Isoindolo[2,1-c]benzo[1,2,4]triazines: A new ring system with antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2006.09.054

A series of isoindolo-benzo-triazines of type 4 was obtained by diazotization of 2-(2-aminoaryl)-1-cyanoisoindoles 3a-j. All the synthesized derivatives were screened by the National Cancer Institute (NCI, Bethesda, USA), for in vitro antitumor activity a

Full text of DOI:10.1016/j.bmc.2006.09.054

Bioorganic & Medicinal Chemistry 15 (2007) 343–349
Isoindolo[2,1-c]benzo[1,2,4]triazines: A new ring system
with antiproliferative activity
Patrizia Diana,* Annamaria Martorana, Paola Barraja, Antonino Lauria,
Alessandra Montalbano, Anna Maria Almerico, Gaetano Dattolo
and Girolamo Cirrincione
`
Dipartimento Farmacochimico Tossicologico e Biologico, Universita degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
Received 19 May 2006; revised 14 September 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—A series of isoindolo-benzo-triazines of type 4 was obtained by diazotization of 2-(2-aminoaryl)-1-cyanoisoindoles 3a–j.
All the synthesized derivatives were screened by the National Cancer Institute (NCI, Bethesda, USA), for in vitro antitumor activity
against a 3-human cancer cell line panel consisting of MCF7 (breast), NCI-H460 (lung), and SF-268 (CNS). Derivatives 4a, f, i, j
were selected to be evaluated in the full panel of about 50 human tumor cell lines derived from nine human cancer cell types and
showed antiproliferative activity generally in the micromolar range. The most sensitive cell lines were: MOLT-4 and SR of the leu-
kemia subpanel, A549/ATCC and EKVX of the nonsmall cell lung subpanel, COLO-205 of the colon cancer subpanel, LOX IMVI
of the Melanoma subpanel, OVCAR-8 of the ovarian cancer subpanel, and MCF7, BT-549 of the breast cancer subpanel.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
pounds that showed IC50 8–500 lg/ml for inhibition of
growth of Hep-2, HeLa, peritoneal carcinoma, and pleu-
rocarcinoma cells.10 In particular, when condensed with
one or more heterocycles, they found application in
many fields such as pharmaceuticals, herbicides, pesti-
cides, and dyes. For instance, pyrrolo[2,1-f][1,2,4]tria-
zines exhibited activity against EGFR or VEGFR-2
kinase and many of them were shown to have substan-
tial activity in cellular assays.11,12 Also indazolylpyrrol-
otriazines showed inhibitory activity against tyrosine
kinase of growth factor receptors such as HER1,
HER2, and HER4, thereby making them useful as anti-
proliferative agents.13
DNA represents one of the most important cellular tar-
gets for several chemotherapeutic drugs. Polycyclic
nitrogen heterocycles with a planar structure such as
acridine, phenanthridine, and actinomycin derivatives
can be good pharmacophores for classes of antitumor
drugs since they can intercalate between the base pairs
of double-strand DNA.1 In connection with our studies
on polycondensed nitrogen heterocycles, containing pyr-
role or indole moieties with antineoplastic activity we
reported the synthesis and the remarkable antiprolifera-
tive activity of indolo[3,2-c]cinnolines and indolo[1,2-
c]triazines.2,3 More recently we reported the synthesis
of pyrrolo[2,1-c][1,2,4]triazines and [1,2,4]triazino[4,3-
a]indoles that showed antitumor activity with GI50 val-
ues in the range 10ꢀ5–10ꢀ6 M.4 Several 1,2,4-triazine
derivatives are well-known compounds endowed with
a wide range of biological activities.5,6 A large number
of synthetic 1,2,4-triazines have shown antineoplastic
activity in in vitro and in in vivo systems.7–9 Benzocon-
densation to the 1,2,4-benzotriazines system led to com-
2. Results and discussion
In our attempts to search for novel antitumor agents, we
extended our interest to the isoindole system and
planned to synthesize isoindolo[2,1-c]benzo[1,2,4]tria-
zines 4a–j with the aim of evaluating their antiprolifera-
tive activity.
2.1. Docking
Keywords: Isoindolo-benzotriazine; Antiproliferative activity; 3D-
Mind.
Preliminary docking studies on the new series of the iso-
indolo[2,1-c]benzo[1,2,4]triazines were performed, using
*
Corresponding author. E-mail: diana@unipa.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.054

344
P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
Table 1. Calculated free energies of binding obtained by AutoDock
AUTODOCK software, in order to evaluate by this fast
in silico screening new potential DNA-interactive drugs.
AutoDock combines a rapid energy evaluation through
precalculated grids of affinity potentials with a variety of
search algorithms to find appropriate binding positions
for a ligand on a given receptor. Several runs of space
search were carried out to find the best ligand–receptor
binding arrangement. We performed automated dock-
ing studies on the isoindolo-triazines 4a–j using
Lamarckian Genetic Algorithm (LGA), which is the
best search global algorithm.14 As biomacromolecular
target, we chose the DNA fragment identified by the
Protein Data Bank (PDB) code 1DSD in which was
originally intercalated the actinomycin D whose X-ray
crystallographic structure has been downloaded from
the PDB web site.15 Before running autodock, it was
necessary to prepare the DNA sequence: the original li-
gand and the water molecules were removed, obtaining
a macro.pdb file, which contained only biomolecule
coordinates.
Compound
DG (kcal/mol)
4a
4b
ꢀ22.77
ꢀ23.88
ꢀ23.10
ꢀ23.72
ꢀ23.25
ꢀ19.96
ꢀ21.74
ꢀ21.21
ꢀ21.75
ꢀ24.47
ꢀ10.36
4c
4d
4e
4f
4g
4h
4i
4j
Actinomycin D
Derivatives 3a–j were prepared in good yield (40–95%)
by a Strecker type synthesis between substituted o-phe-
nylendiamines 1a–g and phthaloyldicarboxaldehyde 2
in the presence of potassium cyanide and sodium
hydrogensulfite. In the case of the monosubstituted
o-phenylendiamines 1b, 1f, and 1g, depending on the
amino group involved in the cyclization to the isoindole
nucleus two isomers were isolated. In particular from 1b
were isolated 3b and 3c; from 1f were isolated 3g and 3h;
from 1g were isolated 3i and 3j. Instead in the case of 1c
(4-methoxy substituted) compound 3d only was isolated
due to the enhanced reactivity of the amino group
placed in para position with respect to the methoxy
substituent. Diazotization of derivatives 3a–j with a stoi-
chiometric amount of sodium nitrite at room tempera-
ture in acetic acid and successive cyclization of the
resulting diazonium group by an intramolecular cou-
pling with the position 3 of the isoindole afforded the
title ring system in excellent yields (84–98%).
The range of calculated free energy is from ꢀ19.96 to
ꢀ24.47 kcal/mol and these values are lower then those
calculated for Actinomycin D (ꢀ10.36 kcal/mol). The li-
gand with the highest affinity was the derivative 4j which
had ꢀ24.47 kcal/mol, and therefore it could be expected
to form the more stable complex with DNA (Table 1).
On the basis of these docking virtual screening results
we supposed that isoindolo[2,1-c]benzo[1,2,4]triazines
may be good candidates as new DNA-interactive
compounds.
2.2. Chemistry
2-(2-Aminoaryl)-1-cyanoisoindoles 3a–j appeared valu-
able and versatile intermediates for the synthesis of the
new ring system isoindolo[2,1-c]benzo[1,2,4]triazines
4a–j.
The structure of all compounds synthesized was con-
firmed by their analytical and spectral data (IR, 1H,
and 13C NMR).
R1
O
O
CN
N
R4
R3
H2N
H2N
R2
R3
H
H
KCN
R2
+
NaHSO3
H2N
3a-j
R1
R4
1a-g
2
NaNO2/AcOH
a
b
c
d
e
f
R1=R2=R3=R4=H
a
b
c
d
e
f
g
h
i
R1=R2=R3=R4=H
R1=R2=R3=H, R4=CH3
R1=CH3, R2=R3=R4=H
R1=R3=R4=H, R2=OCH3
R1=R4=H, R2=R3=CH3
R1=R4=H, R2=R3=Cl
R1=R3=R4=H, R2=Cl
R1=R2=R4=H, R3=Cl
R1=R3=R4=H, R2=CF3
R1=R2=R4=H, R3=CF3
R1=CH3, R2=R3=R4=H
R1=R3=R4=H, R2=OCH3
R1=R4=H, R2=R3=CH3
R1=R4=H, R2=R3=Cl
R1=R3=R4=H, R2=Cl
R1=R3=R4=H, R2=CF3
CN
R4
R3
N
R2
N
N
R1
g
4a-j
j

P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
345
Table 3. Overview of the results of the in vitro antitumor screening for
compounds 4a, f, i, ja
2.3. Biology
c
All the synthesized derivatives have been evaluated, by
the National Cancer Institute (NCI, Bethesda, USA),
in the 3-cell line (MCF7-breast, NCI-H460-non-small
cell lung, SF-268-CNS), one dose primary anti-cancer
assay (Table 2). A compound is considered active when
it reduces the growth of any of the cell lines to 32% or
less (negative numbers indicate cell kill).
Compound
Nb
pGI50
nd
Range
MG_MIDe
4a
4f
4i
4j
42
53
42
53
35
42
32
47
5.19–4.00
4.70–4.00
4.97–4.00
>8.00–4.00
4.47
4.31
4.27
4.46
a Data obtained from NCI’s in vitro disease-oriented human tumour
cells screen.
The compounds, which passed the criteria set by the
NCI for activity in this assay, were scheduled automat-
ically for evaluation against the full panel of tumor cell
lines.16 Four isoindolo-benzo-triazine derivatives of type
4 were selected for evaluation against the full NCI panel
of about 50 human cancer cell lines.
b N is the number of cell lines investigated.
c pGI50 is the ꢀlog of the molar concentration that inhibits 50% net cell
growth.
d n is the number of cell lines giving positive pGI50
.
e MG_MID = mean graph midpoint = arithmetical mean value for all
tested cancer cell lines. If the identical effect was not attainable within
the used concentrated interval, the highest tested concentration was
used for the calculation.
This panel is organized into subpanels derived from nine
different human cancer types: leukemia, melanoma,
lung, colon, renal, ovarian, breast, prostate, and CNS.
The compounds were tested at five concentrations at
10-fold dilution the highest being 10ꢀ4 M and the others
10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M. The following parameters
were determined for every cell line: GI50 (concentration
inhibiting 50% net cell growth), TGI (concentration that
totally inhibited net cell growth), and LC50 (concentra-
tion leading to 50% net cell death). For each of these
parameters the average values of mean graph midpoint
(MG_MID) were calculated.17
range 4.15–6.67. The most sensitive cell lines are
MOLT-4 (pGI50 = 4.98) and SR (pGI50 = 6.67). Excel-
lent response was obtained in the nonsmall lung cancer;
in fact the calculated pGI50 MG_MID values for the sub-
panel related to the active compounds are always higher
than that of the over all cell lines (DMG_MID = 0.51)
The most sensitive cell lines resulted in EKVX and
NCI-522 (pGI50 P 8.00 and 4.77, respectively). Deriva-
tive 4a was selectively active with respect to the nonsmall
lung cancer and especially active against EKVX and
A549/ATCC (pGI50 = 4.90 and 4.87, respectively). It
also showed selectivity with respect to COLO-205
(pGI50 = 5.03) of colon cancer, LOX IMVI and M14
(pGI50 = 5.19 and 4.83, respectively) of melanoma sub-
panel, OVCAR-8 (pGI50 = 4.95) of ovarian cancer, and
MCF7 (pGI50 = 4.85), BT-549 (pGI50 = 4.92) of breast
cancer. Derivative 4i had low pGI50 mean value
(pGI50 = 4.90) but showed a selectivity concerning the
breast cancer MCF7 cell line (pGI50 = 4.97).
An evaluation of the data reported in Table 3 pointed
out that compounds 4a, f, i, and j exhibited antineoplas-
tic activity against most of the human cell lines. The
most active compounds are 4a and4j in terms either of
pGI50 (mean value 4.47 and 4.46, respectively) and per-
centage of sensitive cell lines out of the total number of
cell lines investigated (83% and 89%, respectively).
Isoindolo-benzo-triazines 4a, f, i, and j showed inhibito-
ry effects in the growth of a wide range of cancer cell lines
and most of susceptible tumors were generally inhibited
at micromolar concentration (see Supporting informa-
tion). Compound 4j, bearing a trifluoromethyl group,
was shown to be selective with respect to the leukemia
subpanel having all the subpanel cell lines pGI50 in the
In order to discern the mechanism of action of isoindolo-
benzo-triazines we performed COMPARE computa-
tions18 for derivatives 4a, f, i, j against the NCI ‘Standard
Agents’ database. All compounds had a Pearson correla-
tion coefficient (PCC) <0.6 suggesting that the antiprolif-
erative activity of isoindolo[2,1-c]benzo[1,2,4] triazines
would be mechanistically unrelated to that of any known
drug. We also utilized the facility 3D-MIND (Drug Dis-
covery and Data Mining Information for New Direc-
tions) projecting our compounds 4a, f, i, j onto the
‘Complete DTP Map’ a self-organizing map (SOM)
based on cytotoxicity measurements from the NCI
anti-cancer drug-screening database (http://spheroid.n-
cifcrf.gov) which cluster these data in the high-dimen-
sional GI50 space and provide a means of its visual
translation into a two dimensional anti-cancer map.
The Complete map is divided in six regions designated
alphabetically (M, mitosis; N, membrane transport and
integrity; P, phosphatase- and kinase cell cycle regula-
tion; S, nucleic acid synthesis; Q, R, arbitrarily regions)
which share the same pattern of growth inhibition, and
which substantially reflect their molecular targets and
modes of action.19 The results of this analysis on project-
ed isoindolo-benzo-triazines indicate their location in the
Table 2. One dose primary anti-cancer assay for compounds 4a
Compound
Breast
MCF7
Non-small
cell-lung
NCI-H460
CNS
SF-268
Compound
selected
4a
4b
4c
4d
4e
4f
26
69
72
63
76
80
109
51
65
83
21
50
118
86
48
17
109
61
6
95
88
Y
N
N
N
N
Y
N
N
Y
Y
105
104
76
68
4g
4h
4i
114
86
82
96
4j
8
a Compounds were tested at the concentration of 10–4 M; exposure
time 48 h.

346
P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
Figure 1. Projection of compound 4j onto complete compound map (molecular activity classes).
S region map where antimetabolites of purine and pyrim-
idine nucleotides are found (Fig. 1).
(8.0 ml) was added and the mixture heated for an addi-
tional 90 min. The mixture was cooled and the solid
formed was filtered and purified by chromatography.
It is our intention to undertake studies directed to eluci-
date the biochemical mechanism of action of this series
of isoindolo[2,1-c]benzo[1,2,4]triazines.
Method B. Differs from Method A in that: (i) the o-phe-
nylendiamine 1e, f were added after dissolution in DMF
(15 ml); (ii) after the addition of KCN the mixture was
heated for 2 h; (iii) the mixture was poured into 200 ml
of water and the solid formed was purified by
chromatography.
3. Experimental
All melting points were taken on a Buchi-Tottoli capil-
¨
lary apparatus and are uncorrected; IR spectra were
determined in bromoform with a Jasco FT/IR 5300
spectrophotometer; 1H and 13C NMR spectra were mea-
sured at 200 and 50.3 MHz, respectively, in DMSO-d6
or CDCl3 solution, using a Bruker AC series 200 MHz
spectrometer (TMS as internal reference). Column chro-
matography was performed with Merck silica gel 230–
400 Mesh ASTM or with Sepacore Buchi apparatus.
Elemental analyses (C, H, and N) were within 0.4%
of the theoretical values.
3.1.1. 1-Cyano-2-(20-aminophenyl)isoindole (3a). Method
A. This compound was purified by flash chromatogra-
phy using dichloromethane as eluent to give a solid:
yield 65%; mp 150–151 ꢁC. IR 3473, 3363 (NH2), 2202
(CN) cmꢀ1 1H NMR (DMSO-d6) d 5.19 (2H, s,
.
NH2), 6.70 (1H, t, J = 7.2 Hz, H-50), 6.94 (1H, d,
J = 7.2 Hz, H-30), 7.15–7.19 (3H, m, H-40, H-5 and
H-60), 7.31 (1H, t, J = 8.2 Hz, H-6), 7.66 (1H, d,
J = 8.2 Hz, H-4), 7.78 (1H, d, J = 8.2 Hz, H-7), 7.86
(1H, s, H-3). 13C NMR (DMSO-d6) d 93.9 (s, C-1),
114.0 (s, CN), 115.8 (d, C-30), 116.1 (d, C-50), 117.5 (d,
C-60), 121.5 (d, C-40), 122.2 (d, C-3), 122.4 (d, C-7),
122.5 (s, C-3a), 124.1 (s, C-10), 125.5 (d, C-4), 127.8
(d, C-5), 130.4 (d, C-6), 131.2 (s, C-7a), 144.1 (s, C-20).
Anal. Calcd for C15H11N3 (MW 233.27): C, 77.23; H,
4.75; N, 18.01%. Found: C, 77.46; H, 4.67; N, 18.12%.
3.1. Synthesis of substituted 1-cyano-2-(20-aminophenyl)
isoindoles (3a–j)
Method A. To a solution of 1.56 g (0.015 mol) of sodium
hydrogen sulfite in water (38 ml) was added phthaloyldi-
carboxaldehyde 2 (2 g, 0.015 mol). The mixture was stir-
red until the solid was dissolved and the appropriate o-
phenylenediamine 1a–d, g (0.015 mol) was added, and
the reaction mixture was heated on a steam bath for
30 min. Then KCN (3.39 g, 0.052 mol) in water
3.1.2. 1-Cyano-2-(20-amino-60-methylphenyl)isoindole (3b).
Method A. This compound was purified by Sepacore
Buchi apparatus using dichloromethane/cyclohexane
(7:3) as eluent to give a solid: yield 45%; mp 87–88 ꢁC.

P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
347
IR 3483, 3390 (NH2), 2202 (CN) cmꢀ1. 1H NMR (CDCl3)
d 1.98 (3H, s, CH3), 3.45 (2H, s, NH2), 6.69 (1H, d,
J = 8.1 Hz, H-30), 6.75 (1H, d, J = 8.1 Hz, H-50), 7.16
(1H, t, J = 8.1 Hz, H-40), 7.20 (1H, t, J = 7.6 Hz, H-5),
7.26 (1H, t, J = 7.6 Hz, H-6), 7.35 (1H, s, H-3), 7.71 (1H,
d, J = 7.6 Hz, H-4), 7.74 (1H, d, J = 7.6 Hz, H-7). 13C
NMR (CDCl3) d 17.0 (q, CH3), 94.7 (s, C-1), 113.6 (s,
CN), 114.1 (d, C-50), 118.5 (d, C-3), 120.0 (d, C-7), 120.3
(d, C-4), 121.0 (d, C-30), 123.2 (d, C-40), 123.6 (s, C-60),
124.8 (s, C-3a), 125.9 (d, C-5), 130.3 (s, C-10), 130.6 (d,
C-6), 136.3 (s, C-7a), 143.0 (s, C-20). Anal. Calcd for
C16H13N3 (MW 247.30): C, 77.71; H, 5.30; N, 16.99%.
Found: C, 77.45; H, 5.60; N, 17.08%.
128.2 (d, C-6), 131.7 (s, C-7a), 139.6 (s, C-40), 139.7 (s,
C-20). Anal. Calcd for C17H15N3 (MW 261.33): C,
78.13; H, 5.79; N, 16.08%. Found: C, 78.34; H, 5.67; N,
15.89%.
3.1.6. 1-Cyano-2-(20-amino-40,50-dichlorophenyl)isoindole
(3f). Method B. This compound was purified by flash
chromatography using dichloromethane as eluent to
give a solid: yield 77%; mp 71–72 ꢁC. IR 3394, 3340
1
(NH2), 2200 (CN) cmꢀ1. H NMR (DMSO-d6) d 5.75
(2H, s, NH2), 7.12 (1H, s, H-30), 7.16 (1H, t,
J = 8.4 Hz, H-5), 7.32 (1H, t, J = 8.4 Hz, H-6), 7.58
(1H, s, H-60), 7.66 (1H, d, J = 8.4 Hz, H-4), 7.78 (1H,
d, J = 8.4 Hz, H-7), 7.91 (1H, s, H-3). 13C NMR
(DMSO-d6) d 94.0 (s, C-1), 113.8 (s, CN), 115.8 (s,
C-10), 116.2 (d, C-30), 117.5 (d, C-60), 121.6 (d, C-3),
121.7 (s, C-3a), 122.4 (d, C-7), 122.6 (d, C-4), 124.2 (s,
C-50), 125.8 (d, C-5), 129.6 (d, C-6), 131.2 (s, C-7a),
132.9 (s, C-40), 144.7 (s, C-20). Anal. Calcd for
C15H9Cl2N3 (MW 302.16): C, 59.63; H, 3.00; N,
13.91%. Found: C, 59.72; H, 3.23; N, 13.81%.
3.1.3. 1-Cyano-2-(20-amino-30-methylphenyl)isoindole (3c).
Method A. This compound was purified by Sepacore Bu-
chi apparatus using dichloromethane/cyclohexane (7:3) as
eluent to give a solid: yield 40%; mp 62–63 ꢁC. IR 3481,
1
3388 (NH2), 2200 (CN) cmꢀ1. H NMR (CDCl3) d 2.25
(3H, s, CH3), 3.55 (2H, s, NH2), 6.80 (1H, t,
J = 7.6 Hz, H-50), 7.10 (1H, d, J = 7.6 Hz, H-60), 7.14
(1H, d, J = 7.6 Hz, H-40), 7.23 (1H, t, J = 8.1 Hz, H-5),
7.31 (1H, t, J = 8.1 Hz, H-6), 7.44 (1H, s, H-3), 7.69
(1H, d, J = 8.1 Hz, H-4), 7.73 (1H, d, J = 8.1 Hz, H-7).
13C NMR (CDCl3) d 17.6 (q, CH3), 92.2 (s, C-1), 113.0
(s, CN), 117.9 (d, C-50), 118.3 (d, C-60), 120.5 (d, C-3),
120.9 (d, C-7), 123.2 (d, C-4), 123.8 (s, C-3a), 124.1 (s,
C-10), 124.6 (s, C-30), 125.5 (d, C-40), 125.9 (d, C-5),
131.8 (s, C-7a), 131.9 (d, C-6), 140.5 (s, C-20). Anal. Calcd
for C16H13N3 (MW 247.30): C, 77.71; H, 5.30; N, 16.99.
Found: C, 77.59; H, 5.45; N, 16.87.
3.1.7. 1-Cyano-2-(20-amino-40-chlorophenyl)isoindole (3g).
Method B. This compound was purified by flash chro-
matography using dichloromethane as eluent to give a
solid: yield 40%; mp 170–171 ꢁC. IR 3475, 3390
1
(NH2), 2202 (CN) cmꢀ1. H NMR (DMSO-d6) d 5.53
(2H, s, NH2), 6.70 (1H, d, J = 8.4 Hz, H-50), 6.93
(1H, s, H-30), 7.15 (1H, t, J = 7.9 Hz, H-5), 7.22 (1H,
d, J = 7.9 Hz, H-4), 7.31 (1H, t, J = 7.9 Hz, H-6),
7.65 (1H, d, J = 8.4 Hz, H-60), 7.79 (1H, d,
J = 7.9 Hz, H-7), 7.86 (1H, s, H-3). 13C NMR
(DMSO-d6) d 94.0 (s, C-1), 113.9 (s, CN), 115.0 (d,
C-30), 115.2 (d, C-50), 117.6 (d, C-3), 121.2 (s, C-10),
121.6 (d, C-7), 122.3 (d, C-4), 122.6 (d, C-60), 124.2
(s, C-3a), 125.7 (d, C-5), 129.7 (d, C-6), 131.3 (s,
C-40), 134.9 (s, C-7a), 145.8 (s, C-20). Anal. Calcd for
C15H10ClN3 (MW 267.72): C, 67.30; H, 3.76; N,
15.70%. Found: C, 67.49; H, 3.55; N, 15.83%.
3.1.4. 1-Cyano-2-(20-amino-40-methoxyphenyl)isoindole
(3d). Method A. This compound was purified by flash
chromatography using dichloromethane as eluent to
give a solid: yield 95%; mp 116–117 ꢁC. IR 3465, 3375
(NH2), 2198 (CN) cmꢀ1 1H NMR (CDCl3) d 3.64
.
(2H, br s, NH2), 3.80 (3H, s, CH3), 6.38 (1H, br s,
H-30), 6.42 (1H, d, J = 7.9 Hz, H-50), 7.12 (1H, d,
J = 7.9 Hz, H-60), 7.14 (1H, t, J = 8.6 Hz, H-5), 7.29
(1H, t, J = 8.6 Hz, H-6), 7.41 (1H, s, H-3), 7.66 (1H,
d, J = 8.6 Hz, H-4), 7.70 (1H, d, J = 8.6 Hz, H-7). 13C
NMR (CDCl3) d 55.4 (q, CH3), 95.4 (s, C-1), 101.4 (d,
C-30), 104.5 (d, C-50), 113.9 (s, CN), 117.5 (s, C-10),
118.2 (d, C-60), 120.8 (d · 2, C-3 e C-7), 123.1 (d, C-4),
124.5 (s, C-3a), 125.8 (d, C-5), 128.7 (d, C-6), 131.7 (s,
C-7a), 143.3 (s, C-20), 161.5 (s, C-40). Anal. Calcd for
C16H13N3O (MW 263.30): C, 72.99; H, 4.98; N,
15.96%. Found: C, 71.89; H, 5.03; N, 16.22%.
3.1.8. 1-Cyano-2-(20-amino-50-chlorophenyl)isoindole (3h).
Method B. This compound was purified by flash chroma-
tography using dichloromethane as eluent to give a solid:
yield 55%; mp 131–132 ꢁC. IR 3471, 3365 (NH2), 2200
1
(CN) cmꢀ1. H NMR (DMSO-d6) d 5.41 (2H, s, NH2),
6.94 (1H, d, J = 8.6 Hz, H-40), 7.17 (1H, t, J = 7.6 Hz,
H-5) 7.20–7.36 (3H, m, H-4, H-6, H-60), 7.67 (1H, d,
J = 8.6 Hz, H-30), 7.79 (1H, d, J = 7.6 Hz, H-7), 7.90
(1H, s, C-3). 13C NMR (DMSO-d6) d 95.3 (s, C-1),
114.0 (s, CN), 117.3 (d, C-30), 117.6 (d, C-3), 118.4 (s,
C-3a), 121.6 (d, C-7), 122.3 (d, C-4), 122.6 (d, C-5),
122.8 (s, C-50), 124.2 (s, C-10), 125.8 (d, C-40), 127.5 (d,
C-6), 130.4 (d, C-60), 131.3 (s, C-7a), 145.5 (s, C-20). Anal.
Calcd for C15H10ClN3 (MW 267.72): C, 67.30; H, 3.76;
N, 15.70%. Found: C, 67.12; H, 3.81; N, 15.63%.
3.1.5. 1-Cyano-2-(20-amino-40,50-dimethylphenyl)isoindole
(3e). Method A. This compound was purified by flash
chromatography using dichloromethane as eluent to give
a solid: yield 85%; mp 119–120 ꢁC. IR 3465, 3375 (NH2),
2202 (CN) cmꢀ1. 1H NMR (CDCl3) d 2.17 (3H, s, CH3),
2.22 (3H, s, CH3), 3.44 (2H, s, NH2), 6.64 (1H, s, H-30),
6.95 (1H, s, H-60), 7.12 (1H, t, J = 7.6 Hz, H-5), 7.27 (1H,
t, J = 7.6 Hz, H-6), 7.38 (1H, s, H-3), 7.64 (1H, d,
J = 7.6 Hz, H-4), 7.68 (1H, d, J = 7.6 Hz, H-7). 13C
NMR (CDCl3) d 18.6 (q, CH3), 19.7 (q, CH3), 95.1 (s,
C-1), 114.0 (s, CN), 118.1 (d, C-30), 118.2 (d, C-60),
120.6 (d, C-3), 120.9 (d, C-7), 121.8 (s, C-3a), 123.1 (d,
C-4), 124.5 (s, C-10), 125.8 (d, C-5), 127.0 (s, C-50),
3.1.9. 1-Cyano-2-(20-amino-40-trifluoromethylphenyl)iso-
indole (3i). Method A. This compound was purified by
Sepacore Buchi apparatus using cyclohexane/ethyl-ace-
tate (9:1) as eluent to give an oil: yield 50%. IR: 3481,
1
3371 (NH2), 2202 (CN) cmꢀ1. H NMR (DMSO-d6) d
5.89 (2H, s, NH2), 7.09(1H, t, J = 8.0 Hz, H-5), 7.21
(1H, s, H-30) 7.26 (1H, d, J = 7.8 Hz, H-50), 7.39 (1H,

348
P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
d, J = 7.8 Hz, H-60), 7.45 (1H, t, J = 8.0 Hz, H-6) 7.49
(1H, d, J = 8.0 Hz, H-4), 7.69 (1H, d, J = 8.0 Hz, H-7),
7.89 (1H, s, H-3). 13C NMR (DMSO-d6) d 93.8 (s,
C-1), 109.2 (d, C-50), 114.0 (s, CN), 115.6 (s, CF3),
116.4 (d, C-3), 117.8 (d, C-30), 119.0 (d, C-7), 121.2 (s,
C-3a), 123.0 (d, C-4), 124.7 (d, C-60), 126.0 (d, C-5),
126.2 (d, C-6), 126.8 (s, C-40), 127.2 (s, C-10), 132.0 (s,
C-7a), 144.8 (s, C-20). Anal. Calcd for C16H10F3N3
(MW 301.27): C, 63.79; H, 3.35; N, 18.92%. Found: C,
63.58; H, 3.18; N, 19.03%.
128.4 (d), 129.7 (d), 130.3 (s), 133.8 (s), 135.6 (d),
137.0 (s), 141.8 (s). Anal. Calcd for C16H10N4 (MW
258.28): C, 74.41; H, 3.90; N, 21.69%. Found: C,
74.35; H, 4.03; N, 21.72%.
3.2.3. 11-Cyano-4-methylisoindolo[2,1-c]benzo[1,2,4]tri-
azine (4c). Yield 98%; mp 255–256 ꢁC. IR 2198 (CN)
cmꢀ1 1H NMR (CDCl3) d 2.37 (3H, s, CH3), 7.71
.
(1H, t, J = 8.4 Hz, H-8), 7.74–7.85 (2H, m, H-9 and
H-3), 7.89 (1H, t, J = 7.8 Hz, H-2), 8.05 (1H, d,
J = 7.8 Hz, H-1), 8.78 (1H, d, J = 8.4 Hz, H-7), 9.03
(1H, d, J = 8.4 Hz, H-10). 13C NMR (CDCl3) d 18.3
(q), 88.3 (s), 113.1 (d), 115.2 (s), 117.4 (d), 120.0 (s),
120.8 (d), 124.5 (s), 126.2 (d), 129.7 (d), 129.8 (d),
131.8 (s), 132.7 (d), 134.7 (s), 138.7 (s), 141.0 (s).
Anal. Calcd for C16H10N4 (MW 258.28): C, 74.41;
H, 3.90; N, 21.69%. Found: C, 74.29; H, 4.00; N,
21.81%.
3.1.10. 1-Cyano-2-(20-amino-50-trifluoromethylphenyl)-
isoindole (3j). Method A. This compound was purified
by Sepacore Buchi apparatus using cyclohexane/ethyl-
acetate (9:1) as eluent to give an oil: yield 44%. IR
3481, 3381 (NH2), 2200 (CN) cmꢀ1
.
1H NMR
(DMSO-d6) d 5.99 (2H, s, NH2), 7.02 (1H, d,
J = 8.9 Hz H-30), 7.17 (1H, t, J = 8.0 Hz, H-5), 7.33
(1H, t, J = 8.0 Hz, H-6), 7.57 (1H, s, H-60), 7.58 (1H,
d, J = 8.0 Hz, H-4), 7.67 (1H, d, J = 8.9 Hz, H-40),
7.79 (1H, d, J = 8.0 Hz, H-7), 7.94 (1H, s, H-3). 13C
NMR (DMSO-d6) d 94.0 (s, C-1), 113.9 (s, CN), 114.9
(s, C-50), 115.5 (s, CF3), 115.9 (d, C-3), 117.6 (d, C-7),
120.2 (s, C-3a), 121.3 (d, C-30), 122.5 (d, C-4), 124.2
(s, C-10), 125.6 (d, C-5), 125.7 (d, C-6), 127.5 (d, C-40),
127.6 131.3 (s, C-7a), 147.8 (s, C-20). Anal. Calcd for
C16H10F3N3 (MW 301.27): C, 63.79; H, 3.35; N,
18.92%. Found: C, 63.84; H, 3.21; N, 18.87%.
3.2.4. 11-Cyano-3-methoxyisoindolo[2,1-c]benzo[1,2,4]tri-
azine (4d). Yield 87%; mp 256–257 ꢁC. IR 2198 (CN)
1
cmꢀ1. H NMR (DMSO-d6) d 4.08 (3H, s, CH3), 7.60–
7.66 (2H, m, H-4 and H-10), 7.77 (1H, t, J = 8.0 Hz,
H-8), 7.78 (1H, t, J = 8.0 Hz, H-9), 7.98 (1H, d,
J = 7.0 Hz, H-2), 8.67 (1H, d, J = 8.0 Hz, H-7), 9.01
(1H, d, J = 7.0 Hz, H-1). 13C NMR (DMSO-d6) d 56.1
(q), 93.3 (s), 109.8 (d), 111.5 (s), 116.5 (d), 117.2 (d),
119.9 (s), 120.4 (d), 123.9(d), 126.0 (d), 126.6 (s), 129.5
(d), 130.6 (s), 132.0 (s), 135.9 (s), 145.5 (s). Anal. Calcd
for C16H10N4O (MW 274.28): C, 70.07; H, 3.67; N,
20.43%. Found: C, 69.91; H, 3.74; N, 20.57%.
3.2. General method for the synthesis of isoindolo[2,1-c]-
benzo[1,2,4]triazines (4a–j)
To a solution of appropriate 1-cyano-2-(20-amino-
phenyl)-isoindole 3a–j (3 mmol) in 5 ml of acetic acid,
a solution of sodium nitrite 3 mmol (210 mg) in water
(1 ml) was added. The mixture was stirred at room
temperature for 24 h then poured onto crushed ice.
The precipitate was filtered, air dried, and purified
by flash chromatography with dichloromethane as
eluent to give:
3.2.5. 11-Cyano-2,3-dimethylisoindolo[2,1-c]benzo[1,2,4]-
triazine (4e). Yield 96%; mp 269–270 ꢁC. IR 2195 (CN)
cmꢀ1 1H NMR (CDCl3) d 2.57 (3H, s, CH3), 2.63
.
(3H, s, CH3), 7.64 (1H, t, J = 7.8 Hz, H-8), 7.75 (1H,
t, J = 7.8 Hz, H-9), 8.00 (1H, d, J = 7.8 Hz, H-7), 8.40
(1H, s, H-1), 8.72 (1H, d, J = 7.8 Hz, H-10), 8.87 (1H,
s, H-4). 13C NMR (CDCl3) d 20.1 (q), 21.3 (q), 87.6
(s), 115.0 (d), 115.3 (s), 117.4 (d), 120.0 (s), 120.8 (d),
122.8 (s), 125.9 (d), 129.5 (d), 130.6 (d), 132.6 (s),
134.7 (s), 139.0 (s), 139.1 (s), 144.6 (s). Anal. Calcd for
C17H12N4 (MW 272.31): C, 74.98; H, 4.44; N, 20.57%.
Found: C, 74.85; H, 4.61; N, 20.40%.
3.2.1. 11-Cyano-isoindolo[2,1-c]benzo[1,2,4]triazine (4a).
Yield 84%; mp 266–267 ꢁC. IR 2200 (CN) cmꢀ1
.
1H
NMR (CDCl3) d 7.66 (1H, t, J = 7.4 Hz, H-8), 7.76
(1H, t, J = 7.4 Hz, H-9), 7.89 (1H, t, J = 8.0 Hz, H-2),
7.93 (1H, t, J = 8.0 Hz, H-3), 7.98 (1H, d, J = 7.4 Hz,
H-7), 8.67 (1H, d, J = 8.0 Hz, H-1), 8.69 (1H, d,
J = 7.4 Hz, H-10), 9.11 (1H, d, J = 8.0 Hz, H-4). 13C
NMR (CDCl3) d 88.6 (s), 114.9 (s), 115.4 (d), 117.4
(d), 120.2 (s), 120.7 (d), 124.4 (s), 126.4 (d), 128.8 (d),
129.7 (d), 131.2 (d), 132.4 (s), 132.9 (d), 134.6 (s),
139.8 (s). Anal. Calcd for C15H8N4 (MW 244.26): C,
73.76; H, 3.30; N, 22.94%. Found: C, 73.67; H, 3.47;
N, 23.04%.
3.2.6. 11-Cyano-2,3-dichloroisoindolo[2,1-c]benzo[1,2,4]-
triazine (4f). Yield 87%; mp 241–242 ꢁC. IR 2200 (CN)
cmꢀ1 1H NMR (CDCl3) d 7.73 (1H, t, J = 8.0 Hz,
.
H-8), 7.82 (1H, t, J = 8.0 Hz, H-9), 8.54 (1H, s, H-1),
8.74 (1H, d, J = 8.0 Hz, H-7), 8.76 (1H, s, H-4); 9.38
(1H, d, J = 8.0 Hz, H-10). 13C NMR (CDCl3) d 88.9
(s), 114.9 (s), 117.0 (s), 117.7 (s), 118.8 (s), 120.9 (d),
125.2 (d), 127.3 (d), 129.7 (s), 130.5 (d), 131.8 (d),
132.2 (d), 132.7 (s), 134.2 (s), 163.6 (s). Anal. Calcd
for C15H6Cl2N4 (MW 313.15): C, 57.53; H, 1.93; N,
22.64%. Found: C, 57.36; H, 2.05;N, 22.75%.
3.2.2. 11-Cyano-1-methylisoindolo[2,1-c]benzo[1,2,4]tri-
azine (4b). Yield 90%; mp 217–218 ꢁC. IR 2201 (CN)
cmꢀ1
.
1H NMR (CDCl3) d 3.14 (3H, s, CH3), 7.68
3.2.7. 3-Chloro-11-cyanoisoindolo[2,1-c]benzo[1,2,4]tri-
azine (4g). Yield 85%; mp 267–268 ꢁC. IR 2200 (CN)
(1H, t, J = 8.0 Hz, H-8), 7.72–7.87 (3H, m, H-9, H-3
and H-2), 8.03 (1H, d, J = 8.0 Hz, H-7), 8.49 (1H, dd,
J = 6.9, 2.4 Hz, H-4), 8.73 (1H, d, J = 8.0 Hz, H-10).
13C NMR (CDCl3) d 23.2 (q), 94.1 (s), 115.3 (s), 117.7
(d), 120.0 (s), 120.7 (d), 124.5 (s), 126.7 (d), 128.3 (d),
1
cmꢀ1. H NMR (DMSO-d6) d 7.78 (1H, t, J = 7.6 Hz,
H-8), 7.89 (1H, t, J = 7.6 Hz, H-9), 8.11 (1H, d,
J = 8.4 Hz, H-2), 8.24 (1H, d, J = 7.6 Hz, H-7), 8.71
(1H, d, J = 8.4 Hz, H-1), 8.79 (1H, s, H-4), 9.00 (1H,

P. Diana et al. / Bioorg. Med. Chem. 15 (2007) 343–349
349
d, J = 7.6 Hz, H-10). 13C NMR (DMSO-d6) d 92.7 (s),
Supplementary data
114.7 (s), 117.3 (d), 117.6 (d), 119.5 (s), 120.3 (d),
123.4 (s), 126.6 (d), 129.0 (d), 129.9 (d), 131.8 (s),
133.1 (d), 140.0 (s), 148.1 (s), 158.9 (s). Anal. Calcd
for C15H7ClN4 (MW 278.70): C, 64.64; H, 2.53; N,
20.10%. Found: C, 64.49; H, 2.77; N, 19.99%.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2006.09.054.
3.2.8. 2-Chloro-11-cyanoisoindolo[2,1-c]benzo[1,2,4]tri-
azine (4h). Yield 84%; mp 222–223 ꢁC. IR 2200 (CN)
References and notes
cmꢀ1 1H NMR (CDCl3) d 7.68–8.01 (4H, m, H-3,
.
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H-4, H-8 and H-9), 8.61 (1H, s, H-1), 8.71 (1H, d,
J = 7.4 Hz, H-7), 9.05 (1H, d, J = 7.4 Hz, H-10). 13C
NMR (CDCl3) d 92.8 (s), 115.2 (s), 120.8 (d · 2),
122.9 (s), 125.6 (d), 128.2 (s), 130.2 (d · 2), 131.6 (s),
132.5 (d), 133.0 (d), 134.9 (s), 137.0 (s), 153.7 (s).
Anal. Calcd for C15H7ClN4 (MW 278.70): C, 64.64;
H, 2.53; N, 20.10%. Found: C, 64.70; H, 2.33; N,
20.02%.
3.2.9. 11-Cyano-3-trifluoromethylisoindolo[2,1-c]benzo-
[1,2,4] triazine (4i). Yield 95%; mp 170–171 ꢁC. IR
2200 (CN) cmꢀ1 1H NMR (CDCl3) d 7.70 (1H, t,
.
J = 8.3 Hz, H-8), 7.80 (1H, t, J = 8.3 Hz, H-9), 8.01
(1H, d, J = 8.0 Hz, H-1), 8.60 (1H, d, J = 8.3 Hz,
H-10), 8.74 (1H, d, J = 8.3 Hz, H-7), 8.77 (1H, d,
J = 8.0 Hz, H-2), 9.38 (1H, s, H-4). 13C NMR (CDCl3)
d 90.0 (s), 113.5 (d), 114.2 (s), 115.4 (s), 116.6 (s),
117.7 (d), 120.7 (d), 124.1 (s), 125.1 (d), 125.5 (s),
127.3 (d), 130.3 (d), 131.2 (s), 132.2 (d), 134.6 (s),
140.32 (s). Anal. Calcd for C16H7F3N4 (MW 312.25):
C, 61.54; H, 2.26; N, 17.94%. Found: C, 61.49; H,
2.41; N, 17.81%.
9. Nantka-Namirski, P.; Rykowski, A. Arch. Immun. et
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10. Stottmeister, U.; Schoenfelder, M. W. H.; Sicker, D.;
Andersch, J. U.S. Patent WO2001046162, 2001; Chem.
Abstr. 2001, 135, 207317.
3.2.10. 11-Cyano-2-trifluoromethylisoindolo[2,1-c]benzo-
[1,2,4]triazine (4j). Yield 86%; mp 135–137 ꢁC. IR
2200 (CN) cmꢀ1 1H NMR (CDCl3) d 7.74 (1H, t,
.
11. Borzilleri, R. M.; Cai, Z.; Ellis, C.; Fargnoli, J.; Fura, A.;
Gerhardt, T.; Goyal, B.; Hunt, J. T.; Mortillo, S.; Qian,
L.; Tokarski, J.; Vyas, V.; Wautlet, B.; Zheng, X.; Bhide,
R. S. Bioorg. Med. Chem. Lett. 2005, 15, 1429.
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Fargnoli, J.; Fink, B.; Han, W.; Mortillo, S.; Vite, G.;
Wautlet, B.; Wong, T.; Yu, C.; Zheng, X.; Bhide, R. S.
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J = 8.0 Hz, H-8), 7.83 (1H, t, J = 8.0 Hz, H-9), 8.06
(1H, d, J = 8.0 Hz, H-7), 8.16 (1H, d, J = 9.0 Hz, H-
3), 8.79 (1H, d, J = 8.0 Hz, H-10); 8.95 (1H, s, H-1),
9.27 (1H, d, J = 9.0 Hz, H-4). 13C NMR (CDCl3) d
92.4 (s), 114.3 (s), 114.5 (s), 116.7 (d), 117.7 (d),
120.3 (s), 120.6 (s), 120.9 (d), 125.7 (s), 126.3 (s),
127.3 (d), 128.6 (d), 128.8 (d), 130.4 (d), 132.8 (s),
138.9 (s). Anal. Calcd for C16H7F3N4 (MW 312.25):
C, 61.54; H, 2.26; N, 17.94%. Found: C, 61.37; H,
2.45; N, 18.04%.
Acknowledgments
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This work was financially supported by Ministero
`
dell’Istruzione dell’Universita e della Ricerca. Prelimin-
ary accounts of this work were presented at the XIX
International Congress of Heterocyclic Chemistry, Fort
Collins (USA), August 2003. We Thank the National
Cancer Institute (Bethesda, MD) and especially Dr V.
L. Narayanan and his team for the antitumor tests
reported in this paper.
19. Rambow, A. A.; Shoemaker, R. H.; Sausville, E. A.;
Covell, D. G. J. Med. Chem. 2002, 45, 818.