Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine with potent antitumor and antimicrobial activity

Article abstract of DOI:10.1021/jm9806087

Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine 5 were synthesized by diazotization of substituted 2-(2-aminophenyl)indoles followed by an intramolecular coupling reaction of the diazonium group with the indole nitrogen. To obtain the

Full text of DOI:10.1021/jm9806087

J . Med. Chem. 1999, 42, 2561-2568
2561
Der iva tives of th e New Rin g System In d olo[1,2-c]ben zo[1,2,3]tr ia zin e w ith
P oten t An titu m or a n d An tim icr obia l Activity
Girolamo Cirrincione,* Anna Maria Almerico, Paola Barraja, Patrizia Diana, Antonino Lauria,
Alessandra Passannanti, Chiara Musiu,§ Alessandra Pani,§ Paola Murtas,§ Carla Minnei,§
M. Elena Marongiu,§ and Paolo La Colla*,§
Istituto Farmacochimico, Universita` degli Studi, Via Archirafi 32, 90123 Palermo, Italy, and Dipartimento di Biologia
Sperimentale, Sezione di Microbiologia, Universita` degli Studi, V.le Regina Margherita 45, 09124 Cagliari, Italy
Received October 29, 1998
Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine 5 were synthesized by
diazotization of substituted 2-(2-aminophenyl)indoles followed by an intramolecular coupling
reaction of the diazonium group with the indole nitrogen. To obtain the indolobenzotriazine
system it was necessary to protect the 3 position of the indole nucleus to avoid cyclization into
the indolo[3,2-c]cinnoline system 4. Indolobenzotriazines 5a -g were evaluated in vitro for
antitumor activity against a panel of leukemia-, lymphoma-, carcinoma-, and neuroblastoma-
derived cell lines. Some compounds inhibited the proliferation of T and B cell lines at
submicromolar concentrations, whereas their activity against solid tumor cell lines was in the
micromolar range. When evaluated for their antifungal potential 5a ,d inhibited some of the
fungi tested, although at concentrations very close to those inhibiting the proliferation of human
cells. On the contrary, all indolobenzotriazines proved fairly potent and selective inhibitors of
Streptococcus and Staphylococcus. In particular 5b,c,g were up to 80 times more potent than
the reference drug streptomycin and inhibited the growth of the above Gram-positive bacteria
at concentrations far lower than those cytotoxic for animal cells.
In tr od u ction
As a result of our interest in heterocycles annelated
with either pyrrole or indole rings as potential interca-
lating agents, we have recently reported a new synthesis
of some derivatives of the indolo[3,2-c]cinnoline ring
system 4 and have presented evidence for their in vitro
antitumor activity.6 We now describe the synthesis of
derivatives of the new ring system indolo[1,2-c]benzo-
[1,2,3]triazine 5 and report on the in vitro evaluation
of their biological activity.
Polycondensed nitrogen heterocycles having a planar
structure can be effective pharmacophore moieties of
drugs endowed with antineoplastic activity because they
can intercalate into double-stranded DNA. Acridine and
phenanthridine derivatives of type 1 and 2 are well-
known compounds possessing such a property whose
principal driving forces are stacking and charge-transfer
interactions as well as hydrogen bonding and electro-
static forces.1 In particular, it has been demonstrated
that the biological activity of 9-acridinylmethanesulfo-
nanilide derivatives 1 (R ) NHSO2Me) correlates with
their DNA association constants, the more active com-
pounds being those that more tightly bind to DNA.2
Ethidium derivatives 2 (R ) Et) and anthracycline
antitumor antibiotics 3 also tightly bind to DNA and
show a strong specificity for GC residues.3 Doxorubicin
(3, R ) OH), in particular, interacts with the 2-amino
group of guanine.4 In addition compounds of type 1 and
3 target the topoisomerase II.5
Ch em istr y
Compounds 6, the key intermediates for the synthesis
of derivatives of the indolobenzotriazine ring system,
were conveniently prepared by an intramolecular Wittig
reaction of suitable ylides, which were easily obtained,
in turn, from 2-aminobenzyltriphenylphosphonium salts
and 2-nitrobenzoyl chlorides (Scheme 1). As recently
pointed out,6 for the preparation of derivative 7f we
preferred the classical Fisher reaction due to its higher
yields. This latter compound was therefore synthesized
starting from 2-aminoacetophenone and 4-chlorophe-
nylhydrazine.
To obtain the indolobenzotriazine system it was
necessary to protect the 3 position of the indole ring in
order to avoid the ring closure leading to the indolocin-
noline system during the diazotization reaction. Since
halogenation with N-halosuccinimides in dimethylfor-
mamide gave excellent yields in monohalopyrroles,7 we
chose this reaction to protect the 3 position of the indole
nucleus. Thus, compound 6a was brominated with NBS
in dimethylformamide to give the corresponding 3-bromo-
2-(2-nitrophenyl)indole (8) in quantitative yield. Reduc-
tion of the nitro group with iron in acetic acid led to
the corresponding amino derivative 9 in 85% yield.
Diazotization of the amino derivative 9 with sodium
nitrite in acetic acid failed to give the expected indolo-
* To whom correspondence should be addressed.
§ Universita` degli Studi, Cagliari.
10.1021/jm9806087 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/24/1999

2562 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Cirrincione et al.
Ta ble 1. Antitumor Activity of Indolo[1,2-c]benzo[1,2,3]tria-
zines
Sch em e 1
IC50 (µM)a
cell line
5a
12
4.4 2.7
7.4 7.0
7.0 5.0
2.3 2.0
1.4 1.7
5b
5c
5d 5e 5f
5g
Doxob
leukemia/lymphoma
L1210
7.4 13
2.4 1.7 0.7
3.7 0.8 0.8 0.2
5.0 2.8 0.7 0.4
7.2 1.8 0.5 0.08
1.3 1.1 0.5 0.2
1.1 0.9 1.3 0.5
9.2 0.23
12
11
8.2 0.1
6.6 0.03
1.2 0.02
Wil2-NS
CCRF-SB
CCRF-CEM
MOLT-4
MT-4
0.02
0.01
carcinoma
CHO-K1
HT-29
45
31
24
>50
>50
9.3 6.5 1.6
42
18
16
0.4
0.05
0.2
0.4
0.02
50
10
12
35
3.1 1.3
HeLa
ACHN
5637
5.4 11
11 14
6.0 2.6 1.9 0.3
9.2 9.5 2.7
7.6 8.7 2.6 >50
4.2 2.1
29
neuroblastoma
IMR-32
3.3 2.3
2.3 2.2 2.8 0.8
12
0.01
a
Compound concentration required to reduce cell multiplication
by 50% under conditions allowing untreated cells to undergo at
least three consecutive rounds of multiplication. Values are the
mean of at least three separate experiments. Variability among
triplicate samples was less than 22%. L1210, mouse leukemia;
Wil2-NS, human splenic B lymphoblastoid cells; CCRF-SB, human
acute B lymphoblastic leukemia; CCRF-CEM and MOLT-4, human
acute T lymphoblastic leukemia; MT-4, human CD4+ T cells
expressing the TAT gene of HTLV-1; CHO-K1, Chinese hamster
ovary; HT-29, human colon adenocarcinoma; HeLa, human cervix
carcinoma; ACHN, human renal adenocarcinoma; 5637, human
b
bladder carcinoma; IMR-32, human neuroblastoma. Doxo, doxo-
rubicin (3, R ) OH).
expected indolo[2,1-c]benzo[1,2,3]triazines 5a -f in very
high yields (80-100%). The indolobenzotriazine 5g was
obtained by direct nitration with potassium nitrate in
sulfuric acid of the unsubstituted indolobenzotriazine
5a (yield 75%).
Biology
Title compounds were evaluated in vitro for antitu-
mor, antiviral, antifungal, and antibacterial activity.
An titu m or Activity. The antitumor activity of in-
dolobenzotriazines 5a -g was evaluated against a panel
of leukemia-, lymphoma-, carcinoma-, and neuroblas-
toma-derived cell lines in comparison with the reference
drug doxorubicin (Doxo) (Table 1). Although less potent
than Doxo, test compounds inhibited the proliferation
of B and T leukemic cell lines at submicromolar/
micromolar concentrations. The most potent indoloben-
zotriazine derivative was 5f (IC50 range ) 0.08-0.7 µM)
followed, in order of decreasing potency, by 5e,d (IC50
range ) 0.5-2.8 µM) and by 5b,c,a ,g (IC50 range ) 1.1-
13 µM). The antiproliferative activity of test compounds
against cell lines derived from solid tumors was again
lower than that of Doxo, but still significant, in par-
ticular in the case of 5f (IC50 range ) 0.3-2.7 µM) and
5e (IC50 range ) 1.9-9.5 µM).
An tip r olifer a tive Activity a ga in st Dr u g-Resis-
ta n t Tu m or Cell Lin es. Drug resistance is a relevant
therapeutic problem caused by the emergence of tumor
cells possessing different mechanisms which confer
resistance to a variety of anticancer drugs. Among the
more common mechanisms are those related to the
overexpression of glycoproteins capable to mediate the
efflux of different drugs such as Doxo, vincristine, and
etoposide or to altered contents of target enzymes
(topoisomerases I and II). Therefore, it was interesting
to investigate whether 5f was inhibitory to drug-
[1,2-c]benzo[1,2,3]triazine, as a result of the intramo-
lecular coupling reaction with the indole nitrogen, but
afforded the indolo[3,2-b]cinnoline 4, through an aza-
dehalogenation which can be envisaged as a J app-
Klingemann reaction with extrusion of bromonium ion,
as previously observed in the pyrrole series.8 Therefore,
to protect the 3 position of the indole ring, it was
necessary to undertake a different approach. To this
end, the nitroso group seemed the most suitable one for
protecting the 3 position, due to the fact that it makes
more acidic the imino proton and more reactive the ring
nitrogen. Moreover, even if tautomerism to the isoni-
troso form (which could originate a 3H-indole structure)
could take place, the indole nitrogen (now a pyridine-
like nitrogen) was deemed capable to undergo an
intramolecular coupling reaction with the diazonium
group.
Thus compounds 6a ,c,e were reduced to the corre-
sponding amino derivatives 7 with hydrogen and Pal-
ladium on charcoal, whereas compounds 6b,d were
reacted with iron and acetic acid to avoid dehalogena-
tion. Compounds 7 were acetylated nearly quantita-
tively (95-99%) to give the corresponding acetylamino
compounds 10 which, in turn, were nitrosated at room
temperature with sodium nitrite in acetic acid to give
3-nitrosoindoles 11 in excellent yields (93-97%). Re-
moval of the protecting group gave the amino com-
pounds 12a -f. Subsequent diazotization afforded the

Derivatives of Indolo[1,2-c]benzo[1,2,3]triazine
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2563
Ta ble 2. Effect of 5f on the Proliferation of Wild-Type and Drug-Resistant KB Cells
IC50 (µM)a
compd
KBwt
KBMDR
KBV20C
KB7D
KBCamp
5f
1.8 ( 0.28
1.1 ( 0.17
0.50 ( 0.14
0.35 ( 0.05
26.2 ( 9.6
0.07 ( 0.14
2.1 ( 0.3
1.7 ( 0.4
1.9 ( 0.25
0.06 ( 0.007
0.005 ( 0.001
1.30
doxorubicin
vincristine
etoposide
0.04 ( 0.015
0.001 ( 0.0005
1.45 ( 0.9
0.4 ( 0.08
0.05 ( 0.018
0.65 ( 0.2
0.035 ( 0.01
>50
>50
camptothecin
0.04 ( 0.005
0.03 ( 0.003
0.035 ( 0.005
1.04
a
Compound concentration required to reduce cell multiplication by 50% under conditions allowing untreated controls to undergo at
least three consecutive rounds of multiplication. Data represent mean values ((SD) for three independent determinations.
Ta ble 3. Cytotoxicity of Indolo[1,2-c]benzo[1,2,3]triazines for
Normal Human Lymphocytes
also proved equally cytotoxic for proliferating and rest-
ing normal PBL and leukemic cell lines.
CC50 (µM)a
An tivir a l Activity. When indolobenzotriazines were
tested against human immunodeficency virus type-1
(HIV-1) in de novo infected cells, they resulted ineffec-
tive at concentrations close to those cytotoxic for unin-
fected MT-4 cells (results not shown). Title compounds
were also inactive against other DNA and RNA viruses
such as herpes simplex type-1, coxsackie, and vesicular
stomatitis viruses (results not shown).
An tim ycotic Activity. Test compounds were then
evaluated for antifungal activity in comparison with
miconazole as reference drug (Table 4). Compounds
5c,e,f were totally devoid of antimycotic activity up to
200 µM, whereas the other derivatives were inhibitory
to some of the fungi tested. The most sensitive species
was Cryptococcus neoformans, one of the agents of
opportunistic infections in AIDS patients, against which
5a ,b,d showed potencies comparable to that of micona-
zole. However, the fact that test compounds inhibited
the fungal growth at concentrations that were in the
same order of magnitude of those cytotoxic for human
cells makes 5a ,b,d ,g antifungal agents endowed with
a very poor selectivity index (ratio CC50/MIC). It is worth
noting, however, that indolobenzotriazines 5e,f, which
showed the most potent antitumor activity in vitro, like
Doxo, lacked antimycotic activity.
An tiba cter ia l Activity. The antibacterial potential
of indolobenzotriazines was evaluated against repre-
sentatives of Gram-negative, Gram-positive, and My-
cobacteria (Table 5) together with that of streptomycin,
used as reference drug. In general, title compounds were
inactive or only marginally active against Salmonella,
Shigella, M. fortuitum, and M. smegmatis. By contrast,
all compounds were inhibitory to Streptococcus and
Staphylococcus, against which some derivatives (5b,c,g)
proved both potent and selective, being active at con-
centrations up to 80 times lower than those effective
against human cells. Noteworthy 5b,c,g were also up
to 80-fold more potent than streptomycin against Strep-
tococcus and Staphylococcus and showed the same
potency against penicillin-resistant strains of S. aureus
(data not shown).
SAR Stu d ies. SAR studies indicate that maximum
antitumor activity in vitro correlates with the presence
of either a chlorine atom at position 10 (5f) or a methyl
group at position 2 (5e), whereas the absence of sub-
stituents at positions 10 and 2 (5a ), the substitution of
a chlorine atom for a methoxy (5c) or nitro (5g) group
at position 10, and the substitution of a methyl group
for a chlorine atom (5b) at position 2 significantly
decrease the activity. Moving the chlorine atom from
position 2 to position 3 (5d ) partially restores the
antitumor activity in vitro.
cell line
5e
5f
Doxob
c
PBLPHA
0.5 ((0.19)
0.6 ((0.25)
0.9
0.2 ((0.08)
0.9 ((0.25)
0.35
0.04 ((0.01)
0.09 ((0.03)
0.06
d
PBLresting
leukemiae
a
Compound concentration required to reduce cell multiplication
by 50%. Values are the mean ((SD) for three separate experi-
b
ments. Doxo, doxorubicin (3, R ) OH). c PBL were stimulated
with PHA and then resuspended in IL2-containing medium in the
d
presence of drugs. PBL were treated with test drugs for 3 days,
then stimulated with PHA, and resuspended in drug-free medium.
e Data are the mean IC50 values obtained with lymphoblastoid cell
lines.
resistant cell lines. Thus, we evaluated its activity
against the following KB subclones (Table 2):
1. KBMDR, obtained by infection of KBwt with a
retroviral vector carrying the human mdr-1 gene and
maintained under continuous treatment with Doxo.
These cells express a membrane glycoprotein (Pgp)
which is responsible for the efflux of many unrelated
drugs (hence the name MDR, or multidrug resistance).
2. KBV20C, selected under continuous treatment with
vincristine. These cells possess an MDR phenotype
related to the overexpression of the mdr-1 gene and, like
the KB7D subclone, mediate the efflux of Doxo, vincris-
tine, and etoposide but are sensitive to camptothecin.
3. KBEtop, selected under continuous treatment with
etoposide, a topoisomerase II inhibitor largely used in
the clinic. In this case resistance is due to overexpres-
sion of the mrp gene, which codes for a membrane
glycoprotein (MRP) capable of mediating the efflux of
etoposide, Doxo, and vincristine. These cells also express
altered levels of topoisomerase II. 4. KBCamp, selected
under continuous treatment with camptothecin, a to-
poisomerase I inhibitor. These cells are characterized
by a low expression of topoisomerase I and increased
expression of topoisomerase II.
Interestingly, 5f proved fully inhibitory to all these
resistant cell lines, thus suggesting that it neither is
subject to the pump mediating the efflux of many
antitumor drugs nor interferes with the DNA synthesis
by affecting the topoisomerase I and II-catalyzed step.
Cytotoxicity for Nor m a l Cells. To obtain more
insights into the cytotoxic potential of test compounds
for normal human cells, the two indolobenzotriazines
endowed with the most potent antiproliferative activity
were assayed in vitro against peripheral blood lympho-
cytes (PBL) from healthy donors. Doxo was run as
reference drug (Table 3). 5e,f proved cytotoxic for both
PHA-stimulated and resting PBL at concentrations close
to those active against lymphoblastoid cell lines. In this
respect, however, they did not differ from Doxo, which

2564 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Ta ble 4. Antifungal Activity of Indolo[1,2-c]benzo[1,2,3]triazines
Cirrincione et al.
MIC (µM)a
species
5a
19
19
19
3.1
9.4
19
5b
5c
5d
12
25
3.0
1.6
50
6.2
5e
5f
5g
66
22
66
7.4
66
Doxob
Micc
C. albicans
150
150
19
0.8
9.4
9.4
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
7.5
0.9
7.5
0.9
0.9
1.9
C. paratropicalis
C. parapsilosis
C. neoformans
T. mentagrophytes
A. fumigatus
>200
a
Minimum inhibitory concentration. Values are the mean of at least three separate experiments. Variability among triplicate samples
was less than 15%. Doxo, doxorubicin (3, R ) OH). c Mic, miconazole.
b
Ta ble 5. Antibacterial Activity of Indolo[1,2-c]benzo[1,2,3]triazines
MIC (µM)a
5d
species
5a
5b
5c
5e
5f
5g
66
Strepb
Salmonella
Shigella
25
25
0.8
0.8
>5
2.5
1.2
4.6
7.4
>200
>200
0.1
0.05
>5
>200
>200
0.1
0.2
>5
>5
2.5
>200
>200
>200
>200
>200
>200
8.6
2.1
2.1
4.2
4.0
5.0
7.4
5.4
2.9
22
Streptococcus
Staphylococcus
M. fortuitum
M. smegmatis
MAC
0.4
0.4
>5
>5
0.6
0.8
0.4
>5
>5
5
9.3
9.2
0.8
0.4
>5
>5
0.6
4.7
g10
0.09
0.09
>5
ND
ND
ND
ND
4.2
2.5
1.8
1.6
M. tuberculosis (ATCC)
M. tuberculosis (clinical isolate 1104)
5.3
4.5
4.4
5.9
a
Minimum inhibitory concentration. Values are the mean of at least three separate experiments. Variability among triplicate samples
b
was less than 18%. Strep, streptomycin.
(TMS as internal reference) at 200 and 50.3 MHz, respectively,
using a Bruker AC series 200-MHz spectrometer. Column
chromatography was performed with Merck silica gel, 230-
400 mesh ASTM. Elemental analyses were within (0.4% of
the theoretical values.
It is worth noting that maximum potency of antifun-
gal activity correlates with the absence of substituents
(5a ) or the presence of a chlorine atom at position 3 (5d ).
5d is the sole compound capable of potently inhibiting
the proliferation of both animal and fungal cells, whereas
the derivatives endowed with the highest antitumor
activity in vitro (5e,f) are totally ineffective on fungal
growth. On the other hand, potent and selective anti-
bacterial activity correlates with the presence of a
chlorine atom at position 2 (5b) or with a methoxy (5c)
or nitro (5g) group at position 10. Interestingly, since
both 5c,g are ineffective as antifungal agents and are
poorly cytotoxic against human cell lines, they can be
considered as antibacterial agents endowed with a
certain degree of selectivity.
Syn th esis of Su bstitu ted 2-(2-Nitr oa r yl)in d oles 6a -e.
Compounds 6a -e were prepared by intramolecular Wittig
reaction of 2-aroylamidobenzyltriphenylphosphonium salts
according to the procedure previously described for 6a -d .6
2-(5-Methyl-2-nitrophenyl)indole (6e) was purified by column
chromatography (eluant: toluene-diethyl ether, 9:1): yield
75%; mp 76 °C; IR 3400 (NH) cm-1 1H NMR δ 2.45 (3H, s,
;
CH3), 6.53 (1H, d, J ) 1.5 Hz, H-3), 7.03 (1H, dt, J ) 7.3 Hz,
J ) 1.0 Hz, H-5), 7.15 (1H, dt, J ) 7.3 Hz, J ) 1.0 Hz, H-6),
7.42 (2H, dd, J ) 7.3 Hz, J ) 1.0 Hz, H-7 and H-4′), 7.56 (1H,
dd, J ) 7.3 Hz, J ) 1.0 Hz, H-4), 7.61 (1H, d, J ) 1.0 Hz,
H-6′), 7.90 (1H, d, J ) 7.3 Hz, H-3′), 10.06 (1H, bs, NH); 13C
NMR δ 20.8 (q), 101.4 (d), 111.5 (d), 119.4 (d), 120.4 (d), 122.0
(d), 124.3 (d), 126.5 (s), 128.1 (s), 129.3 (d), 131.8 (d), 132.9
(s), 137.0 (s), 138.4 (s), 143.3 (s). Anal. (C15H12N2O2) C, H, N.
Syn th esis of Su bstitu ted 2-(2-Am in oa r yl)in d oles 7a -
f. P r oced u r e A: Compounds 7a ,c,e were prepared by cata-
lytic reduction of the corresponding nitro derivatives 6 over
10% Pd on charcoal in a Parr apparatus according to the
procedure previously described for 7a ,c.6 2-(2-Amino-5-meth-
ylphenyl)indole (7e) was recrystallized from ethanol: yield
86%; mp 199-200 °C; IR 3377 and 3302 (NH2), 3229 (NH)
cm-1; 1H NMR δ 2.23 (3H, s, CH3), 4.99 (2H, s, NH2), 6.66 (1H,
d, J ) 2.0 Hz, H-3), 6.74 (1H, d, J ) 7.8 Hz, H-3′), 6.89 (1H,
dd, J ) 7.8 Hz, J ) 2.0 Hz, H-4′), 7.00 (1H, dt, J ) 6.8 Hz, J
) 2.0 Hz, H-5), 7.08 (1H, dt, J ) 6.8 Hz, J ) 2.0 Hz, H-6),
7.21 (1H, d, J ) 2.0 Hz, H-6′), 7.40 (1H, dd, J ) 6.8 Hz, J )
2.0 Hz, H-7), 7.52 (1H, dd, J ) 6.8 Hz, J ) 2.0 Hz, H-4), 10.36
(1H, bs, NH); 13C NMR δ 20.2 (q), 99.9 (d), 111.1 (d), 116.1 (d),
117.1 (s), 119.0 (d), 119.7 (d), 121.0 (d), 124.9 (s), 128.6 (2×s),
129.0 (2×d), 136.2 (s), 143.2 (s). Anal. (C15H14N2) C, H, N.
P r oced u r e B: Compounds 7b,d , bearing a halogen, were
prepared by reduction with iron in acetic acid of the corre-
sponding nitro compounds 6b,d , according to the procedure
previously described.6
Con clu sion s
The data reported herein indicate that indolobenzo-
triazines are endowed with a broad spectrum of biologi-
cal activities, ranging from antitumor to antifungal and
antibacterial. Interestingly, their spectrum and potency
can be modulated by introducing certain substituents
at different positions of the benzene moieties, and this
allows to obtain compounds which show activities
specific for different cell types. Therefore, whereas
compounds 5e,f are endowed with potent antitumor
activity, compounds 5a ,d are the most potent antifungal
agents, and derivatives 5b,c,g are the most potent
against bacteria.
On the basis of these results, experiments aimed at
defining the targets and the mechanisms of the inhibi-
tory effect against eukaryotic and prokaryotic cells are
in progress.
Exp er im en ta l Section
P r oced u r e C: 2-(2-Aminophenyl)-5-chloroindole (7f) was
prepared by a Fisher indole synthesis refluxing for 3 h a
mixture of 2′-aminoacetophenone hydrochloride (4.05 g, 30
mmol), 4-chlorophenylhydrazine hydrochloride (6.37 g, 30
mmol), and sodium acetate (3.2 g, 40.0 mmol) in ethanol (34
(A) Ch em istr y. All melting points were taken on a Buchi-
Tottoli capillary apparatus and are uncorrected; IR spectra
were determined with a J asco FT/IR 5300 spectrophotometer;
1H and 13C NMR spectra were measured in DMSO-d6 solutions

Derivatives of Indolo[1,2-c]benzo[1,2,3]triazine
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2565
mL) and acetic acid (12 mL). After cooling the reaction mixture
was poured onto crushed ice, and the hydrazone precipitated
was filtered off, air-dried, and employed without further
purification. Thus a mixture of the hydrazone (7.78 g, 30 mmol)
and polyphosphoric acid (40 g) was heated at 130 °C for 10
min. After cooling at 90 °C, cold water (300 mL) was added
and the suspension was neutralized with aqueous sodium
hydroxide (5%). The solid was filtered off, air-dried, and
recrystallized from ethanol: yield 80%; mp 148-149 °C; IR
3381 and 3308 (NH2), 3225 (NH) cm-1; 1H NMR δ 5.23 (2H, s,
NH2), 6.68 (1H, dt, J ) 7.4 Hz, J ) 1.2 Hz, H-5′), 6.69 (1H, d,
J ) 1.8 Hz, H-3), 6.85 (1H, dd, J ) 7.4 Hz, J ) 1.2 Hz, H-3′),
7.07 (1H, dd, J ) 8.5 Hz, J ) 1.8 Hz, H-6), 7.08 (1H, dt, J )
7.4 Hz, J ) 1.2 Hz, H-4′), 7.35 (1H, dd, J ) 7.4 Hz, J ) 1.2
Hz, H-6′), 7.40 (1H, d, J ) 8.5 Hz, H-7), 7.56 (1H, d, J ) 1.8
Hz, H-4), 11.46 (1H, bs, NH); 13C NMR δ 99.7 (d), 112.5 (d),
115.9 (d), 116.5 (s), 116.6 (d), 118.8 (d), 120.9 (d), 123.6 (s),
128.8 (d), 128.9 (d), 129.8 (s), 134.8 (s), 138.0 (s), 145.8 (s).
Anal. (C14H11N2Cl) C, H, N.
H-3′ and H-6′), 9.43 (1H, s, NHAc), 11.35 (1H, bs, NH); 13C
NMR δ 23.4 (q), 101.3 (d), 111.2 (d), 119.1 (d), 120.0 (d), 121.3
(d), 125.3 (d), 126.6 (d), 127.0 (s), 127.6 (d), 128.3 (d), 128.9
(s), 134.8 (s), 134.9 (s), 136.5 (s), 168.6 (s). Anal. (C16H14N2O)
C, H, N.
2-(2-Acetylam in o-5-ch lor oph en yl)in dole (10b): yield 96%;
1
mp 213 °C; IR 3383 (NHAc), 3354 (NH), 1670 (CO) cm-1; H
NMR δ 2.04 (3H, s, CH3), 6.77 (1H, d, J ) 1.0 Hz, H-3), 7.00
(1H, dt, J ) 7.5 Hz, J ) 1.6 Hz, H-5), 7.11 (1H, dt, J ) 7.5 Hz,
J ) 1.6 Hz, H-6), 7.37 (1H, dd, J ) 8.5 Hz, J ) 1.8 Hz, H-4′),
7.40 (1H, dd, J ) 7.5 Hz, J ) 1.6 Hz, H-7), 7.56 (1H, dd, J )
7.5 Hz, J ) 1.6 Hz, H-4), 7.62 (1H, d, J ) 8.5 Hz, H-3′), 7.67
(1H, d, J ) 1.8 Hz, H-6′), 9.52 (1H, s, NHAc), 11.45 (1H, bs,
NH); 13C NMR δ 23.7 (q), 102.5 (d), 111.7 (d), 119.6 (d), 120.6
(d), 122.2 (d), 127.5 (d), 128.4 (d), 128.6 (d), 128.6 (s), 129.0
(s), 129.6 (s), 133.5 (s), 134.1 (s), 136.9 (s), 169.1 (s). Anal.
(C16H13N2OCl) C, H, N.
2-(2-Acetyla m in op h en yl)-5-m eth oxyin d ole (10c): yield
95%; mp 197 °C; IR 3350 (NHAc), 3277 (NH), 1645 (CO) cm-1
;
1H NMR δ 2.06 (3H, s, CH3), 3.76 (3H, s, OCH3), 6.64 (1H, d,
J ) 1.5 Hz, H-3), 6.76 (1H, dd, J ) 8.7 Hz, J ) 2.4 Hz, H-6),
7.07 (1H, d, J ) 2.4 Hz, H-4), 7.28 (1H, dt, J ) 8.3 Hz, J ) 1.8
Hz, H-5′), 7.30 (1H, d, J ) 8.7 Hz, H-7), 7.31 (1H, dt, J ) 8.3
Hz, J ) 1.8 Hz, H-4′), 7.60 (1H, dd, J ) 8.3 Hz, J ) 1.8 Hz,
H-6′), 7.63 (1H, dd, J ) 8.3 Hz, J ) 1.8 Hz, H-3′), 9.44 (1H, s,
NHAc), 11.21 (1H, bs, NH); 13C NMR δ 23.8 (q), 55.5 (q), 101.6
(d), 101.7 (d), 112.1 (d), 112.3 (d), 125.6 (d), 126.9 (d), 127.4
(s), 127.8 (d), 129.1 (d), 129.1 (s), 132.0 (s), 135.1 (s), 135.7 (s),
153.8 (s), 169.0 (s). Anal. (C17H16N2O2) C, H, N.
Syn th esis of 3-Br om o-2-(2-n itr op h en yl)in d ole (8). To
a solution of 6a (1.2 g, 5 mmol) in anhydrous dimethylforma-
mide (20 mL) was added a solution of N-bromosuccinimide (0.9
g, 5 mmol) in dimethylformamide (10 mL) dropwise at room
temperature. After 2 h the reaction mixture was poured onto
crushed ice; the solid precipitated was filtered off and air-dried
to give 8, which was pure enough to give satisfactory analytical
and spectral data: yield 100%; mp 146 °C; IR 3368 (NH) cm-1
;
1H NMR δ 7.21 (1H, dt, J ) 7.3 Hz, J ) 1.2 Hz, H-5), 7.30
(1H, dt, J ) 7.3 Hz, J ) 1.2 Hz, H-6), 7.49 (2H, dd, J ) 7.3
Hz, J ) 1.2 Hz, H-4 and H-7), 7.81 (2H, dt, J ) 7.5 Hz, J )
1.3 Hz, H-4′ and H-5′), 7.91 (1H, dd, J ) 7.5 Hz, J ) 1.3 Hz,
H-6′), 8.23 (1H, dd, J ) 7.5 Hz, J ) 1.3 Hz, H-3′), 12.02 (1H,
s, NH); 13C NMR δ 112.2 (d), 118.6 (d), 120.6 (d), 123.4 (d),
125.1 (d), 125.9 (s), 126.9 (s), 130.7 (d), 131.6 (s), 137.6 (d),
133.7 (d), 133.7 (s), 135.8 (s), 148.9 (s). Anal. (C14H9N2O2Br)
C, H, N.
2-(2-Acetylam in o-4-ch lor oph en yl)in dole (10d): yield 97%;
1
mp 215 °C; IR 3350 (NHAc), 3317 (NH), 1684 (CO) cm-1; H
NMR δ 2.08 (3H, s, CH3), 6.73 (1H, d, J ) 1.6 Hz, H-3), 7.02
(1H, dt, J ) 7.2 Hz, J ) 1.4 Hz, H-5), 7.12 (1H, dt, J ) 7.2 Hz,
J ) 1.4 Hz, H-6), 7.32 (1H, dd, J ) 8.0 Hz, J ) 1.6 Hz, H-5′),
7.43 (1H, d, J ) 8.0 Hz, H-6′), 7.57 (1H, dd, J ) 7.2 Hz, J )
1.4 Hz, H-7), 7.62 (1H, dd, J ) 7.2 Hz, J ) 1.4 Hz, H-4), 7.82
(1H, d, J ) 1.6 Hz, H-3′), 9.57 (1H, s, NHAc), 11.43 (1H, bs,
NH); 13C NMR δ 23.9 (q), 102.1 (d), 111.7 (d), 119.5 (d), 120.4
(d), 121.9 (d), 125.1 (d), 125.5 (d), 125.5 (s), 128.6 (s), 130.8
(d), 132.0 (s), 134.0 (s), 136.5 (s), 137.0 (s), 169.2 (s). Anal.
(C16H13N2OCl) C, H, N.
Syn th esis of 2-(2-Am in op h en yl)-3-br om oin d ole (9). A
solution of 8 (0.95 g, 3 mmol) in acetic acid (30 mL) was heated
at 40 °C; then iron powder (0.85 g, 15 mmol) was added over
30 min. After the addition was complete, the mixture was kept
at 40 °C overnight and then poured onto crushed ice. The
precipitate was collected, air-dried, and recrystallized from
ethanol: yield 85%; mp 154-155 °C; IR 3414 and 3343 (NH2),
3130 (NH) cm-1; 1H NMR δ 5.55 (2H, s, NH2), 6.80 (1H, dd, J
) 7.5 Hz, J ) 1.6 Hz, H-3′), 6.88 (1H, dt, J ) 7.5 Hz, J ) 1.6
Hz, H-5′), 7.21-7.26 (3H, m, H-4′, H-4 and H-7), 7.34 (2H, dt,
J ) 7.4 Hz, J ) 1.6 Hz, H-5 and H-6), 7.60 (1H, dt, J ) 7.5
Hz, J ) 1.6 Hz, H-6′), 8.45 (1H, s, NH); 13C NMR δ 91.3 (s),
111.1 (d), 116.2 (d), 118.6 (d), 119.2 (d), 120.8 (d), 123.3 (d),
128.0 (s), 130.3 (d), 131.3 (d), 131.3 (s), 133.0 (s), 135.2 (s),
144.8 (s). Anal. (C14H11N2Br) C, H, N.
Dia zotiza tion of 2-(2-Am in op h en yl)-3-br om oin d ole (9).
To a stirred solution of the amine 9 (0.57 g, 2 mmol) in acetic
acid (30 mL) was added sodium nitrite (0.14 g, 2 mmol)
dissolved in the minimum amount of water dropwise at 0-5
°C. The reaction mixture was kept at the same temperature
for 3 h. It was then poured onto crushed ice and neutralized
with aqueous sodium hydroxide (5%). The precipitate was
filtered off, air-dried, and recrystallized from ethanol to give
the indolo[3,2-c]cinnoline 4 (yield 70%) which had analytical
and spectral data (IR, NMR) identical to an authentic sample.6
2-(2-Acetyla m in o-5-m eth ylp h en yl)in d ole (10e): yield
96%; mp 168 °C; IR 3381 (NHAc), 3362 (NH), 1672 (CO) cm-1
;
1H NMR δ 2.04 (3H, s, CH3), 2.37 (3H, s, CH3), 6.70 (1H, d, J
) 1.3 Hz, H-3), 7.00 (1H, dt, J ) 7.4 Hz, J ) 1.3 Hz, H-5),
7.12 (1H, dt, J ) 7.4 Hz, J ) 1.3 Hz, H-6), 7.15 (1H, dd, J )
7.7 Hz, J ) 1.3 Hz, H-4′), 7.40 (1H, d, J ) 1.3 Hz, H-6′), 7.44
(1H, dd, J ) 7.4 Hz, J ) 1.3 Hz, H-7), 7.48 (1H, d, J ) 7.7 Hz,
H-3′), 7.56 (1H, dd, J ) 7.4 Hz, J ) 1.3 Hz, H-4), 9.34 (1H, s,
NHAc), 11.33 (1H, bs, NH); 13C NMR δ 20.6 (q), 23.5 (q), 101.3
(d), 111.3 (d), 119.1 (d), 120.0 (d), 121.4 (d), 126.9 (d), 127.1
(s), 128.2 (d), 128.4 (s), 129.2 (d), 132.4 (s), 134.6 (s), 135.0 (s),
136.5 (s), 170.6 (s). Anal. (C17H16N2O) C, H, N.
2-(2-Acetylam in oph en yl)-5-ch lor oin dole (10f): yield 99%;
mp 170-171 °C; IR 3366 (NHAc), 3265 (NH), 1680 (CO) cm-1
;
1H NMR δ 2.04 (3H, s, CH3), 6.69 (1H, d, J ) 1.4 Hz, H-3),
7.10 (1H, dd, J ) 8.4 Hz, J ) 2.1 Hz, H-6), 7.30 (2H, dt, J )
7.1 Hz, J ) 1.6 Hz, H-4′ and H-5′), 7.42 (1H, d, J ) 8.4 Hz,
H-7), 7.60 (2H, dd, J ) 7.1 Hz, J ) 1.6 Hz, H-3′ and H-6′),
7.61 (1H, d, J ) 2.1 Hz, H-4), 9.48 (1H, s, NHAc), 11.55 (1H,
bs, NH); 13C NMR δ 22.0 (q), 101.1 (d), 112.8 (d), 119.2 (d),
121.3 (s), 121.3 (d), 123.7 (s), 125.5 (d), 126.8 (d), 128.1 (d),
129.1 (d), 129.6 (s), 135.1 (s), 135.2 (s), 136.8 (s), 168.8 (s). Anal.
(C16H13N2OCl) C, H, N.
Syn th esis of Su bstitu ted 2-(2-Acetyla m in op h en yl)-
in d oles 10a -f. 2-(2-Aminophenyl)indoles 7a -f (7 mmol) were
dissolved in acetic anhydride (30 mL) and stirred at room
temperature for 2 h. The solution was then poured onto
crushed ice. The solid precipitated was filtered off, air-dried,
and recrystallized from ethanol.
Syn th esis of Su bstitu ted 3-Nitr osoin d oles 11a -f. To a
solution of the acetylamino derivatives 10a -f (6 mmol) in
acetic acid (30 mL) was added sodium nitrite (0.41 g, 6 mmol)
dissolved in the minimum amount of water dropwise at 0-5
°C with stirring. After 1 h, an orange precipitate was formed.
The mixture was poured onto crushed ice; the solid precipi-
tated was filtered off, air-dried, and recrystallized from etha-
nol.
2-(2-Acetyla m in op h en yl)in d ole (10a ): yield 98%; mp 150
1
°C; IR 3383 (NHAc), 3354 (NH), 1670 (CO) cm-1; H NMR δ
2.05 (3H, s, CH3), 6.70 (1H, d, J ) 1.4 Hz, H-3), 7.00 (1H, dt,
J ) 7.0 Hz, J ) 2.0 Hz, H-5), 7.11 (1H, dt, J ) 7.0 Hz, J ) 2.0
Hz, H-6), 7.31 (2H, dt, J ) 6.3 Hz, J ) 1.8 Hz, H-4′ and H-5′),
7.42 (1H, dd, J ) 7.0 Hz, J ) 2.0 Hz, H-7), 7.56 (1H, dd, J )
7.0 Hz, J ) 2.0 Hz, H-4), 7.63 (2H, dd, J ) 6.3 Hz, J ) 1.8 Hz,
2-(2-Acetyla m in op h en yl)-3-n itr osoin d ole (11a ): yield
97%; mp 228-230 °C; IR 3120 (NHAc), 2820 (very broad NH),

2566 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Cirrincione et al.
1
1645 (CO) cm-1; H NMR δ 2.15 (3H, s, CH3), 7.20 (1H, dt, J
hydroxide (15%, 10 mL). The reaction mixture was refluxed
for 2 h. After cooling, the solution was neutralized with
hydrochloric acid (1 N). The solid was filtered off, air-dried,
and recrystallized from ethanol.
) 7.6 Hz, J ) 2.0 Hz, H-5), 7.37 (1H, dt, J ) 7.6 Hz, J ) 2.0
Hz, H-6), 7.50 (1H, dt, J ) 7.5 Hz, J ) 1.2 Hz, H-4′), 7.53 (1H,
dt, J ) 7.5, J ) 1.2 Hz, H-5′), 7.63 (1H, dd, J ) 7.6 Hz, J )
2.0 Hz, H-7), 8.17 (1H, dd, J ) 7.6 Hz, J ) 2.0 Hz, H-4), 8.34
(1H, dd, J ) 7.5 Hz, J ) 1.2 Hz, H-6′), 8.45 (1H, dd, J ) 7.5
Hz, J ) 1.2 Hz, H-3′), 11.85 (1H, s, NHAc), 13.72 (1H, broad
NH); 13C NMR δ 24.8 (q), 119.2 (s), 120.1 (d), 120.6 (d) 122.5
(d), 126.6 (d), 127.6 (d), 131.2 (d), 131.3 (s), 131.4 (d), 132.5
(s), 132.5 (d), 139.1 (2×s), 154.4 (s), 168.4 (s). Anal. (C16H13N3O2)
C, H, N.
2-(2-Am in op h en yl)-3-n itr osoin d ole (12a ): yield 90%; mp
1
205 °C; IR 3351 and 3241 (NH2), 3167 (NH) cm-1; H NMR δ
6.61 (1H, dt, J ) 8.1 Hz, J ) 1.9 Hz, H-5′), 6.87 (1H, dd, J )
8.1 Hz, J ) 1.9 Hz, H-3′), 7.22 (1H, dt, J ) 8.1 Hz, J ) 1.9 Hz,
H-4′), 7.30 (1H, dt, J ) 7.4 Hz, J ) 1.5 Hz, H-5), 7.50 (1H, dt,
J ) 7.4 Hz, J ) 1.5 Hz, H-6), 7.53 (1H, dd, J ) 7.4 Hz, J ) 1.5
Hz, H-7), 7.63 (2H, s, NH2), 8.19 (1H, dd, J ) 7.4 Hz, J ) 1.5
Hz, H-4), 8.48 (1H, dd, J ) 8.1, Hz, J ) 1.9 Hz, H-6′), 13.75
(1H, bs, NH); 13C NMR δ 112.8 (s), 115.0 (d), 116.2 (d), 119.8
(d), 120.6 (s), 126.7 (d), 127.3 (d), 131.7 (d), 132.0 (d), 132.4
(d), 150.9 (s), 154.2 (s), 155.7 (s), 165.0 (s). Anal. (C14H11N3O)
C, H, N.
2-(2-Acetylam in o-5-ch lor oph en yl)-3-n itr osoin dole (11b):
yield 96%; mp 250 °C; IR 3120 (NHAc), 2847 (very broad NH),
1
1668 (CO) cm-1; H NMR δ 2.14 (3H, s, CH3), 7.37 (1H, dt, J
) 6.8 Hz, J ) 1.5 Hz, H-5), 7.51 (1H, dd, J ) 8.1 Hz, J ) 2.2
Hz, H-4′), 7.54 (1H, dt, J ) 6.8 Hz, J ) 1,5 Hz, H-6), 7.59 (1H,
dd, J ) 6.8 Hz, J ) 1.5 Hz, H-7), 8.13 (1H, dd, J ) 6.8 Hz, J
) 1.5 Hz, H-4), 8.42 (1H, d, J ) 2.2 Hz, H-6′), 8.49 (1H, d, J
2-(2-Am in o-5-ch lor op h en yl)-3-n itr osoin d ole (12b): yield
94%; mp 217 °C; IR 3321 and 3239 (NH2), 3219 (NH) cm-1; 1H
NMR δ 6.87 (1H, d, J ) 8.9 Hz, H-3′), 7.20 (1H, dd, J ) 8.9
Hz, J ) 2.2 Hz, H-4′), 7.29 (1H, dt, J ) 7.0 Hz, J ) 1.7 Hz,
H-5), 7.47 (1H, dt, J ) 7.0 Hz, J ) 1.7 Hz, H-6), 7.50 (1H, dd,
J ) 7.0 Hz, J ) 1.7 Hz, H-7), 7.79 (2H, bs, NH2), 8.14 (1H, dd,
J ) 7.0 Hz, J ) 1.7 Hz, H-4), 8.53 (1H, d, J ) 2.2 Hz, H-6′),
13.87 (1H, bs, NH); 13C NMR δ 113.4 (s), 118.0 (d), 118.5 (s),
120.2 (d), 120.6 (s), 127.3 (d), 127.4 (d), 130.9 (d), 131.5 (d),
132.2 (d), 149.5 (s), 153.9 (s), 155.5 (s), 163.7 (s). Anal.
(C14H10N3OCl) C, H, N.
2-(2-Am in op h en yl)-5-m et h oxy-3-n it r osoin d ole (12c):
yield 90%; mp 206 °C; IR 3337 and 3231 (NH2), 3148 (NH)
cm-1; 1H NMR δ 3.84 (3H, s, CH3), 6.61 (1H, dt, J ) 7.5 Hz, J
) 1.6 Hz, H-5′), 6.85 (1H, dd, J ) 7.5 Hz, J ) 1.6 Hz, H-3′),
7.06 (1H, dd, J ) 8.4 Hz, J ) 2.4 Hz, H-6), 7.19 (1H, dt, J )
7.5 Hz, J ) 1.6 Hz, H-4′), 7.44 (1H, d, J ) 8.4 Hz, H-7), 7.49
(2H, bs, NH2), 7.75 (1H, d, J ) 2.4 Hz, H-4), 8.42 (1H, dd, J )
7.5 Hz, J ) 1.6 Hz, H-6′), 13.58 (1H, bs, NH); 13C NMR δ 56.0
(q), 113.3 (s), 113.7 (d), 115.2 (d), 116.3 (d), 116.6 (d), 120.6
(d), 121.7 (s), 131.5 (d), 132.2 (d), 147.8 (s), 150.5 (s), 155.9 (s),
158.7 (s), 163.5 (s). Anal. (C15H13N3O2) C, H, N.
) 8.1 Hz, H-3′), 12.05 (1H, s, NH), 14,13 (1H, broad NH); 13
C
NMR δ 24.9 (q), 129.2 (s), 120.5 (d), 121.8 (s), 121.8 (d), 126.2
(s), 126.7 (d), 128.0 (d), 130.8 (d), 131.4 (d), 131.6 (d), 131.6
(s), 134.6 (s), 138.3 (s), 154.3 (s), 168.6 (s). Anal. (C16H12N3O2-
Cl) C, H, N.
2-(2-Acet yla m in op h en yl)-5-m et h oxy-3-n it r osoin d ole
(11c): yield 95%; mp 250 °C; IR 3120 (NHAc), 2839 (very broad
NH), 1626 (CO) cm-1; H NMR δ 1.91 (3H, s, CH3), 3.83 (3H,
1
s, OCH3), 7.10 (1H, dd, J ) 7.1 Hz, J ) 1.5 Hz, H-6), 7.18 (1H,
dt, J ) 8.5 Hz, J ) 2.1 Hz, H-5′), 7.47 (1H, dt, J ) 8.5 Hz, J
) 2.1 Hz, H-4′), 7.55 (1H, d, J ) 7.1 Hz, H-7), 7.73 (1H, d, J
) 1.5 Hz, H-4), 8.32 (1H, dd, J ) 8.5 Hz, J ) 2.1 Hz, H-6′),
8.44 (1H, dd, J ) 8.5 Hz, J ) 2.1 Hz, H-3′), 11.90 (1H, s, NH),
12.45 (1H, broad NH); 13C NMR δ 21.0 (q), 55.7 (q), 113.0 (d),
114.6 (d), 116.2 (d), 118.4 (s), 120.4 (d), 121.1 (s), 121.2 (s),
122.5 (d), 130.9 (d), 132.2 (d), 138.9 (s), 145.9 (s), 154.4 (s),
159.1 (s), 168.5 (s). Anal. (C17H15N3O3) C, H, N.
2-(2-Acetylam in o-4-ch lor oph en yl)-3-n itr osoin dole (11d):
yield 95%; mp 232 °C; IR 3146 (NHAc), 2859 (very broad NH),
1
1618 (CO) cm-1; H NMR δ 2.17 (3H, s, CH3), 7.23 (1H, dd, J
) 8.7 Hz, J ) 2.2 Hz, H-5′), 7.37 (1H, dt, J ) 7.4 Hz, J ) 1.2
Hz, H-5), 7.51 (1H, dt, J ) 7.4 Hz, J ) 1.2 Hz, H-6), 7.58 (1H,
dd, J ) 7.4 Hz, J ) 1.2 Hz, H-7), 8.14 (1H, dd, J ) 7.4 Hz, J
) 1.2 Hz, H-4), 8.43 (1H, d, J ) 8.7 Hz, H-6′), 8.59 (1H, d, J
) 2.2 Hz, H-3′), 12.28 (1H, s, NHAc), 14.30 (1H, broad NH);
13C NMR δ 25.2 (q), 117.5 (s), 119.6 (d), 120.1 (s), 120.6 (d),
122.5 (d), 127.0 (s), 127.0 (d), 128.1 (d), 131.8 (d), 134.0 (d),
136.3 (s), 140.8 (s), 140.9 (s), 154.7 (s), 169.2 (s). Anal.
(C16H12N3O2Cl) C, H, N.
2-(2-Acetylam in o-5-m eth ylph en yl)-3-n itr osoin dole (11e):
yield 96%; mp 246 °C; IR 3131 (NHAc), 2854 (NH), 1636 (CO)
cm-1; 1H NMR δ 2.12 (3H, s, CH3), 2.31 (3H, s, CH3), 7.31 (1H,
dd, J ) 8.1 Hz, J ) 1.4 Hz, H-4′), 7.36 (1H, dt, J ) 7.2 Hz, J
) 1.2 Hz, H-5), 7.52 (1H, dt, J ) 7.2 Hz, J ) 1.2 Hz, H-6),
7.60 (1H, dd, J ) 7.2 Hz, J ) 1.2 Hz, H-7), 8.13 (1H, d, J )
1.4 Hz, H-6′), 8.16 (1H, dd, J ) 7.2 Hz, J ) 1.2 Hz, H-4), 8.32
(1H, d, J ) 8.1 Hz, H-3′), 11.70 (1H, s, NHAc), 14.01 (1H, broad
NH); 13C NMR δ 20.5 (q), 24.8 (q), 119.3 (s), 120.2 (s), 120.6
(s), 120.8 (s), 126.7 (s), 127.6 (d), 127.8 (d), 131.4 (d), 131.8
(d), 131.8 (d), 132.5 (d), 132.6 (d), 136.9 (s), 154.5 (s), 168.2
(s). Anal. (C17H15N3O2) C, H, N.
2-(2-Am in o-4-ch lor op h en yl)-3-n itr osoin d ole (12d ): yield
90%; mp 243 °C; IR 3393 and 3261 (NH2), 3132 (NH) cm-1; 1H
NMR δ 6.64 (1H, dd, J ) 8.3 Hz, J ) 2.2 Hz, H-5′), 6.97 (1H,
d, J ) 2.2 Hz, H-3′), 7.29 (1H, dt, J ) 7.2 Hz, J ) 1.7 Hz,
H-5), 7.48 (1H, dt, J ) 7.2 Hz, J ) 1.7 Hz, H-6), 7.53 (1H, dd,
J ) 7.2 Hz, J ) 1.7 Hz, H-7), 7.90 (2H, s, NH2), 8.19 (1H, dd,
J ) 7.2 Hz, J ) 1.7 Hz, H-4), 8.52 (1H, d, J ) 8.3 Hz, H-6′),
13.89 (1H, bs, NH); 13C NMR δ 111.7 (s), 115.0 (d), 115.0 (d),
120.0 (d), 120.6 (s), 126.9 (d), 127.3 (d), 131.9 (s), 134.0 (d),
136.5 (d), 151.9 (s), 153.9 (s), 155.5 (s), 164.2 (s). Anal.
(C14H10N3OCl) C, H, N.
2-(2-Am in o-5-m eth ylph en yl)-3-n itr osoin dole (12e): yield
95%; mp 190-191 °C; IR 3344 and 3213 (NH2), 3120 (NH)
1
cm-1; H NMR δ 2.20 (3H, s, CH3), 6.76 (1H, d, J ) 8.3 Hz,
H-3′), 7.02 (1H, dd, J ) 8.3 Hz, J ) 2.2 Hz, H-4′), 7.26 (1H, dt,
J ) 7.3 Hz, J ) 1.4 Hz, H-5), 7.40 (2H, bs, NH2), 7.45 (1H, dt,
J ) 7.3 Hz, J ) 1.4 Hz, H-6), 7.49 (1H, dd, J ) 7.3 Hz, J ) 1.6
Hz, H-7), 8.14 (1H, dd, J ) 7.3 Hz, 1.4 Hz, H-4), 8.26 (1H, d,
J ) 2.2 Hz, H-6′), 13.74 (1H, bs, NH); 13C NMR δ 20.3 (q),
112.6 (s), 116.2 (d), 119.5 (d), 120.4 (s), 122.9 (s), 126.4 (d),
127.1 (d), 131.7 (d), 131.9 (d), 132.7 (d), 140.0 (s), 148.6 (s),
155.6 (s), 162.6 (s). Anal. (C15H13N3O) C, H, N.
2-(2-Acetylam in oph en yl)-5-ch lor o-3-n itr osoin dole (11f):
yield 93%; mp 255-257 °C; IR 3120 (NHAc), 2795 (NH), 1649
(CO) cm-1; 1H NMR δ 2.10 (3H, s, CH3), 7.16 (1H, dt, J ) 7.8
Hz, J ) 1.2 Hz, H-4′), 7.48 (1H, dt, J ) 7.8 Hz, J ) 1.2 Hz,
H-5′), 7.50 (1H, dd, J ) 8.5 Hz, J ) 2.0 Hz, H-6), 7.56 (1H, d,
J ) 8.5 Hz, H-7), 8.05 (1H, d, J ) 2.0 Hz, H-4), 8.25 (1H, dd,
J ) 7.8 Hz, J ) 1.2 Hz, H-6′), 8.38 (1H, dd, J ) 7.8 Hz, J )
2-(2-Am in op h en yl)-5-ch lor o-3-n itr osoin d ole (12f): yield
95%; mp 228-230 °C; IR 3395 and 3231 (NH2), 3123 (NH)
1
cm-1; H NMR δ 6.59 (1H, dt, J ) 7.4 Hz, J ) 1.4 Hz, H-5′),
6.85 (1H, dd, J ) 7.4 Hz, J ) 1.4 Hz, H-3′), 7.19 (1H, dt, J )
7.4 Hz, J ) 1.4 Hz, H-4′), 7.48 (1H, d, J ) 7.0 Hz, H-7), 7.53
(1H, dd, J ) 7.0 Hz, J ) 1.4 Hz, H-6), 7.63 (2H, bs, NH2), 8.12
(1H, d, J ) 1.4 Hz, H-4), 8.43 (1H, dd, J ) 7.4 Hz, J ) 1.4 Hz,
H-6′), 14.01 (1H, bs, NH); 13C NMR δ 112.4 (s), 112.9 (d), 116.2
(d), 120.8 (d), 121.6 (s), 126.5 (d), 130.3 (s), 131.2 (d), 131.8
(d), 132.1 (d), 150.8 (s), 152.5 (s), 154.8 (s), 165.0 (s). Anal.
(C14H10N3OCl) C, H, N.
1.2 Hz, H-3′), 11.68 (1H, s, NHAc), 14.26 (1H, broad NH); 13
C
NMR δ 24.8 (q), 119.1 (s), 120.7 (d), 121.1 (d), 122.6 (d), 125.9
(d), 126.2 (s), 130.8 (d), 131.5 (d), 131.6 (2×s), 132.5 (d), 139.2
(2×s), 153.8 (s), 168.5 (s). Anal. (C16H12N3O2Cl) C, H, N.
Syn t h esis of Su b st it u t ed In d olo[1,2-c]b en zo[1,2,3]-
tr ia zin es 5a -g. Hyd r olysis of Com p ou n d s 11a -f. To a
solution of acetylamino derivatives 11a -f (1.6 mmol) in
ethanol (10 mL) was added an aqueous solution of potassium
Dia zotiza tion of Su bstitu ted 2-(2-Am in op h en yl)-3-n i-
tr osoin d oles 12a -f. Compounds 12a -f were diazotized fol-
lowing the procedure described for compound 9. The solid

Derivatives of Indolo[1,2-c]benzo[1,2,3]triazine
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2567
precipitated was filtered off, air-dried, and pure enough to give
satisfactory analytical and spectral data.
H9/IIIB, MT-4, and C8166 (grown in RPMI 1640 containing
10% fetal calf serum (FCS), 100 U/mL penicillin G, and 100
µg/mL streptomycin) cells were used for anti-HIV assays. Cell
cultures were checked periodically for the absence of myco-
plasma contamination with a MycoTect kit (Gibco).
Vir u ses. Human immunodefieciency virus type-1 (HIV-1,
IIIB strain) was obtained from supernatants of persistently
infected H9/IIIB cells. HIV-1 stock solutions had a titer of 5 ×
107 cell culture infectious dose fifty (CCID50)/mL.
An tivir a l Assa ys. Activity of compounds against the HIV-1
multiplication in acutely infected cells was based on inhibition
of virus-induced cytopathogenicity in MT-4 cells. Briefly, 50
µL of RPMI 10% FCS containing 1 × 104 cells was added to
each well of flat-bottomed microtiter trays containing 50 µL
of medium and serial dilution of test compounds; 20 µL of an
HIV-1 suspension containing 100 CCID50 was added. After a
4-day incubation at 37 °C, the number of viable cells was
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) method.9,10 Cytotoxicity of com-
pounds, based on the viability of mock-infected cells, as
monitored by the MTT method, was evaluated in parallel with
their antiviral activity.
An titu m or Assa ys. Exponentially growing leukemia and
lymphoma cells were resuspended at a density of 1 × 105 cells/
mL in RPMI containing serial dilutions of the test drugs. Cell
viability was determined after 4 days at 37 °C by the MTT
method. Activity against cell lines derived from solid tumors
was evaluated in exponentially growing cultures seeded at 5
× 104 cells/mL which were allowed to adhere for 16 h to culture
plates before addition of the drugs. Cell viability was deter-
mined by the MTT method 4 days later.
12-Nitr osoin d olo[1,2-c]ben zo[1,2,3]tr ia zin e (5a ): yield
80%; mp 210 °C; IR 1471 (NO), 1448 (NdN) cm-1; 1H NMR δ
7.77 (1H, dt, J ) 7.3 Hz, J ) 1.5 Hz, H-10), 7.82 (1H, dt, J )
7.3 Hz, 1.5 Hz, H-9), 8.35 (1H, dt, J ) 8.4 Hz, J ) 1.9 Hz,
H-3), 8.37 (1H, dt, J ) 8.4 Hz, J ) 1.9 Hz, H-2), 8.43 (1H, dd,
J ) 7.3 Hz, J ) 1.5 Hz, H-11), 8.56 (1H, dd, J ) 8.4 Hz, J )
1.9 Hz, H-4), 8.75 (1H, dd, J ) 7.3 Hz, J ) 1.5 Hz, H-8), 9.46
(1H, dd, J ) 8.4 Hz, J ) 1.9 Hz, H-1). Anal. (C14H8N4O) C, H,
N.
2-Ch lor o-12-n it r osoin d olo[1,2-c]b en zo[1,2,3]t r ia zin e
(5b): yield 80%; mp 207 °C; IR 1466 (NO), 1448 (NdN) cm-1
;
1H NMR δ 7.75 (2H, dt, J ) 7.7 Hz, J ) 2.0 Hz, H-9 and H-10),
8.32 (1H, dd, J ) 7.7 Hz, J ) 2.0 Hz, H-8), 8.35 (1H, dd, J )
7.7 Hz, J ) 2.0 Hz, H-11), 8.49 (1H, dd, J ) 8.8 Hz, J ) 2.3
Hz, H-3), 8.70 (1H, d, J ) 8.8 Hz, H-4), 9.25 (1H, d, J ) 2.3
Hz, H-1). Anal. (C14H7N4OCl) C, H, N.
10-Met h oxy-12-n it r osoin d olo[1,2-c]b en zo[1,2,3]t r ia -
zin e (5c): yield 85%; mp 208 °C; IR 1473 (NO), 1458 (NdN)
1
cm-1; H NMR δ 7.36 (1H, dd, J ) 9.0 Hz, J ) 2.4 Hz, H-9),
7.93 (1H, d, J ) 2.4 Hz, H-11), 8.33 (2H, dt, J ) 6.8 Hz, J )
1.4 Hz, H-2 and H-3), 8.45 (1H, d, J ) 9.0 Hz, H-8), 8.71 (1H,
dd, J ) 6.8 Hz, J ) 1.4 Hz, H-4), 9.40 (1H, dd, J ) 6.8 Hz, J
) 1.4 Hz, H-1). Anal. (C15H10N4O2) C, H, N.
3-Ch lor o-12-n it r osoin d olo[1,2-c]b en zo[1,2,3]t r ia zin e
(5d ): yield 83%; mp 207 °C; IR 1464 (NO), 1446 (NdN) cm-1
;
1H NMR δ 7.79 (1H, dt, J ) 7.3 Hz, J ) 1.6 Hz, H-10), 7.82
(1H, dt, J ) 7.3 Hz, J ) 1.6 Hz, H-9), 8.39 (1H, dd, J ) 7.3
Hz, J ) 1.6 Hz, H-11), 8.41 (1H, dd, J ) 7.3 Hz, J ) 1.6 Hz,
H-8), 8.55 (1H, dd, J ) 8.7 Hz, J ) 2.1 Hz, H-2), 8.91 (1H, d,
J ) 2.1 Hz, H-4), 9.41 (1H, d, J ) 8.7, H-1). Anal. (C14H7N4-
OCl) C, H, N.
Lin ea r Regr ession An a lysis. Tumor cell growth at each
drug concentration was expressed as percentage of untreated
controls, and the concentration resulting in 50% (EC50, IC50
)
2-Met h yl-12-n it r osoin d olo[1,2-c]b en zo[1,2,3]t r ia zin e
(5e): yield 100%; mp 190-191 °C; IR 1479 (NO), 1448 (NdN)
cm-1; 1H NMR δ 2.70 (3H, s, CH3), 7.73 (2H, dt, J ) 7.4 Hz, J
) 1.5 Hz, H-9 and H-10), 8.15 (1H, dd, J ) 8.8 Hz, J ) 2.5 Hz,
H-3), 8.36 (1H, dd, J ) 7.4 Hz, J ) 1.5 Hz, H-8), 8.47 (1H, dd,
J ) 7.4 Hz, J ) 1.5 Hz, H-11), 8.57 (1H, d, J ) 8.8 Hz, H-4),
9.18 (1H, d, J ) 2.5 Hz, H-1). Anal. (C15H10N4O) C, H, N.
growth inhibition was determined by linear regression analy-
sis.
Cytotoxicity Assa ys. Peripheral blood lymphocytes (PBL)
from HIV-negative donors were obtained by separation on
Fycoll-Hypaque gradients. After extensive washings, cells were
resuspended (1 × 106 cells/mL) in RPMI-1640 with 10% FCS
and incubated overnight.
For cytotoxicity evaluations in proliferating PBL cultures,
nonadherent cells were resuspended at 1 × 106 cells/mL in
growth medium, stimulated with PHA (2.5 µg/mL) for 24 h
before dilution to 1 × 105 cells/mL in medium containing PHA
(2.5 µg/mL), IL-2 (50 U/mL), and various concentrations of the
test compounds. Viable cell number was determined 6 days
later. Under these conditions, untreated PBL were able to
undergo exponential growth up to four cell cycles, as deter-
mined by viable cell counts.
For cytotoxicity evaluations in resting PBL cultures, non-
adherent cells were resuspended at high density (1 × 106 cells/
mL) and treated for as long as 3 days with the test compounds.
Then, the cells were extensively washed to remove the inhibi-
tors and were stimulated with PHA for 24 h before being
diluted to 1 × 105 cells/mL in medium containing PHA and
IL-2. Cell viability was determined after incubation at 37 °C
for 6 days.
An tiba cter ia l Assa ys. S. aureus, group D Streptococcus,
Salmonella spp., and Shigella spp. were recent clinical isolates.
Assays were carried out in nutrient broth, pH 7.2, with an
inoculum at 103 bacterial cells/tube. Minimum inhibitory
concentrations (MIC) were determined after incubation at 37
°C for 18 h in the presence of serial dilutions of the test
compounds.
10-Ch lor o-12-n it r osoin d olo[1,2-c]b e n zo[1,2,3]t r ia -
zin e (5f): yield 100%; mp 210 °C; IR 1475 (NO), 1448 (NdN)
1
cm-1; H NMR δ 7.76 (1H, dd, J ) 8.8 Hz, J ) 2.0 Hz, H-9),
8.31 (2H, dt, J ) 7.8 Hz, J ) 1.6 Hz, H-2 and H-3), 8.34 (1H,
d, J ) 2.0 Hz, H-11), 8.53 (1H, d, J ) 8.8 Hz, H-8), 8.70 (1H,
dd, J ) 7.8 Hz, J ) 1.6 Hz, H-4), 9.36 (1H, dd, J ) 7.8 Hz, J
) 1.6 Hz, H-1). Anal. (C14H7N4OCl) C, H, N.
10-Nitr o-12-n itr osoin dolo[1,2-c]ben zo[1,2,3]tr iazin e (5g).
This compound was prepared by nitration of the unsubstituted
indolobenzotriazine 5a . Thus to a solution of 5a (0.5 g, 2 mmol)
in sulfuric acid (96%, 12 mL) was added potassium nitrate (0.2
g, 2 mmol) dissolved in sulfuric acid (96%, 5 mL) at 0 °C
dropwise with stirring. The reaction mixture was allowed to
reach room temperature, kept under stirring overnight, and
poured onto crushed ice. The solid precipitate was filtered off,
air-dried, and pure enough to give satisfactory analytical and
spectral data: yield 60%; mp 215 °C; IR 1525 (NO2), 1494 (NO),
1455 (NdN) cm-1; 1H NMR δ 8.30 (1H, dt, J ) 7.4 Hz, J ) 1.8
Hz, H-3), 8.39 (1H, dt, J ) 7.4 Hz, J ) 1.8 Hz, H-2), 8.53 (1H,
dd, J ) 8.9 Hz, J ) 2.4 Hz, H-9), 8.73 (1H, d, J ) 8.9 Hz,
H-8), 8.75 (1H, dd, J ) 7.4 Hz, J ) 1.8 Hz, H-4), 9.02 (1H, d,
J ) 2.4 Hz, H-11), 9.43 (1H, dd, J ) 7.4 Hz, J ) 1.8 Hz, H-1).
Anal. (C14H7N5O3) C, H, N.
(B) Biology. Com p ou n d s. Test compounds were dissolved
in DMSO at an initial concentration of 200 µM and then were
serially diluted in culture medium.
An tim ycotic Assa ys. Yeast inocula were obtained by
properly diluting cultures incubated at 37 °C for 30 h in
Sabouraud dextrose broth to obtain 5 × 103 cells/mL. On the
contrary, dermatophyte inocula were obtained from cultures
grown at 37 °C for 5 days in Sabouraud dextrose broth by
finely dispersing clumps with a glass homogenizer before
diluting to 0.05 OD590/mL. Then, 20 µL of the above suspen-
sions was added to each well of flat-bottomed microtiter trays
containing 80 µL of medium with serial dilutions of test
Cells. Cell lines were from American Type Culture Collec-
tion (ATCC). The human nasopharyngeal carcinoma KB cell
line and the drug-resistant subclones KBMDR, KBV20C, KB70
,
and KBCamp were a generous gift of Prof. Y. C. Cheng, Yale
University. Bacterial and fungal strains were either clinical
isolates (obtained from Clinica Dermosifilopatica, University
of Cagliari) or collection strains from ATCC.

2568 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Cirrincione et al.
(2) Feigon, J .; Denny, W. A.; Leupin, W.; Kearns, D. R. Interaction
of Antitumor Drugs with Natural DNA: 1H NMR Study of
Binding Mode and Kinetics. J . Med. Chem. 1984, 27, 450-465.
(3) Watson, J . D.; Hopkins, N. H.; Roberts, J . W.; Steitz, J . A.;
Weiner, A. M. In Molecular Biology of the Gene, 4th ed.;
Benjamin/Cummings Publishing Co.: Reading, MA, 1987; Vol.
I, p 255.
(4) Myers, C. E., J r.; Chabner, B. A. In Cancer Chemotherapy:
Principles and Practice; Chabner, B. A., Collins, J . M., Eds.;
Lippincott: Philadelphia, PA, 1990; p 356.
(5) Hertzberg, R. P. Agents Interfering with DNA Enzymes. In
Comprehensive Medicinal Chemistry; Sammes, P. G., Ed.; Per-
gamon Press: Oxford, U.K., 1990; Vol. 2, pp 753-791.
(6) Cirrincione, G.; Almerico, A. M.; Barraja, P.; Diana, P.; Gri-
maudo, G.; Mingoia, F.; Dattolo, G.; Aiello, E. Polycondensed
Nitrogen Heterocycles. Part 27. Indolo[3,2-c]cinnoline. Synthesis
and Antileukemic Activity. Farmaco 1995, 50, 849-852.
(7) Aiello, E.; Dattolo, G.; Cirrincione, G.; Almerico, A. M.; D’Asdia,
I. Preparation of Monohalopyrroles. J . Heterocycl. Chem. 1982,
19, 977-979.
(8) Cirrincione, G.; Almerico, A. M.; Aiello, E.; Dattolo, G. Polycon-
densed Nitrogen Heterocycles. Part 23. Pyrrolo[3,2-c]cinnolines
by a J app-Klingemann Type Reaction. J . Heterocycl. Chem. 1990,
27, 983-985.
compounds, and the trays were incubated at 37 °C. Growth
controls were visually determined after 2 days (yeast) or 3 days
(dermatophytes).
MIC was defined as the compound concentration at which
no macroscopic signs of fungal growth were detected. The
minimal germicidal concentration (MBC or MFC) was deter-
mined by subcultivating in Sabouraud dextrose agar samples
from cultures with no apparent growth.
An tim ycoba cter ia l Assa ys. M. smegmatis, M. avium
complex (MAC), and M. tuberculosis 27294 were ATCC strains,
whereas M. fortuitum and M. tuberculosis 1104 were clinical
isolates. MICs were assessed in microtiter plates by adding
20-µL aliquots of a Mycobacterium suspension (whose turbidity
was equal to that of a 1 McFarland standard containing 108
CFU/mL) to 80 µL of Middlebrood 7II9 medium containing
0.5% glicerol and 10% OADC and various concentrations of
the test compounds. The plates were then incubated for 1 day
(M. smegmatis and M. fortuitum) or 9 days (MAC and M.
tuberculosis) at 37 °C. At the end of incubation, the number
of viable Mycobacteria was determined by the MTT method.
Ack n ow led gm en t. This work was supported by
grants from Ministero dell’Universita` e della Ricerca
Scientifica e Tecnologica, by Consiglio Nazionale delle
Ricerche (Rome), and by Assessorato Igiene e Sanita`,
Regione Autonoma della Sardegna.
(9) Denizot, F.; Lang, R. Rapid colorimetric assay for cell growth
and survival. J . Immunol. Methods 1986, 89, 271-277.
(10) Pauwels, R.; Balzarini, J .; Baba, M.; Snoeck, R.; Scholds, D.;
Herdewijn, P.; Desmyter, J .; De Clercq, E. Rapid and automated
tetrazolium-based colorimetric assay for the detection of anti-
HIV compounds. J . Virol. Methods 1988, 20, 309-321.
Refer en ces
(1) Wakelin, L. P. G.; Waring, M. J . DNA Intercalating Agents. In
Comprehensive Medicinal Chemistry; Sammes, P. G., Ed.; Per-
gamon Press: Oxford, U.K., 1990; Vol 2, pp 703-724.
J M9806087