filtered, dried and purified by chromatography using
dichloromethane as eluent.
3
.1.4.1. 3,3′-(1H-Pyrrole-2,5-diyl)bis(1-methyl-1H-indole) (1a)
−
1 1
Green solid; yield: 65%; mp: 175 °C; IR 3438 (NH) cm ; H NMR (200 MHz, DMSO-d ) δ: 3.82 (s,
6
3
7
1
H), 6.46 (s, 1H), 7.12 (t, 1H, J = 7.2 Hz), 7.21 (t, 1H, J = 7.2 Hz), 7.45 (d, 1H, J = 7.6 Hz), 7.68 (s, 1H),
13
.89 (d, 1H, J = 7.6 Hz), 10.80 (s, 1H); C NMR (50.3 MHz, DMSO-d ) δ: 32.5 (CH ), 105.3 (CH),
6
3
08.7 (C), 109.8 (CH), 119.2 (CH), 119.9 (CH), 121.4 (CH), 125.0 (C), 125.1 (CH), 125.8 (C), 136.8 (C).
Anal. Calcd for C H N : C, 81.20; H, 5.89; N, 12.91. Found: C, 80.94; H, 5.53; N, 13.22.
2
2
19
3

Mar. Drugs 2013, 11
650
3
.1.4.2. 3,3′-(1H-Pyrrole-2,5-diyl)bis(1,5-dimethyl-1H-indole) (1b)
−
1 1
Green solid; yield: 60%; mp: 177–178 °C; IR 3454 (NH) cm ; H NMR (200 MHz, DMSO-d )
6
δ: 2.45 (s, 3H), 3.80 (s, 3H), 6.43 (s, 1H), 7.03 (d, 1H, J = 8.4 Hz), 7.34 (d, 1H, J = 8.4 Hz), 7.61 (s, 1H),
1
3
7
1
.67 (s, 1H), 10.80 (s, 1H); C NMR (50.3 MHz, DMSO-d ) δ: 21.3 (CH ), 32.5 (CH ), 105.2 (CH),
6 3 3
08.2 (C), 109.5 (CH), 119.5 (CH), 123.0 (CH), 125.2 (CH), 125.3 (C), 125.8 (C), 127.7 (C), 135.3 (C).
Anal. Calcd for C H N : C, 81.55; H, 6.56; N, 11.89. Found: C, 81.26; H, 6.28; N, 11.60.
2
4
23
3
3
.1.4.3. 3,3′-(1H-Pyrrole-2,5-diyl)bis(5-methoxy-1-methyl-1H-indole) (1c)
−
1 1
Green solid; yield: 60%; mp: 148–150 °C; IR 3452 (NH) cm ; H NMR (200 MHz, DMSO-d )
6
δ: 3.80 (s, 3H), 3.83 (s, 3H), 6.41 (s, 1H), 6.86 (dd, 1H, J = 2.4, 9.0 Hz), 7.30 (d, 1H, J = 2.4 Hz), 7.37 (d,
1
3
1
1
1
H, J = 9.0 Hz), 7.60 (s, 1H), 10.80 (s, 1H); C NMR (50.3 MHz, DMSO-d ) δ: 32.7 (CH ), 55.4 (CH ),
6 3 3
01.6 (CH), 105.1 (CH), 108.3 (C), 110.6 (CH), 111.4 (CH), 125.3 (C), 125.7 (C), 125.8 (CH), 132.1 (C),
53.7 (C). Anal. Calcd for C H N O : C, 74.78; H, 6.01; N, 10.90. Found: C, 74.99; H, 5.75; N, 11.14.
24
23
3
2
3
.1.4.4. 3,3′-(1H-Pyrrole-2,5-diyl)bis(5-chloro-1-methyl-1H-indole) (1d)
−
1 1
Green solid; yield: 50%; mp: 159–160 °C; IR 3444 (NH) cm ; H NMR (200 MHz, DMSO-d )
6
δ: 3.84 (s, 3H), 6.42 (s, 1H), 7.21 (d, 1H, J = 8.6 Hz), 7.51 (d, 1H, J = 8.6 Hz), 7.72 (s, 1H), 7.83 (s, 1H),
1
3
1
0.90 (s, 1H); C NMR (50.3 MHz, DMSO-d ) δ: 32.7 (CH ), 105.7 (CH), 108.4 (C), 111.5 (CH), 118.9
6 3
(
CH), 121.3 (CH), 124.1 (C), 125.2 (C), 125.9 (C), 127.0 (CH), 135.3 (C). Anal. Calcd for C H Cl N :
22 17 2 3
C, 67.01; H, 4.35; N, 10.66. Found: C, 66.77; H, 4.08; N, 10.37.
3
.1.4.5. 3,3′-(1H-Pyrrole-2,5-diyl)bis(5-bromo-1-methyl-1H-indole) (1e)
−
1 1
Green solid; yield: 50%; mp: 137 °C; IR 3446 (NH) cm ; H NMR (200 MHz, DMSO-d ) δ: 3.84 (s,
6
3
H), 6.41 (s, 1H), 7.32 (dd, 1H, J = 1.9, 8.6 Hz), 7.48 (d, 1H, J = 8.6 Hz), 7.70 (s, 1H), 7.97 (d, 1H,
13
J = 1.9 Hz), 11.00 (s, 1H); C NMR (50.3 MHz, DMSO-d ) δ: 32.7 (CH ), 105.8 (CH), 108.3 (C), 112.0
6
3
(CH), 112.0 (C), 121.9 (CH), 123.9 (CH), 125.2 (C), 126.6 (C), 126.9 (CH), 135.5 (C). Anal. Calcd for
C H Br N : C, 54.68; H, 3.55; N, 8.70. Found: C, 54.48; H, 3.45; N, 8.33.
22
17
2
3
3
.2. Biology
3
.2.1. In Vitro Antitumor Activity towards Permanent Growing Human Tumor Cell Lines
Antitumor activity of the compounds was tested in a monolayer cell survival and proliferation assay
using human tumor cell lines. Studies using panels of human tumor cell lines of different
origin/histotype allow for analysis of potency and tumor selectivity of test compounds and to identify
active compounds that qualify for further preclinical evaluation.
3
.2.1.1. Cell Lines
2
4 out of the 42 cell lines as tested were established at Oncotest from patient-derived human tumor
xenografts passaged subcutaneously in nude mice [41]. The origin of the donor xenografts was

Mar. Drugs 2013, 11
651
described [34,42]. The other cell lines were obtained from ATCC (Rockville, MD, USA), DSMZ
Braunschweig, Germany) were kindly provided by the National Cancer Institute (Bethesda, MA, USA).
(
Cells were cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum and 0.1 mg/mL
gentamicin under standard conditions (37 °C, 5% CO ). Authenticity of all cell lines was proven by STR
2
analysis at the DSMZ.
3
.2.1.2. Cytotoxicity Assay (Monolayer Assay)
A modified propidium iodide assay was used to assess the compounds’ activity toward human tumor
cell lines [29]. Briefly, cells were harvested from exponential phase cultures by trypsinization, counted
and plated in 96-well flat-bottom microtiter plates at a cell density dependent on the cell line
(
4000–20,000 cells/well). After 24 h recovery period to allow the cells to adhere and resume exponential
growth, test compounds were added at 10 concentrations in half-log increments and left for further
days. The inhibition of proliferation was determined by measuring the DNA content using an aqueous
4
propidium iodide solution (7 μg/mL). Fluorescence was measured using the Cytofluor micro-plate
reader (excitation λ = 530 nm, emission λ = 620 nm), providing a direct relationship to the total viable
cell number. In each experiment, all data points were determined in triplicates. Relative IC values were
5
0
®
determined by non-linear regression using the analysis software GraphPad Prism (GraphPad software
Inc., La Jolla, CA, USA).
3
.2.2. Ex-Vivo Antitumor Activity towards Tumor Xenografts
Effects of the test compounds on clonogenicity of tumor cells were investigated in a clonogenic
assay. Tumor xenografts were derived from patient tumors engrafted as a subcutaneously growing tumor
in NMRI nu/nu mice obtained from Oncotest’s breeding facility [35,36]. Details of the test procedure
have been described earlier [38]. Briefly, solid human tumor xenografts were removed from mice under
sterile conditions, mechanically disaggregated and subsequently incubated with an enzyme cocktail
consisting of collagenase type IV (41 U/mL), DNase I (125 U/mL), hyaluronidase type III (100 U/mL)
and dispase II (1.0 U/mL) in RPMI 1640-Medium at 37 °C for 60 min. Cells were passed through sieves
of 200 μm and 50 μm mesh size and washed twice with sterile PBS-buffer. The percentage of viable cells
was determined in a Neubauer-hemocytometer using trypan blue exclusion. The assay contained 3 layers
of equal volume. The bottom layer consisted of 0.2 mL/well Iscove’s Modified Dulbecco’s Medium
(
IMDM, Life Technologies), supplemented with 20% (v/v) fetal calf serum (Sigma), 0.01% (w/v)
4
4
gentamicin (Life Technologies) and 0.75% (w/v) agar (BD Biosciences). 1.5 × 10 to 4 × 10 cells were
added to 0.2 mL of the same culture medium supplemented with 0.4% (w/v) agar and plated in
2
4-multiwell dishes onto the bottom layer. The test compounds were applied by continuous exposure
(drug overlay) in 0.2 mL of culture medium. Every dish included six untreated control wells and
drug-treated groups in triplicate at 6 concentrations. Cultures were incubated at 37 °C and 7.5% CO in a
2
humidified atmosphere for 7–20 days and monitored closely for colony growth using an inverted
microscope. Within this period, in vitro tumor growth led to the formation of colonies with a diameter of
>50 μm. At the time of maximum colony formation, counts were performed with an automatic image
analysis system (Bioreader 5000-Wα, Biosys GmbH). 24 h prior to evaluation, vital colonies were

Mar. Drugs 2013, 11
652
stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium
chloride (1 mg/mL, 100 μL/well). Relative IC values were calculated as described in 3.2.1.2.
5
0
4
. Conclusions
In the present study, the synthesis and characterization of five substituted 2,5-bis(3′-indolyl)pyrroles
of type 1, nortopsentin analogues, in which the imidazole ring spacer of the natural product is replaced
by the pyrrole ring, was described. Among them, 1a and 1b showed antitumor activity in the low
micromolar or even sub-micromolar range towards a panel of human tumor cell lines in vitro.
Furthermore, in the ex-vivo clonogenic assay, pronounced tumor selectivity was detected, in particular
for 1b, whereas sensitive tumors were scattered among various tumor histotypes.
Acknowledgments
This work was financially supported by Ministero dell’Istruzione dell’Università e della Ricerca (MIUR).
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2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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(