Synthesis of the new ring system bispyrido[4',3':4,5]pyrrolo [1,2-a:1',2'-d]pyrazine and its deaza analogue

Article abstract of DOI:10.3390/molecules190913342

Derivatives of the new ring systems bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d] pyrazine-6,13-dione and its deaza analogue pyrido[4",3":4',5']pyrrolo-[1',2':4,5]pyrazino [1,2-a]indole-6,13-dione were conveniently synthesized through a four-step sequence. Symmetrical derivatives of the former ring system were obtained through self condensation. On the other hand, condensation of 6-azaindole carboxylic acid with indole 2-carboxylic acid afforded the deaza analogue ring system. Derivatives of the title ring system were tested by the National Cancer Institute (Bethesda, MD, USA) and four of them exhibited modest activity against MCF7 (a breast cancer cell line) and/or UO-31 (a renal cancer cell line).

Full text of DOI:10.3390/molecules190913342

Molecules 2014, 19, 13342-13357; doi:10.3390/molecules190913342
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of the New Ring System Bispyrido[4',3':4,5]pyrrolo
1,2-a:1',2'-d]pyrazine and Its Deaza Analogue
[
Barbara Parrino, Virginia Spanò, Anna Carbone, Paola Barraja, Patrizia Diana,
Girolamo Cirrincione and Alessandra Montalbano *
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
*
Author to whom correspondence should be addressed; E-Mail: alessandra.montalbano@unipa.it;
Tel.: +39-091-2389-6822; Fax: +39-091-2386-0854.
Received: 24 July 2014; in revised form: 18 August 2014 / Accepted: 20 August 2014 /
Published: 29 August 2014
Abstract: Derivatives of the new ring systems bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]
pyrazine-6,13-dione and its deaza analogue pyrido[4'',3'':4',5']pyrrolo-[1',2':4,5]pyrazino
[1,2-a]indole-6,13-dione were conveniently synthesized through a four-step sequence.
Symmetrical derivatives of the former ring system were obtained through self
condensation. On the other hand, condensation of 6-azaindole carboxylic acid with indole
2
-carboxylic acid afforded the deaza analogue ring system. Derivatives of the title ring
system were tested by the National Cancer Institute (Bethesda, MD, USA) and four of
them exhibited modest activity against MCF7 (a breast cancer cell line) and/or UO-31
(
a renal cancer cell line).
Keywords: diketopiperazines; plinabulin A; bispyrido-pyrrolo-pyrazine; pyrido-pyrrolo-
pyrazino-indole; antiproliferative activity
1
. Introduction
Piperazine-2,5-diones represent a very interesting class of compounds because this heterocyclic
system is found in many unique natural products [1]. In recent years there has been a growing
awareness of the diversity and biological roles played by many diketopiperazines among the over
one-hundred found in Nature. Many derivatives have antiviral (e.g., the gliotoxins and sporidesmins),
phytotoxic (e.g., cyclo(Pro-Tyr)) and antibiotic (e.g., bicyclomycin) properties, whereas other

Molecules 2014, 19
13343
compounds show antineoplastic activity, in particular phenylahistin (1, Figure 1), a fungal metabolite
isolated from culture broths of Aspergillus ustus NFC-F038, which is a result of a condensation
between L-phenylalanine and an isoprenylated dehydrohistidine residue with a quaternary carbon
at C-5 of the imidazole ring [2].
Figure 1. Chemical structures of diketopiperazine derivatives 1‒6.
O
O
1
3
13
1
5
15
18
9
2
9
2
1
4
8
3
14
8
3
16
16
17
NH
N
NH
N
1
0
1
NH
1
1
0
NH
4
4
17
19
HN
HN
11
7
5
19
7
1
1
5
1
2
6
12
18
6
2
2
2
0
20
23
23
O
O
21
21
1
2
24
22
O
10
4
11
5
O
3
9
14
13
R
6
1
2
N
2
X
8
1
N
4
7
N
8
1
9
1
Y
N
5
2
7
R
6
13
10
O
4
3
O
12
11
4
6
,5 X=N Y=N
X=N Y=CH
3
It is a colchicine-like microtubule binding agent endowed with cytotoxic activity against a wide
variety of tumor cell lines [2–4], since it is able to competitively inhibit the binding site of colchicine
to tubuline [3]. Phenylahistin derivatives were synthesized [5] with the aim of finding new antineoplastic
derivatives, but also to understand the structural features necessary for the anti-microtubule activity.
One of the most interesting compounds was revealed to be plinabulin (2, Figure 1) [6] a potent
microtubule-targeting agent; it showed cytotoxic activity (IC50 = 15 nM) against human colon
adenocarcinoma HT-29 cell line and it is currently in phase II clinical trials [7]. SAR studies revealed
that the hydrogen bond between N8-H and N3 is crucial, allowing the formation of a rigid uniplanar
pseudo-three-ring structure necessary for the binding to the microtubules.
Considering also that some properly decorated 6H,13H-pyrazino[1,2-a:4,5-a']diindole-6,13-diones
3
that are indolo-diketopiperazines showed cytotoxic activity in the µM range against L1210 cell
line [8–10] and, in particular, that 2,9-dimethoxy derivative gave complete inhibition of erythrocyte
differentiation, whether spontaneous or induced by haemin, in leukemia K562 cell line at 50 µM, we
decided to further explore the biological potential of these compounds. Considering the experience
acquired in the course of our research on polycyclic nitrogen systems bearing pyrrole [11–13],
indole [14–18], isoindole [19–22] and indazole [23] moieties with antitumor activity, we have decided
to synthetize diaza- and aza-analogues of the ring system 3 bearing two (compounds 4, 5) or one
(
compound 6) nitrogen atoms in the aromatic moiety in order to verify the antineoplastic properties of
this new heterocyclic ring system.

Molecules 2014, 19
13344
Considering that the new compounds have the diketopiperazine core, capable of a colchicine-like
microtubule binding, molecular docking studies were performed in order to investigate the potential
binding ability of compounds 4–6 on tubulin. For this purpose, all compounds were docked in two
different tubulin crystal structures (PDB ID code: 1SA0 [24] and 3HKD [25]) that represent two
potential binding mode for colchicine site ligands.
In the 1SA0 crystal structure, colchicine, a tubulin assembly inhibitor, is the co-crystallized ligand
and its binding site is located at the α,β interface of tubulin subunits [24]. In the crystal structure
3HKD, TN-16, a pyrrolidine-2,4-dione derivative, is the co-crystallized ligand. It inhibits microtubule
assembly by competing with colchicine for tubulin binding [25,26]. The TN-16 binding pocket is
located on the interface between the α and β subunits of the tubulin dimer and slightly extended out of
the β subunit [25,27]. The X-ray crystal structures were prepared using Protein Preparation Wizard.
Docking was carried out using Glide software SP mode default parameters [28].
An evaluation of the docking score results indicated that compounds 4–6 showed the best Glide
docking score values in 3HKD (Glide score values between −9.739 and −8.927), compared to those
obtained in the 1SA0 structure (Glide score values between −6.888 and −4.832) in which they did not
show a good superimposition to colchicine. The only exception was for compound 4d, that was not
docked by Glide in 3HKD (Table 1).
Table 1. Derivatives 4a–d, 5a–e and 6a–d docking scores for 3HKD and 1SA0.
Compound
3HKD
−8.927
1SA0
−6.643
−6.675
−6.007
−6.455
−6.661
−6.700
−4.832
−6.477
−6.122
−6.705
−6.150
−6.380
−6.888
4
a
4
b
−9.562
4
c
−9.354
4d
5a
5b
nd
−9.289
−9.641
5
c
−9.203
5
d
−9.739
5
6
e
−9.653
a
−9.299
6
b
−9.648
6
c
−9.690
6
d
−9.718
nd: Not determined.
Analyzing the binding mode of the planned compounds in 3HKD, they showed H-bond interactions
between the Glu 200 residue and one of the two carbonyl groups, interacting with the binding site in a
way similar to the native ligand TN-16 (Figure 2). Although all compounds showed similar docking
score values (Table 1), unsubstituted compounds 4a and 6a showed lower docking score values than
the corresponding substituted derivatives. Generally the presence of a methoxy group in one of the two
indole or aza-indole moieties seems to stabilize the tubulin-ligand complex through hydrophobic
interactions with the Val 238 residue. On the basis of the docking studies we planned the synthesis of
derivatives 4–6 in order to verify whether they were endowed with interesting biological properties.

Molecules 2014, 19
13345
Figure 2. Wall eyed superimposition of compounds 4a–d, 5a–e and 6a–d with
TN-16 (red).
2
. Results and Discussion
The key intermediates of the synthetic pathway for the pentacyclic new ring systems are 1H-pyrrolo
2,3-c]pyridine-2-carboxylic acids 10a–d (Scheme 1). Commercially available pyridines 7a,b were
reacted with diethyl oxalate using potassium ethoxide as the base to give the corresponding derivatives
a,b in 50 and 45% yield, respectively [29]; pyridines 7c,d were synthetized from the suitable 2-chloro
[
8
derivatives through nucleophilic substitution with sodium methoxide [30,31]. The so-obtained
methoxypyridines were reacted with diethyl oxalate using t-BuOK as the base allowing the isolation of
compounds 8c [30] and 8d in good yields (72%–75%). The latter compound was isolated as the enolic
4
tautomer. Derivatives 8a,b were reduced with iron in saturated aqueous NH Cl and THF to avoid
halogen displacement. On the other hand compounds 8c,d were dissolved in EtOH and hydrogenated
over 10% Pd on charcoal. After an appropriate work-up of the reaction mixture, derivatives 9a–d were
obtained in good yields (60%–85%). Carboxylic acid derivatives 10a–d were obtained in excellent
yields (71%–95%) through alkaline hydrolysis of the corresponding ethyl esters.
Derivatives 10a–d were cyclized at room temperature in anhydrous THF with 4-dimethylaminopyridine
(
DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) as activating agents to give the
new pentacyclic ring systems. Symmetrical derivatives 4a–d were obtained by self-condensation of the
corresponding 6-aza-indole carboxylic acids 10a–d (Table 2).

Molecules 2014, 19
13346
Scheme 1. Synthesis of derivatives 4a–d, 5a–e and 6a–d.
Reagents and conditions: (i) Diethyl oxalate, potassium ethoxide in diethyl ether and EtOH, rt, 15–72 h
8a,b) or t-BuOK, in diethyl ether and ethanol, reflux, 4 h then 24 h rt (8c,d); (ii) Fe, saturated aqueous
NH Cl, THF, EtOH, reflux, 2 h (9a,b) or H /Pd-C, EtOH (9c,d); (iii) NaOH 2M, EtOH, reflux 1–2 h;
(
4
2
(
iv) DMAP, EDCI, THF, rt, 48 h; (v) Indole-2-carboxylic acid, DMAP, EDCI, THF, rt, 48 h.
Table 2. Derivatives 4a–d, 5a–e and 6a–d.
Compound
R
1
R
2
R
3
R
4
Yields(%)
4
a
H
Cl
H
H
H
H
25
30
20
28
40
55
42
44
45
33
65
65
30
4
b
H
Cl
4
c
OCH3
H
H
H
OCH3
4d
5a
5b
OCH3
H
OCH3
H
H
OCH3
H
OCH3
H
H
OCH3
H
5
c
H
H
OCH3
5
d
Cl
H
OCH3
H
5
6
e
Cl
H
H
-
OCH3
a
H
H
-
-
-
-
6
b
Cl
H
-
6
c
OCH3
H
H
-
6
d
OCH3
-
For the synthesis of the asymmetrical compounds 5a–e, the activation of the proper acid 10a–d with
EDCI was followed by the addition of the suitable carboxylic acid and a further addition of EDCI in

Molecules 2014, 19
13347
order to allow the intramolecular cyclization. In particular, 5a–e were obtained from the condensation
of 10a with 10d; 10c with 10d; 10a with 10c; 10b with 10d, and 10b with 10c, respectively (Table 2).
The reaction mixture was particularly difficult to purify because of the presence not only of the
asymmetrical desired derivatives 5a–e, but also of 4%–6% of the symmetrical ones 4a–d as
byproducts of the reaction.
Moreover, through the synthetic pathway previously described it was possible to synthesize the
deaza-analogues 6a–d (Table 2), from the reaction between derivatives 10a–d and commercially
available indole-2-carboxylic acid (Scheme 1). Also in this case, not only the desired compounds 6a–d
were isolated from the reaction mixture, but also the symmetrical ones 4a–d (3%–6%) as byproducts
of the reaction together with 6H,13H-pyrazino[1,2-a:4,5-a']diindole-6,13-dione deriving from the
indole-2-carboxylic acid self-condensation (7%–9%).
All the synthesized derivatives of the new ring system 6H,13H-bispyrido[4',3':4,5]pyrrolo
[1,2-a:1',2'-d]pyrazine-6,13-dione 4a–d, 5a–e and their deaza-analogues 6a–d, were submitted to the
National Cancer Institute (Bethesda, MD, USA) for screening. All derivatives were prescreened
−5
according to the NCI protocol at 10 M dose on the full panel of 60 human cancer cell lines derived
from nine human cancer cell types that have been grouped in disease sub-panels including leukemia,
non-small-cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate and breast
tumour cell lines.[32]
None of the prescreened derivatives were selected for the five dose screening (NCI-60 DTP Human
Tumor Cell Line Screen), since only derivatives 5a and 6a, 6c and 6d showed moderate antineoplastic
activity at micromolar concentrations. In particular derivative 5a exhibited modest activity against the
UO-31 renal cancer sub-panel cell line with a growth inhibitory percentage of 47.0; unsubstituted
deaza analogue 6a and 9-methoxy substituted derivative 6c were shown to be selective against the
MCF7 breast cancer cell line with growth inhibitory percentages of 50.6 and 39.5, respectively. More
interesting results were obtained from the 11-methoxy substituted compound 6d which was shown to
be selective against both the UO-31 renal cancer sub-panel and the MCF7 breast cancer sub-panel cell
lines with growth inhibitory percentages of 46.6 and 50.9, respectively.
3. Experimental Section
3.1. Chemistry
Anhydrous organic solvents were prepared by the appropriate procedures prior to use. The other
organic solvents were reagent grade and used as received. Analytical TLC was performed on Merck
Kieselgel 60-F254 plates. Column chromatography was performed with Merck silica gel 230–400 mesh
ASTM or with a Büchi Sepacor prepacked cartridge system chromatography module.
All melting points were taken on a Buchi-Tottoli capillary apparatus and are uncorrected; IR spectra
1
13
were determined in CHBr , with a Shimadzu FT/IR 8400S spectrophotometer; H- and C-NMR
3
6 3
spectra were measured in DMSO-d or CDCl solutions, at 200 and 50.3 MHz, respectively, using a
Bruker Avance II series 200 MHz spectrometer. Elemental analyses (C, H, N) were within 0.4% of the
theoretical values and were recorded with a VARIO EL III elemental analyzer.

Molecules 2014, 19
13348
3
.1.1. General Procedure for the Preparation of 2-Methoxy-pyridines 7c,d
These compounds were synthesized according to the previously described procedure [30,31].
2
-Methoxy-4-methyl 5-nitropyridine (7c). This compound was obtained in 95% yield. Analytical and
spectroscopic data are in accordance to those reported in literature [30].
2
-Methoxy-4-methyl 3-nitropyridine (7d). This compound was obtained in 80% yield. Analytical and
spectroscopic data are in accordance to those reported in literature [31].
3
.1.2. General Procedure for the Preparation of Ethyl-3-(nitropyridin-4-yl)-2-oxopropanoates 8a,b
These compounds were synthesized according to the previously described procedure [29].
Ethyl-3-(3-nitropyridin-4-yl)-2-oxopropanoate (8a). This compound was obtained in 50% yield.
Analytical and spectroscopic data are in accordance to those reported in literature [29].
Ethyl-3-(2-chloro-5-nitropyridin-4-yl)-2-oxopropanoate (8b). This compound was obtained in 45%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [29].
3
.1.3. General Procedure for the Preparation of Ethyl-3-(nitropyridin-4-yl)-2-oxopropanoates 8c,d
To a stirred solution of t-BuOK (2.4 mmol) in anhydrous EtOH (1 mL) and diethyl ether (10 mL)
diethyl oxalate (2.4 mmol, 0.3 mL) was added under a nitrogen atmosphere. The reaction mixture was
kept at room temperature for 15 min, then a solution of the suitable pyridine 7c,d (2.4 mmol) was
added and the reaction mixture was refluxed for 4 h and stirred at room temperature 24 h. The orange
residue thus obtained was shaken in diethyl ether, filtered off and air dried. Water (9.2 mL) was added
and acetic acid was added until pH 4.0. The desired product was filtered off, and dried in the desiccator
to afford the desired products as cream solids.
Ethyl 3-(2-methoxy-5-nitropyridin-4-yl)-2-oxopropanoate (8c). This compound was obtained in 72%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [30,33].
Ethyl 3-(2-methoxy-3-nitropyridin-4-yl)-2-oxopropanoate (8d). Title compound 8d was isolated as the
1
enolic tautomer. Rf = 0.33 (CH
2
Cl
2
); mp 78.4–79.6 °C; yield 75%; IR: 3426 (OH), 1706 (CO) cm ;
1
H-NMR (DMSO-d
6 3 3
) δ: 1.28 (3H, t, J = 6.0 Hz, CH ), 3.97 (3H, s, OCH ), 4.28 (2H, q, J = 6.0 Hz,
CH
2
), 6.00 (1H, s, CH), 7.86 (1H, d, J = 6.00 Hz, H-5), 8.34 (1H, d, J = 6.0 Hz, H-6), 11.30 (1H, bs,
13
OH). C-NMR (DMSO-d
48.8 (s), 154.3 (s), 163.0 (s). Anal. Calcd for C11
Found: C, 49.21; H, 4.75; N, 10.16.
6
) δ: 13.9 (q), 54.4 (q), 62.2 (t), 97.2 (d), 116.4 (d), 132.7 (s), 136.3 (s), 148.3 (d),
1
12 2 6
H N O
(268.22): C, 49.26; H, 4.51; N, 10.44.
3
.1.4. General Procedure for the Preparation of Ethyl 1H-pyrrolo[2,3-c]pyridine-2-carboxylates 9a,b
These compounds were synthesized according to the previously described procedure [29,34].

Molecules 2014, 19
13349
Ethyl 1H-pyrrolo[2,3-c]pyridine-2-carboxylate (9a). This compound was obtained in 60% yield.
Analytical and spectroscopic data are in accordance to those reported in literature [29].
Ethyl 5-chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (9b). This compound was obtained in 60%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [34].
3
.1.5. General Procedure for the Preparation of Ethyl 1H-pyrrolo[2,3-c]pyridine-2-carboxylates 9c,d
Derivatives 8c,d (2.9 mmol) were dissolved in EtOH (40 mL) and hydrogenated over 10% Pd on
charcoal. The catalyst was removed by filtration under argon and the solvent was evaporated in vacuo.
Ethyl 5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (9c). This compound was obtained in 85%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [33].
Ethyl 7-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (9d). Title compound 9d was purified by
flash-chromatography using CH Cl /ethyl acetate 96:4. Rf = 0.63 (CH Cl /ethyl acetate 95:5) as a
2
2
2
2
1
1
white powder; mp 134.1–135.0 °C; yield 75%; IR: 3435 (NH), 1708 (CO) cm ; H-NMR (CDCl ) δ: 1.41
3
(
3H, t, J = 6.0 Hz, CH
H-4), 7.77 (1H, d, J = 6.0 Hz, H-5), 9.61 (1H, bs, NH). C-NMR (CDCl
07.7 (d), 110.8 (d), 122.3 (s), 129.3 (s), 133.0 (s), 135.9 (d), 151.9 (s), 161.4 (s). Anal. Calcd for
(220.22): C, 59.99; H, 5.49; N, 12.72. Found: C, 60.14; H, 5.66; N, 12.57.
3
), 4.09 (3H, s, OCH
3
), 4.43 (2H, q, J = 6.0 Hz, CH
2
), 7.13–7.17 (2H, m, H-3,
13
3
) δ: 14.3 (q), 53.3 (q), 61.4 (t),
1
11 12 2 3
C H N O
3
.1.6. General Procedure for the Preparation of 1H-pyrrolo[2,3-c]pyridine-2-carboxylic Acids 10a–d
To a stirred solution of 9a–d (1.3 mmol) in EtOH (12 mL) 2M NaOH was added (1.7 mmol, 1.1 mL).
The reaction mixture was heated under reflux for 1h (10a) or 2h (10b) and the solvent was evaporated.
Water (10 mL) was added and acetic acid was added until pH 4.0. The desired product was filtered off,
dried into the desiccators to afford the desired product.
1H-Pyrrolo[2,3-c]pyridine-2-carboxylic acid (10a). This compound was obtained in 95% yield.
Analytical and spectroscopic data are in accordance to those reported in literature [29].
5-Chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (10b). This compound was obtained in 71%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [29].
5
-Methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (10c). This compound was obtained in 80%
yield. Analytical and spectroscopic data are in accordance to those reported in literature [35].
7
-Methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (10d). This compound was obtained after 1 h
reflux as a white powder. Rf = 0.40 (CH Cl /MeOH 9:1); mp 269.3–271.1 °C; yield 82%; IR: 3550
NH), 3311 (OH), 1718 (CO) cm ; H-NMR (DMSO-d ) δ: 4.02 (3H, s, OCH ), 7.07 (1H, s, H-3),
.21 (1H, d, J = 6.0 Hz , H-4), 7.68 (1H, d, J = 6.0 Hz, H-5), 9.61(1H, bs, NH), 12.30 (1H, bs, OH).
2
2
−1 1
(
6
3
7
13
6
C-NMR (DMSO-d ) δ: 52.7 (q), 106.8 (d), 110.5 (d), 122.0 (s), 131.5 (s), 132.6 (s), 134.8 (d), 151.6 (s),
1
62.3 (s). Anal. Calcd for C H N O
9 8 2 3
(192.17): C, 56.25; H, 4.20; N, 14.58. Found: C, 56.29; H, 4.24;
N, 14.37.

Molecules 2014, 19
13350
3
.1.7. General Procedure for the Preparation of 6H,13H-Bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]
pyrazine-6,13-diones 4a–d
To a stirred solution of 10a–d (2.3 mmol) in anhydrous THF (20 mL) dimethylaminopyridine
(
DMAP, 2.3 mmol) was added, followed by EDCI (4.8 mmol) addition after 10 min; the reaction
mixture was stirred for 48h at room temperature. The solid was collected by filtration and
recrystallizated from CH Cl and MeOH, affording the desired products as yellow solids. Compounds
2
2
1
4
a–d were characterized only by H-NMR spectroscopy. The poor solubility of the title compounds
1
3
prevented the C-NMR spectra from being recorded.
6
H,13H-Bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (4a). Rf = 0.34 (CH
2
Cl
2
/MeOH
1
1
9
5:5); mp 352.3–353.9 °C; yield 25%; IR: 1722 (CO) cm ; H-NMR (DMSO-d ) δ: 7.91 (2H, d,
6
J = 6.0 Hz, H-4 and H-11), 7.92 (2H, s, H-5 and H-12), 8.60 (2H, d, J = 6.0 Hz, H-3 and H-10), 9.71
2H, s, H-1 and H-8). Anal. Calcd for C16 (288.26): C, 66.67; H, 2.80; N, 19.44. Found: C,
(
8 4 2
H N O
6
6.62; H, 2.84; N, 19.39.
3
,10-Dichloro-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (4b). Rf = 0.60
1
1
(CH Cl /MeOH 98:2); mp 380.6–381.9 °C; yield 30%; IR: 1716 (CO) cm ; H-NMR (DMSO-d ) δ: 7.88
2 2 6
(
2H, s, H-4 and H-11), 8.05 (2H, s, H-5 and H-12), 9.47 (2H, s, H-1 and H-8). Anal. Calcd for
Cl (357.15): C, 53.81; H, 1.69; N, 15.69. Found: C, 53.89; H, 1.78; N, 15.97.
C
16
H
6
2 4 2
N O
3
,10-Dimethoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (4c). Rf = 0.57
−1 1
(CH
2
Cl
2
/MeOH 98:2); mp 343.0–344.2 °C; yield 20%; IR: 1710 (CO) cm ; H-NMR (DMSO-d
), 7.26 (2H, s, H-4 and H-11), 7.73 (2H, s, H-5 and H-12), 9.27 (2H, s, H-1 and H-8).
(348.31): C, 62.07; H, 3.47; N, 16.09. Found: C, 61.92; H, 3.53; N, 15.95.
6
) δ:
3.95 (3H, s, OCH
3
12 4 4
Anal. Calcd for C18H N O
1
,8-Dimethoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (4d). Rf = 0.45
−1 1
(CH
2 2 6
Cl /MeOH 98:2); mp 380.6–381.9 °C; yield 28%; IR: 1723 (CO) cm ; H-NMR (DMSO-d ) δ:
4
.06 (3H, s, OCH ), 7.44 (2H, d, J = 6.0 Hz, H-4 and H-11), 7.79 (2H, s, H-5 and H-12), 8.12 (2H, d,
3
12 4 4
J = 6.0 Hz, H-3 and H-10). Anal. Calcd for C18H N O (348.31): C, 62.07; H, 3.47; N, 16.09. Found:
C, 61.83; H, 3.66; N, 16.05.
3
.1.8. General Procedure for the Preparation of 6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]
pyrazine-6,13-diones 5a–e
To a stirred solution of 10a–d (2.3 mmol) in anhydrous THF (20 mL) dimethylaminopyridine
(DMAP, 2.3 mmol) was added, followed by EDCI (1.2 mmol) after 10 min; the reaction mixture was
stirred at room temperature for 1h. The suitable acid 10a–d (1.0 mmol) and EDCI (1.2 mmol) were
added and the reaction mixture was stirred for 48 h. The solid was collected by filtration, purified by
flash chromatography using CH
2 2 2 2
Cl /MeOH 98:2 and recrystallized from CH Cl and MeOH, affording
1
the desired product as a yellow solid. Compounds 5a–e were characterized only by H-NMR spectroscopy.
1
3
The poor solubility of the title compounds prevented C-NMR spectra from being recorded.

Molecules 2014, 19
13351
8-Methoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (5a). This product
was obtained by reaction of 10a with 10d. Rf = 0.46 (CH Cl /MeOH 98:2); mp 328.4–329.0 °C; yield
2
2
1
1
4
6 3
0%; IR: 1712 (CO), 1694 (CO) cm ; H-NMR (DMSO-d ) δ: 4.06 (3H, s, OCH ), 7.45 (1H, d,
J = 6.0 Hz, H-11), 7.82 (1H, s, H-12), 7.89–7.92 (2H, m, H-5 and H-4), 8.14 (1H, d, J = 6.0 Hz,
H-10), 8.60 (1H, d, J = 4.0 Hz, H-3), 9.67 (1H, s H-1). Anal. Calcd for C H N O (318.29): C, 64.15;
17
10
4
3
H, 3.17; N, 17.60. Found: C, 63.87; H, 3.13; N, 17.75. From this reaction derivatives 4a (yield 4%) and
4
d (yield 6%) were also isolated.
1,10-Dimethoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (5b). This product
was obtained by reaction of 10c with 10d. Rf = 0.34 (CH Cl /MeOH 95:5); mp 309.1–309.4 °C; yield
2
2
−1
1
5
5%; IR: 1712 (CO), 1689 (CO) cm ; H-NMR (DMSO-d ) δ: 3.94 (3H, s, OCH ), 4.05 (3H, s,
6 3
3
OCH ), 7.25 (1H, s, H-12), 7.42 (1H, d, J = 4.0 Hz, H-4), 7.79 (1H, s, H-11), 7.84 (1H, s, H-5), 8.12
(
1H, d, J = 4.0 Hz, H-3), 9.24 (1H, s, H-8). Anal. Calcd for C H N O (348.31): C, 62.07; H, 3.47;
18 12 4 4
N, 16.09. Found: C, 62.20; H, 3.42; N, 16.25. From this reaction derivatives 4c (yield 5%) and 4d
yield 6%) were also isolated.
(
3-Methoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione (5c). This product
was obtained by reaction of 10a with 10c. Rf = 0.37 (CH Cl /MeOH 98:2); mp 271.1–271.8 °C; yield
2
2
−
1 1
4
7
9
2%; IR: 1718 (CO), 1707 (CO) cm ; H-NMR (DMSO-d
6
3
) δ: 3.95 (3H, s, OCH ), 7.26 (1H, s, H-12),
.78 (1H, s, H-5), 7.87 (1H, s, H-4), 7.90 (1H, d, J = 6.0 Hz, H-11), 7.59 (1H, d, J = 6.0 Hz, H-10),
.28 (1H, s, H-8), 9.68 (1H, s, H-1). Anal. Calcd for C17 (318.29): C, 64.15; H, 3.17; N, 17.60.
10 4 3
H N O
Found: C, 64.06; H, 3.08; N, 17.89. From this reaction derivatives 4a (yield 4%) and 4c (yield 5%)
were also isolated.
1
0-Chloro-1-methoxy-6H,13H-bispyrido[4',3':4,5]pyrrolo-[1,2-a:1',2'-d]pyrazine-6,13-dione
This product was obtained by reaction of 10b with 10d. Rf = 0.47 (CH Cl /MeOH 98:2); mp
92.2–293.0 °C; yield 44%; IR: 1712 (CO), 1690 (CO) cm ; H-NMR (DMSO-d ) δ: 4.06 (3H, s,
(5d).
2
2
−1 1
2
6
OCH
1H, d, J = 6.0 Hz, H-3), 9.44 (1H, s, H-8). Anal. Calcd for C17
N, 15.88. Found: C, 57.60; H, 2.48; N, 15.96. From this reaction derivatives 4b (yield 6%) and 4d
yield 5%) were also isolated.
3
), 7.45 (1H, d J = 6.0 Hz, H-4), 7.75 (1H, s, H-5), 7.92 (1H, s, H-12), 8.05 (1H, s, H-11), 8.14
(
H
9
ClN (352.73): C, 57.89; H, 2.57;
4 3
O
(
3-Chloro-10-methoxy-6H,13H-bispyrido[4',3':4,5]-pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione
(5e).
This product was obtained by reaction of 10b with 10c. Rf = 0.56 (CH Cl /MeOH 98:2); mp
2
2
−1 1
3
12.0–312.5 °C; yield 45%; IR: 1720 (CO), 1705 (CO) cm ; H-NMR (DMSO-d
OCH ), 7.26 (1H, s, H-11), 7.80 (1H, s, H-12), 7.82 (1H, s, H-5), 8.04 (1H, s, H-4), 9.27 (1H, s, H-8),
.46 (1H, s, H-1). Anal. Calcd for C17 ClN (352.73): C, 57.89; H, 2.57; N, 15.88. Found: C, 57.80;
H, 2.49; N, 16.16. From this reaction derivatives 4b (yield 5%) and 4c (yield 5%) were also isolated.
6
) δ: 3.96 (3H, s,
3
9
H
9
4 3
O

Molecules 2014, 19
13352
3.1.9. General Procedure for the Preparation of 6H,13H-Pyrido[4'',3'':4',5']pyrrolo[1',2':4,5]pyrazino
[1,2-a]indole-6,13-diones 6a–d
To a stirred solution of the suitable 10a–d (1.2 mmol) in anhydrous THF (20 mL)
dimethylaminopyridine (DMAP) (1.2 mmol) was added, followed by EDCI (1.2 mmol) after 10 min;
the reaction mixture was stirred at room temperature for 1h. Indole 2-carboxylic acid (1.0 mmol) and
EDCI (1.2 mmol) were added and the reaction mixture was stirred for 48 h. The solid was collected by
filtration, purified by flash chromatography using using CH Cl /MeOH 98:2 and recrystallized with
2
2
CH
2
Cl
2
and MeOH, affording the desired products as yellow solid. Compounds 6a–d were
1
characterized only by H-NMR spectroscopy. The poor solubility of the title compounds prevented
1
3
C-NMR spectra from being recorded.
6
H,13H-Pyrido[4'',3'':4',5']pyrrolo[1',2':4,5]pyrazino[1,2-a]indole-6,13-dione (6a). Rf = 0.28
−1 1
(
CH
δ: 7.48 (1H, td, J = 6.0 2.0 Hz, H-9), 7.67 (1H, td, J = 6.0 2.0 Hz, H-10), 7.85 (1H, s, H-12), 7.88–7.93
3H, m, H-4, H-5 and H-8), 8.48 (1H, d, J = 6.0 Hz, H-11), 8.58 (1H, d, J = 6.0 Hz, H-3), 9.71 (1H, s,
H-1). Anal. Calcd for C H N O (287.27): C, 71.08; H, 3.16; N, 14.63. Found: C, 71.29; H, 3.29; N,
2 2 6
Cl /MeOH 98:2); mp 347.4–347.8 °C; yield 33%; IR: 1701 (broad, CO) cm ; H-NMR (DMSO-d )
(
17
9
3
2
1
4.84. From this reaction derivatives 4a (yield 5%) and 6H,13H-pyrazino[1,2-a:4,5-a']diindole-6,13-dione
yield 8%) whose analytical and spectroscopic data are in accordance to those reported in literature
were also isolated [36].
(
3
-Chloro-6H,13H-pyrido[4'',3'':4',5']pyrrolo[1',2':4,5]pyrazino[1,2-a]indole-6,13-dione (6b). Rf = 0.63
−1
1
(CH
(DMSO-d
(1H, d, J = 8.0 Hz, H-8), 7.95 (1H, s, H-5), 8.02 (1H, s, H-4), 8.48 (1H, d, J = 8.0 Hz, H-11),
2
Cl
2
/MeOH 98:2); mp 306.3–306.7 °C; yield 65%; IR: 1700 (broad, CO) cm ; H-NMR
6
) δ: 7.49 (1H, t, J = 8.0 Hz, H-9), 7.67 (1H, t, J = 8.0 Hz, H-10), 7.77 (1H, s, H-12), 7.92
9
6
6
8 3 2
.48 (1H, s, H-1). Anal. Calcd for C17H ClN O (321.72): C, 63.47; H, 2.51; N, 13.06. Found: C,
3.68; H, 2.46; N, 13.30. From this reaction were also isolated derivatives 4b (yield 3%) and
H,13H-pyrazino[1,2-a:4,5-a']diindole-6,13-dione (yield 7%) whose analytical and spectroscopic data
are in accordance to those reported in literature [36].
3
-Methoxy-6H,13H-pyrido[4'',3'':4',5']pyrrolo[1',2':4,5]pyrazino[1,2-a]indole-6,13-dione (6c). Rf = 0.65
−1
1
(CH
2
Cl
2
/MeOH 98:2); mp 279.0–279.4 °C; yield 65%; IR: 1727 (CO), 1702 (CO) cm ; H-NMR
) δ: 3.95 (3H, s, OCH ), 7.24 (1H, s, H-4), 7.47 (1H, t, J = 8.0 Hz, H-9), 7.65 (1H, t, J = 8.0 Hz,
(DMSO-d
6
3
H-10), 7.71 (1H, s, H-12), 7.87 (1H, s, H-5), 7.91 (1H, d, J = 8.0 Hz, H-8), 8.46 (1H, d, J = 8.0 Hz, H-11),
9
6
6
11 3 3
.29 (1H, s, H-1). Anal. Calcd for C18H N O (317.30): C, 68.14; H, 3.49; N, 13.24. Found: C,
8.09; H, 3.70; N, 13.13. From this reaction were also isolated derivatives 4c (yield 4%) and
H,13H-pyrazino[1,2-a:4,5-a']diindole-6,13-dione (yield 7%) whose analytical and spectroscopic data
are in accordance to those reported in literature [36].
1
-Methoxy-6H,13H-pyrido[4'',3'':4',5']pyrrolo[1',2':4,5]pyrazino[1,2-a]indole-6,13-dione (6d). Rf = 0.63
−1
1
(CH
2 2
Cl /MeOH 98:2); mp 283.8–283.9 °C; yield 30%; IR: 1712 (CO), 1690 (CO) cm ; H-NMR
(
DMSO-d ) δ: 4.05 (3H, s, OCH ), 7.41–7.50 (2H, m, H-4 and H-9), 7.64 (1H, t, J = 8.0 Hz, H-10), 7.81
6
3
(2H, s, H-5 and H-12), 7.90 (1H, d, J = 8.0 Hz, H-8), 8.10 (1H, d, J = 6.0 Hz, H-3), 8.43 (1H, d, J = 8.0 Hz,

Molecules 2014, 19
13353
H-11). Anal. Calcd for C H N O (317.30): C, 68.14; H, 3.49; N, 13.24. Found: C, 68.39; H, 3.45; N,
1
8
11
3
3
12.95. From this reaction were also isolated derivatives 4d (yield 6%) and 6H,13H-pyrazino[1,2-a:4,5-a']
diindole-6,13-dione (yield 9%) whose analytical and spectroscopic data are in accordance to those
reported in literature [36].
3
.2. Docking
Docking studies were performed for all designed compounds by Glide 5.9 (Schrödinger Inc.,
New York, NY, USA, 2013). The X-ray crystallographic structures of tubulin (PDB code 3HKD [24]
and 1SA0 [23]) were downloaded from Protein Data Bank [37]. For Glide docking studies, the
stathmin-like domain and chains B, C were removed. The proteins were minimized by Protein
Preparation Wizard. Partial atomic charges were assigned according to the OPLS_2005 force field. A
radius of 20 Å was selected for active site cavity during receptor grid generation with the center
defined by the co-crystallized ligand TN-16 and colchicine. All compounds used in the docking study
with Glide were built within Maestro by using the build module of Schrödinger Inc. (2013). Docking
calculations were performed using standard mode of Glide Program. To validate the Glide docking
protocol, TN-16 was redocked into the binding site. The docking structure was compared to the crystal
structure showing that this protocol successfully reproduces the crystal TN-16 tubulin complex.
3
.3. Biology
Methodology of the in Vitro Cancer Screen
−5
In vitro cancer screens were done according to the NCI protocol at 10 M dose on the full panel of
6
0 human cancer cell lines derived from nine human cancer cell types that have been grouped in
disease sub-panels including leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR),
non-small-cell lung (A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M,
NCI-H460, NCI-H522), colon (COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620),
central nervous system (SF-268, SF-295, SF-539, SNB-19, SNB-75, U251), melanoma (LOX IMVI,
MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62),
ovarian (IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), renal
(786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), prostate (PC-3, DU-145) and
breast tumour (MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468) cell lines [32].
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5000 to
4
0,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 °C, 5% CO , 95% air and 100% relative humidity for 24 h prior to
2
addition of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at the time of drug addition (Tz).
Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final maximum test concentration with complete

Molecules 2014, 19
13354
medium containing 50 µg/mL gentamicin. Aliquots of 100 µL of drug are added to the appropriate
microtiter wells already containing 100 µL of medium, resulting in the required final drug
concentration. Following drug addition, the plates are incubated for an additional 48 h at 37 °C, 5%
CO , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition
2
of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA (final
concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant is discarded, and the
plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µL)
at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 min at room
temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and
the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the
absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the
methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the
wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA). Using the seven
absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug
(
Ti)], the percentage growth is calculated. Percentage growth inhibition is calculated as:
ꢁꢂꢃ − ꢂꢄꢅ⁄ꢁꢆ − ꢂꢄꢅꢇ × 100 for concentrations for which Ti ≥ Tz
ꢁꢂꢃ − ꢂꢄꢅ⁄ꢂꢄꢇ × 100 for concentrations for which Ti ˂ Tz
ꢀ
(1)
(2)
ꢀ
For further information to see NCI website [38].
4
. Conclusions
In conclusion, we have reported the synthesis of derivatives of the new ring systems
6H,13H-bispyrido[4',3':4,5]pyrrolo[1,2-a:1',2'-d]pyrazine-6,13-dione 4, 5 and 6H,13H-pyrido[4'',3'':4',5']-
pyrrolo[1',2':4,5]pyrazino[1,2-a]indole-6,13-dione 6 using a simple and versatile synthetic pathway.
−5
All derivatives were prescreened according to the NCI protocol at 10 M dose on the full panel of 60
human cancer cell lines derived from nine human cancer cell types. Only derivatives 5a and 6a, 6c and
6
d showed a moderate antineoplastic activity at micromolar concentration. In particular derivative 5a
exhibited modest activity against the UO-31 renal cancer sub-panel cell line; deaza analogue 6a and
the 9-methoxy substituted derivative 6c were shown to be selective against the MCF7 breast cancer
cell line. More interesting results were obtained from the 11-methoxy substituted compound 6d which
showed selectivity against both the UO-31 renal cancer sub-panel and the MCF7 breast cancer sub-panel
cell lines. Unfortunately the moderate activity showed by derivatives 5a and 6a, 6c and 6d against a
limited number of cell lines could not allow a reliable SAR evaluation. However, the antiproliferative
activity shown by derivatives 5a and 6a, 6c and 6d, although modest, encourages further studies
directed toward the synthesis of new compounds with an improved growth inhibitory effect.
Acknowledgments
This work was financially supported by Ministero dell’Istruzione dell’Università e della Ricerca.
We thank the National Cancer Institute (Bethesda, MD) for the antitumour tests reported in this paper.

Molecules 2014, 19
13355
Author Contributions
Girolamo Cirrincione, Patrizia Diana, Alessandra Montalbano and Paola Barraja designed research;
Barbara Parrino performed docking studies and analyzed the data, Alessandra Montalbano,
Anna Carbone and Virginia Spanò performed research and analyzed the data; Girolamo Cirrincione,
Patrizia Diana, Alessandra Montalbano, Barbara Parrino and Paola Barraja wrote the paper. All
authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1
.
.
Hilton, S.; Rossiter, S. Three heterocyclic ring fused (5-6-5). In Comprehensive Heterocyclic
Chemistry III; Jones, K., Ed.; Elsevier Ltd.: Oxford, UK, 2008; Volume 12, pp. 747–751.
Kanoh, K.; Kohno, S.; Asari, T.; Harada, T.; Katada, J.; Muramatsu, M.; Kawashima, H.; Sekiya, H.;
Uno, I. (−)-Phenylahistin: A new mammalian cell cycle inhibitor produced by Aspergillus ustus.
Bioorg. Med. Chem. Lett. 1997, 7, 2847–2852.
2
3
4
5
6
.
.
.
.
Kanoh, K.; Kohno, S.; Katada, J.; Takahashi, J.; Uno, I. (−)-Phenylahistin arrests cells in mitosis
by inhibiting tubulin polymerization. J. Antibiot. 1999, 52, 134–141.
Kanoh, K.; Kohno, S.; Katada, J.; Hayashi, Y.; Muramatsu, M.; Uno, I. Antitumor activity of
phenylahistin in vitro and in vivo. Biosci. Biotechnol. Biochem. 1999, 63, 1130–1133.
Kanoh, K.; Kohno, S.; Katada, J.; Takahashi, J.; Uno, I.; Hayashi, Y. Synthesis and biological
activities of phenylahistin derivatives. Bioorg. Med. Chem. 1999, 7, 1451–1457.
Yamazaki, Y.; Sumikura, M.; Hidaka, K.; Yasui, H.; Kiso, Y.; Yakushiji, F.; Hayashi, Y.
Anti-microtubule “plinabulin” chemical probe KPU-244-B3 labeled both α- and β-tubulin.
Bioorg. Med. Chem. 2010, 18, 3169–3174.
7
.
Yamazaki, Y.; Tanaka, K.; Nicholson, B.; Deyanat-Yazdi, G.; Potts, B.; Yoshida, T.; Oda, A.;
Kitagawa, T.; Orikasa, S.; Kiso, Y.; et al. Synthesis and structure-activity relationship study of
antimicrotubule agents Phenylahistin derivatives with a didehydropiperazine-2,5-dione structure.
J. Med. Chem. 2012, 55, 1056–1071.
8
9
1
1
.
Boger, D.L.; Fink, B.E.; Hedrick, M.P. A new class of highly cytotoxic diketopiperazines.
Bioorg. Med. Chem. Lett. 2000, 10, 1019–1020.
.
Paleni, D. Preparation of Dioxopyrazinodiindoles as Neoplasm Inhibitors. FR 2650590, 8
February 1991.
0. Paleni, D. Preparation and Formulation of Dioxopyrazinodiindoles as Cell Differentiation
Inhibitors. DE 4102921, 6 August 1992.
1. Barraja, P.; Caracausi, L.; Diana, P.; Carbone, A.; Montalbano, A.; Cirrincione, G.; Brun, P.;
Palù, 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,
4
830–4843.

Molecules 2014, 19
13356
1
1
1
1
1
2. 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.
3. Carbone, A.; Parrino, B.; Barraja, P.; Spanò, V.; Cirrincione, G.; Diana, P.; Maier, A.; Kelter, G.;
Fiebig, H.-H. Synthesis and antiproliferative activity of 2,5-bis(3'-indolyl)pyrroles, analogues of
the marine alkaloid nortopsentin. Mar. Drugs 2013, 11, 643–654.
4. Barraja, P.; Caracausi, L.; Diana, P.; Spanò, 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.
5. Diana, P.; Carbone, A.; Barraja, P.; Montalbano, A.; Parrino, B.; Lopergolo, A.; Pennati, M.;
Zaffaroni, N; Cirrincione, G. Synthesis and antitumor activity of 3-(2-phenyl-1,3-thiazol-4-yl)-
1
H-indoles and 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindoles. ChemMedChem 2011, 6, 1300–1309.
6. Carbone, A.; Pennati, M.; Parrino, B.; Lopergolo, A.; Barraja, P.; Montalbano, A.; Spanò, V.;
Sbarra, S.; Doldi, V.; de Cesare, M.; et al. Novel 1H-pyrrolo[2,3-b]pyridine derivatives nortopsentin
analogues: Synthesis and antitumor activity in peritoneal mesothelioma experimental models.
J. Med. Chem. 2013, 56, 7060–7072.
1
7. Carbone, A.; Pennati, M.; Barraja, P.; Montalbano, A.; Parrino, B.; Spanò, V.; Lopergolo, A.;
Sbarra, S.; Doldi, V.; Zaffaroni, N.; et al. Synthesis and antiproliferative activity of substituted
3
-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-pyrrolo[3,2-b]pyridines, marine alkaloid nortopsentin
analogues. Curr. Med. Chem. 2014, 21, 1654–1666.
1
1
8. Barraja, P.; Diana, P.; Montalbano, A.; Dattolo, G.; Cirrincione, G.; Viola, G.; Vedaldi, D.;
Dall’Acqua, F. Pyrrolo[2,3-h]quinolinones: A new ring system with potent photoantiproliferative
activity. Bioorg. Med. Chem. 2006, 14, 8712–8728.
9. Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G.; Vedaldi, D.; Salvador A.; Viola, G.;
Dall’Acqua F. Pyrano[2,3-e]isoindol-2-ones, a new angelicin heteroanalogues. Bioorg. Med.
Chem. Lett. 2009, 19, 1711–1714.
2
0. Barraja, P.; Spanò, 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.
2
1. 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.
2
2
2
2. Spanò, V.; Montalbano, A.; Carbone, A.; Parrino, B.; Diana, P.; Cirrincione, G.; Castagliuolo, I.;
Brun, P.; Issinger, O.-G.; Tisi, S.; et al. Synthesis of a new class of pyrrolo[3,4-h]quinazolines
with antimitotic activity. Eur. J. Med. Chem. 2014, 74, 340–357.
3. 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
2
013, 69, 6474–6477.
4. Ravelli, R.B.G.; Gigant, B.; Curmi, P.A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M.
Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature
2
004, 428, 198–202.

Molecules 2014, 19
13357
2
5. Dorléans, A.; Gigant, B.; Ravelli, R.B.G.; Mailliet, P.; Mikol, V.; Knossow, M. Variations in the
colchicine-binding domain provide insight into the structural switch of tubulin. Proc. Natl. Acad.
Sci. USA 2009, 106, 13775–13779.
2
2
6. Arai, T. Inhibition of microtubule assembly in vitro by TN-16, a synthetic antitumor drug. FEBS Lett.
1
983, 155, 273–276.
7. Barbier, P.; Dorléans, A.; Devred, F; Sanz, L.; Allegro, D.; Alfonso, C.; Knossow, M.; Peyrot, V.;
Andreu, J.M. Stathmin and interfacial microtubule inhibitors recognize a naturally curved
conformation of tubulin dimmers. J. Biol. Chem. 2010, 285, 31672–31681.
2
2
8. Glide, version 5.9; Schrödinger, LLC: New York, NY, USA, 2013.
9. Bradley, S.E.; Krulle, T.M.; Murray, P.J.; Procter, M.J.; Rowley, R.J.; Sambrook, S.C.P.; Thomas, G.H.
Preparation of Pyrrolopyridine-2-Carboxylic Acid Amide as Inhibitors of Glycogen Phosphorylase.
WO 2004104001, 2 December 2004.
3
3
3
0. Casara, P.; le Diguarher, T.; Durand, D.; Geneste, O.; Hickman, J. New Tricyclic Derivatives,
Their Preparation as Pro-Apoptotic and Antitumor Agents and Their Pharmaceutical Compositions
Containing Them. WO 2010007248, 21 January 2010.
1. Evans, G.B.; Furneaux, R.H.; Hutchison, T.L.; Kezar, H.S.; Morris, P.E., Jr.; Schramm, V.L.;
Tyler, P.C. Addition of lithiated 9-deazapurine derivatives to a carbohydrate cyclic imine:
Convergent synthesis of the aza-C-nucleoside immucillins. J. Org. Chem. 2001, 66, 5723–5730.
2. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.;
Cronise, P.; Vaigro-Wolff, A.; et al. Feasibility of a high-flux anticancer drug screen using a
diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83, 757–766.
3. Frydman, B.; Despuy, M.E.; Rapoport, H. Pyrroles from azaindoles. A synthesis of porphobilinogen.
J. Am. Chem. Soc. 1965, 3530–3531.
3
3
3
3
4. Dubois, L.; Evanno, Y.; Malanda, A. Preparation Of 1-(Arylalkyl)-1H-Pyrrolopyridine-2-Carboxamide
Derivatives as VR1 Type Capsaicin Receptor Antagonists. WO 2007010138, 25 January 2007.
5. Frydman, B.; Reil, S.J.; Boned, J.; Rapoport, H. Synthesis of substituted 4- and 6-azaindoles.
J. Org. Chem. 1968, 33, 3762–3766.
6. Qiao, G.G.; Meutermans, W.; Wong, M.W.; Traeubel, M.; Wentrup, C. (Cyanovinyl)ketenes from
azafulvenones. An apparent retro-Wolff rearrangement. J. Am. Chem. Soc. 1996, 118, 3852–3861.
7. Protein Data Bank. Available on line: http://www.rcsb.org/pdb (accessed on 28 August 2014).
8. NCI-60 DTP Human tumor cell line screen. Available online: http://dtp.nci.nih.gov/branches/
btb/ivclsp.html (accessed on 20 July 2012).
3
3
Sample Availability: Not available.
©
2014 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
http://creativecommons.org/licenses/by/3.0/).
(