Novel pyridazino[4,3-b]indoles with dual inhibitory activity against Mycobacterium tuberculosis and monoamine oxidase

Article abstract of DOI:10.1021/jm030479g

Tuberculosis is one of the most common infectious diseases known to man. About 37% of the world's population (about 1.86 billion people) are infected with Mycobacterium tuberculosis. According to the World Health Organization, every year approximately 8 million people develop active tuberculosis and almost 2 million of those die from the disease. The incidence of multidrug-resistant tuberculosis (MDR-TB) is increasing. The present drug regimen for treating tuberculosis has been in existence for 30 years. New drugs that will shorten total treatment duration, improve the treatment of MDR-TB, and address latent tuberculosis are the most urgent need of tuberculosis control programs. A new series of synthetic 3-amino-4-arylpyridazino[4,3-b]indoles (pyridazinoindoles) were identified as inhibitors of Mycobacterium tuberculosis. The design, synthesis, and antimycobacterial activity of these compounds are described. While the most active compounds are still not comparable to the front-line drugs rifampicin and isoniazid, they do show promise. Most of the pyridazinoindoles with appreciable antituberculosis activity also inhibit monoamine oxidase, suggestive of a novel inhibitory effect on mycobacterial redox reactions.

Full text of DOI:10.1021/jm030479g

J. Med. Chem. 2004, 47, 3455-3461
3455
Novel Pyridazino[4,3-b]indoles with Dual Inhibitory Activity against
Mycobacterium tuberculosis and Monoamine Oxidase
Valeriya S. Velezheva,*,† Patrick J. Brennan,*,‡ Vladimir Yu. Marshakov,† Dmitrij V. Gusev,†
Inessa N. Lisichkina,† Alexander S. Peregudov,† Larisa N. Tchernousova,§ Tatiana G. Smirnova,§
Sofia N. Andreevskaya,§ and Alexei E. Medvedev|
A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Street,
119991 Moscow, Russia, Department of Microbiology, Immunology and Pathology, Colorado State University,
Fort Collins, Colorado 80523-1682, Central TB Research Institute, Russian Academy of Medical Sciences,
2 Yauzskaya Alley, 107564 Moscow, Russia, and Institute of Biomedical Chemistry,
Russian Academy of Medical Sciences, 10 Pogodinskaya Street, 119832 Moscow, Russia
Received September 24, 2003
Tuberculosis is one of the most common infectious diseases known to man. About 37% of the
world’s population (about 1.86 billion people) are infected with Mycobacterium tuberculosis.
According to the World Health Organization, every year approximately 8 million people develop
active tuberculosis and almost 2 million of those die from the disease. The incidence of
multidrug-resistant tuberculosis (MDR-TB) is increasing. The present drug regimen for treating
tuberculosis has been in existence for 30 years. New drugs that will shorten total treatment
duration, improve the treatment of MDR-TB, and address latent tuberculosis are the most
urgent need of tuberculosis control programs. A new series of synthetic 3-amino-4-arylpy-
ridazino[4,3-b]indoles (pyridazinoindoles) were identified as inhibitors of Mycobacterium
tuberculosis. The design, synthesis, and antimycobacterial activity of these compounds are
described. While the most active compounds are still not comparable to the front-line drugs
rifampicin and isoniazid, they do show promise. Most of the pyridazinoindoles with appreciable
antituberculosis activity also inhibit monoamine oxidase, suggestive of a novel inhibitory effect
on mycobacterial redox reactions.
Introduction
specific essential redox enzymes in the tubercle bacil-
lus.8 For instance, some derivatives of hydrazine, such
as INH, are known to destroy catalase-peroxidase
enzyme redox metabolic pathways of the pathogen.9
Also, the dual activity of N,N-containing heterocycles
as substrates for M. tuberculosis peroxidases and inhibi-
tors of mycobacterial growth is well-known.10 In the
1960s, Pershin et al. reported that hydrazine derivatives
with appreciable antituberculosis activity also inhibited
monoamine oxidase (MAO) activity.11,12 The MAO in-
hibitory action of INH and particularly iproniazid was
described long ago.13 Iproniazid, initially used as a drug
in the treatment of tuberculosis, is a central nervous
stimulant because of a mild inhibitory effect on MAO.13
Several original Russian antidepressants and reversible
MAO inhibitors, such as pirlindole and tetrindol, ex-
hibited marked antituberculosis activity.14 The MAO
inhibitor isocarboxazide (marplan), used in psychiatry,
and its close analogue 5-methylisoxazole-3-carboxylic
acid hydrazide were once employed for treating leprosy
caused by Mycobacterium leprae.15
Tuberculosis is one of the most important public
health problems in Russia, other East European coun-
tries, and worldwide. Since 1985 and particularly in the
1990s, a search for new antituberculosis substances has
ranked among the priority areas of chemotherapeutic
research.1,2 The highly effective combined therapy regi-
men of isoniazid (INH), rifampicin (RIF), and pyrazin-
amide (PZA) forms the basis of the current DOTS
(directly observed therapy, short course), which is now
being vigorously implemented in FSR (Former Soviet
Republic) countries. These three drugs with significantly
different chemical structures affect different essential
metabolic pathways of Mycobacterium tuberculosis.3
Nevertheless, the emergence of monodrug-resistant and
multidrug-resistant strains of M. tuberculosis in FSR
and elsewhere, even in the course of successful DOTS
programs, emphasizes the need for new drug leads of
new structural classes and with novel mechanisms of
action. Recently, antituberculosis activity was revealed
among new types of nitrogen-containing heterocycles,2,4
alkaloids,5,6 and amide derivatives of sulfonyl fatty acids
with alkyl tails.7
Pyridazine and fused pyridazine derivatives have long
attracted the interest of Russian researchers as anti-
tuberculosis agents; antituberculosis compounds were
found in the series of 3-pyridazinones16 and pyridazino-
[3,4-b]quinoxalines.17 There are other examples.18-20
Some members of the 3-amino-4-arylpyridazino[4,3-b]-
indole family displayed activity against M. tuberculosis
H37Rv and M. fortuitum with MICs of 23 and 32 µg/
mL, respectively,21 and did not inhibit the growth of
Gram-positive or Gram-negative bacteria. Besides those,
antituberculosis bicyclic isatin-3-hydrazono derivatives
Some antituberculosis drugs introduced into therapy
have been shown to exert their action by inhibition of
* To whom correspondence should be addressed. For V.S.V.: phone,
7-095-1359253; fax, 7-095-1355085; e-mail, vel@ineos.ac.ru.
For P.J.B.: phone, (970) 491-6700; fax, (970) 491-1815; e-mail:
Patrick.Brennan@ColoState.edu.
† A. N. Nesmeyanov Institute of Organoelement Compounds.
‡ Colorado State University.
§ Central TB Research Institute.
| Institute of Biomedical Chemistry.
10.1021/jm030479g CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/15/2004

3456 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Scheme 1. General Synthetic Methods
Velezheva et al.
(hydrazono-1H-2-indolinones)22,23 have some structural
features resembling the pyridazinoindoles. Endogenous
isatin and some of its analogues, 2-arylidene-3-indoli-
nones (indogenides) and 2-R-cyanoarylmethylene-3-in-
dolinones (cyanoindogenides), in addition to their anti
M. tuberculosis activity, act as inhibitors of MAO in rat
brain mitochondrial fractions.24 M. tuberculosis H37Rv
contains a putative flavine-containing MAO, Rv3170,25
with high homology to mitochrondrial MAO, and so it
is possible that the inhibition of mycobacterial growth
results from the inhibition of mycobacterial MAO.
Accordingly, we speculate that antituberculosis agents
will be found among cyclic derivatives of hydrazine, such
as fused pyridazines, with MAO inhibitory effects.
In continuation of our work on indole-fused com-
pounds,21,26,27 we choose to search for antituberculosis
agents in the new series of 3-amino-4-arylpyridazino-
[4,3-b]indoles (pyridazinoindoles) that are tricyclic hy-
drazine derivatives.
or acetone cyanohydrin in the presence of a base. The
reaction proceeds both in protic and aprotic solvents
(DMSO, DMF, MeOH)21 (Scheme 1), and the net result
is the replacement of a vinyl hydrogen in the in-
dogenides by a cyano group. The N(1)-unsubstituted
indogenides 1 are better substrates than their N(1)-
substituted counterparts in this reaction. The starting
indogenides 1, substituted in the 1,2-(substituted phen-
yl)- and 5-positions, were prepared according to a
procedure published earlier.28
In the present case, the synthesis of cyanoindogenides
2 was carried out in DMSO at 20-30 °C for 3-5 days
in the presence of a 3-fold excess of KCN. The yields of
the parent cyanoindogenide 2a, 3- or 4-substituted
phenyl analogues 2b-f, and 2j-m were 75-85% under
these conditions. Although N(1)-alkylindogenides and
2-substituted phenylindogenides 1g,h also gave cy-
anoindogenides 3 and 2g,h, respectively, the yields were
no greater than 20% under the above reaction condi-
tions. Good yields of N(1)-alkylcyanoindogenides 3 were
obtained by alkylation of the corresponding N(1)-un-
substituted cyanoindogenides with alkyl halides in the
presence of strong bases such as sodium alkoxides. Then
cyanoindogenides 2 and N(1)-alkylcyanoindogenides 3
were converted to pyridazinoindoles 4 and 5 in 60-90%
yield by cyclocondensation with hydrazine hydrate in
boiling acetic acid.
The purpose of this study was to synthesize new
potent antituberculosis pyridazinoindole analogues and
test them for inhibition of the growth of M. tuberculosis.
We also studied MAO inhibition of the pyridazinoindoles
to gain a better understanding of the relationships
between antituberculosis activity and MAO inhibition.
Chemistry
In the synthesis of the 3-amino-4-arylpyridazino[4,3-
b]indoles, we employed a new reaction involving the
transformation of 2-arylidene-3-indolinones (indogenides)
1 to 2-R-cyanoarylmethylene-3-indolinones (cyanoin-
dogenides) 2 under the action of alkali metal cyanides
The synthesis of 3-(2H)pyridazinones 6 was carried
out via diazotization of pyridazinoindoles 4a, 5a, and
5d followed by hydrolysis of the intermediate diazonium
salts with H2SO4. The yields of pyridazinones 6 were
80-87% (Scheme 2).

Pyridazino[4,3-b]indoles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3457
Scheme 2. Synthesis of Compounds 6
Table 1. In Vitro Activity against M. tuberculosis H37Rv and
MAO Inhibitory Action of Pyridazinoindoles 4-7 Bearing
Various Substituents in the Ring System
Scheme 3. Synthesis of Compounds 7
percent inhibition of
M. tuberculosis
inhibition of
MAO activity
PIa
at 20
µg/mL
PIa
at 2.0
µg/mL
PIa
at 0.2
µg/mL
IC50
MAO A
(µM)
IC50
MAO B
(µM)
b
MIC50
compd
(µg/mL)
4a
4b
4c
4d
4e
4f
4g
4h
4j
28.5
35.1
40.5
61.9
30.2
66.0
40.3
35.0
32.0
91.4
88.5
40.2
62.3
88.0
45.3
3.1
25.5
0.0
2.3
0.0
0.0
8.7
0.0
5.2
0.0
0.0
0.0
9.2
7.1
2.3
0.0
21.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
NIc
NIc
NIc
NIc
0.0
16.49
12.63
14.86
31.6
>1000
158
>1000
32.9
0.0
10.0
0.0
0.0
0.0
44.3
56.3
25.5
0.0
79.4
631
316
∼1000
NIc
NIc
4k
4l
4.17
1.77
63.1
63.0
28.2
25.1
79.0
∼1000
5a
5c
5d
5g
5h
5j
63.6
0.0
1.42
14.1
NIc
316
41.6
∼1000
100
0.0
∼1000
60.1
54.2
60.7
40.5
40.3
30.1
94.3
15.9
20.0
20.4
20.7
50.8
0
0.0
63.1
We also tried to obtain N(3)-acylamino-substituted
pyridazinoindoles, but this proved to be difficult. The
acylation of the primary amino group did not proceed
under mild conditions, and the products of N,N-diacy-
lation or mixtures of different products were usually
obtained under severe conditions. We did manage to
obtain 3-N-formylamino-3-N,N-diacetylamino and 3-N-
phthaloylamino derivatives 7a-c (Scheme 3).
5k
5l
0.0
0.0
17.12
10.48
5m
5n
5o
5p
5q
5r
0.0
0.0
NIc
72
11
15.8
100
0.0
NIc
10.5
0.0
100
>1000
0.0
5s
5t
0.0
1000
17.8
NIc
0.0
5u
6a
6b
6c
7a
7b
7c
RIF
INH
0.0
35.4
Estimation of Antimycobacterial Activity of
Compounds. Antimycobacterial activity of the py-
ridazinoindoles 4-7 was estimated by their effects on
the inhibition of the selective incorporation of radiola-
beled 5,6-[3H]-uracil into viable M. tuberculosis H37Rv.29
The assay was based on data demonstrating that
parallel estimations of mycobacterial colony-forming
units (CFU), conducted as described,30 and 5,6-[3H]-
uracil uptake under identical cultural conditions and
dynamics showed correlation coefficients higher than
0.9, thereby validating the usage of the latter, less
laborious, and less variable method.29,31 The results of
incorporation of 5,6-[3H]-uracil into growing M. tuber-
culosis are expressed as counts per minute (cpm) ( SD
for triplicate cultures. The target compounds (Table 1)
and reference drugs RIF and INH were tested at
concentrations of 20, 2.0, and 0.2 µg/mL on 25 × 104 of
M. tuberculosis strain H37Rv (Pasteur). The activity of
compounds was estimated as percent of inhibition (PI),
which was determined as the percent difference between
the incorporation of 5,6-[3H]-uracil into growing M.
tuberculosis in the presence and the absence of the
compounds over 42 h of incubation. The MIC50 was
defined as the lowest concentration of drug resulting
in 50% inhibition of [3H]-uracil incorporation into the
organism.
0
0
0
0
0
94.3
68.7
91.7
59.1
54.8
49.7
0.10
0.25
a PI: percent of inhibition based on the difference in incorpora-
tion of [3H]-uracil into growing M. tuberculosis (25 × 104 CFU)
over 42 h at the 20, 2.0, and 0.2 µg/mL concentration of the
compounds compared to the nontreated cultures. b MIC50
: con-
centration of compounds resulting in 50% reduction in [3H]-uracil
incorporation compared to the nontreated cultures. c NI: no
inhibition observed at the highest concentration tested (1000 µM).
B from rat liver mitochondria were determined by
methods described previously.32
Results
We designed and tested a large congeneric series of
3-, 4-, 5-, and 8-substituted pyridazino[4, 3-b]indole
derivatives (34 compounds) in order to improve the
antituberculosis activity of previously synthesized py-
ridazinoindoles 4a, 4b, 4d, 4e, 4g, 5a and to gain an
understanding of their structure-activity relationships
in the context of anti-M. tuberculosis and anti-MAO
activities (Table 1).
Measurement of MAO Inhibition. The inhibitory
effects of compounds on activities of MAO A and MAO
The compounds tested for antituberculosis activity
were divided into four series. Series A comprised N(5)-

3458 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Velezheva et al.
unsubstituted compounds 4a-j, derived from the parent
structure 4a with various substituents in positions 2-4
of the phenyl ring, and 8-bromosubstituted compounds
4k,l. Series B comprised N(5)-alkylated compounds,
both monosubstituted 5a,n,t and disubstituted 5c-k,o-
s,u and trisubstituted pyridazinoindoles 5l,m with
variations in positions 4, 5, and 8 of the ring system.
Series C and D involved N(5)-unsubstituted 3-(2H)-
pyridazinone 6a and its mono- and disubstituted ana-
logues 6b and 6c, and also 5-methyl-3-N-acylamino-
substituted pyridazinoindoles 7a-c, respectively.
Antimycobacterial activity of the pyridazinoindoles
4-7 was estimated from their effects on the inhibition
of incorporation of radiolabeled 5,6-[3H]-uracil into
viable M. tuberculosis H37Rv. The results (Table 1)
demonstrate a reduction in 5,6-[3H]-uracil uptake in the
range of 20-94% at 20 µg/mL for most of the compounds
tested compared to about 94% and 69% for RIF and
INH, respectively. Nine of the pyridazinoindoles (4d, 4f,
4k, 4l, 5c, 5d, 5j, 5l, 5p) exhibited the greatest
antituberculosis activity with PI g 60% at 20 µg/mL.
Only compounds 4l and 5d at 2.0 µg/mL were as active
as INH at the same concentration in impairing [3H]-
uracil incorporation. However, at 0.2 µg/mL, all py-
ridazinoindoles were poorly active, whereas RIF and
INH inhibited [3H]-uracil incorporation by about 50%.
Compounds 5d and 4l had MIC50 values of 1.42 and 1.77
µg/mL, respectively, compared to MIC50 values of 0.10
and 0.25 µg/mL for RIF and INH, respectively. Thus,
they were the most active pyridazinoindoles tested.
These two did not inhibit the growth of Staphylococcus
aureus or Escherichia coli even at 10-fold higher con-
centrations.
ment of steric and electronic factors in influencing
antituberculosis activity of compounds 4-7. Further-
more, lipophilicity may also play a role on the effect of
the compounds tested.
Twenty pyridazinoindoles were tested as inhibitors
of rat liver mitochondrial MAO A and/or MAO B (Table
1).32,33 Two pyridazinoindoles (4a and 4b) did not inhibit
MAO A or MAO B even at the highest concentration
tested (1000 µM). Six other analogues (4g, 4h, 5g, 5n,
5o, and 5s) were active against only one of the isoforms.
Nearly half of all compounds studied had IC50 values
in the range of 10-80 µM with respect to MAO A and/
or MAO B. These IC50 values were higher than those of
known MAO inhibitors (e.g., IC50 values for MAO
inhibition by fused indoles such as pirlindole and
tetrindol are 0.2 and 0.05 µM, respectively32,33). Within
the present series, compounds 5d and 5p were among
the most potent and selective inhibitors of MAO A and
MAO B, respectively. Compound 5u was rather potent
and appeared to be a nonselective inhibitor of both MAO
A and MAO B with IC50 values of 17.8 and 35.4 µM,
respectively. Among the nine compounds (see above)
with the greatest antituberculosis activity (PI g 60%
at 20 µg/mL), seven (4d, 4f, 4k, 4l, 5d, 5j, 5p) were
tested for MAO A inhibition, and for six of them (4f,
4k, 4l, 5d, 5j, 5p), the IC50 values with respect to MAO
A were e100 µM. It is noteworthy that the two most
active compounds against M. tuberculosis, 5d (PI ) 88%
at 20 µg/mL; PI ) 63.6% at 2.0 µg/mL) and 4l (PI )
88.5% at 20 µg/mL; PI ) 56.3% at 2.0 µg/mL), had MAO
A IC50 inhibitory values of 14.1 and 63 µM, respectively;
i.e., they are effective MAO inhibitors (Table 1).
Discussion
In series A, the parent pyridazinoindole 4a and its
analogues 4b, 4c, 4e, 4g-j substituted in positions 2,
3, and 4 of the 4-phenyl ring displayed only weak
antituberculosis activity. The replacement of the para
hydrogen with an isopropyl or dimethylamino substitu-
ent or the insertion of bromine into position 8 led to the
more active compounds 4d, 4f, 4k, 4l. In series B, N(5)-
alkylated derivatives 5c, 5d, 5j, 5l, and 5p exhibited
the greatest potency. Irrespective of the substituted aryl,
an N(5)-alkyl (methyl, ethyl) substituent at the ring
nitrogen was highly favorable for antituberculosis activ-
ity (compare 4a and 5a, 4c and 5c, 4d and 5d, 4d and
5p). In the 4-(4-isopropyl)phenyl series represented by
5p, increasing the bulk of the indole substituent to
n-propyl (5r), dimethylaminoethyl (5s), or benzyl (5t)
was detrimental to antimycobacterial activity. The
replacement of hydrogen in position 8 with bromine was
beneficial in series A (compare 4a and 4k, 4d and 4l)
but had a variable effect in series B (compare 5a and
5k, 5d and 5l). Trisubstituted compounds 5l and 5m
showed modest levels of antituberculosis activity. Con-
version of the 3-amino group to a hydroxyl, as in the
pyridazinones 6 of series C, or N-acylation of the
3-amino group, as in compounds 7 of series D, destroyed
antituberculosis activity. Overall, members of the 4-(4-
isopropyl)phenyl series with either an N(5)-methyl (5d)
or an 8-bromo (4l) substituent exhibited the greatest
antituberculosis activity. Thus, the level of antituber-
culosis activity depends to a large extent on the nature
and the specific combination of substituents in the 4-,
5-, and 8-positions. These results suggest the involve-
We have long considered that the relationship be-
tween the antituberculosis and the MAO inhibitory
action of such compounds as pyridazinoindoles is not
fortuitous in that simultaneous manifestation of anti-
psychotic, anti-MAO, and antituberculosis activities is
typical for some classes of N,N-containing compounds
and fused indoles.11-14,32,33 However, there is no con-
sistent correlation between inhibition of M. tuberculosis
and MAO activity. In the case of the bromo-substituted
pyridazinoindoles such as 4l and 5l, 4k and 5k, we
conclude that the insertion of a bromo substituent
changes not only electronic effects but also topographical
features of the structures. The insertion of a 4-isopropyl
group in the aryl substituent of pyridazinoindoles
sometimes increases both antibacterial and MAO in-
hibitory activities. For example, the insertion of this
group into compound 5a converted it into 5d, resulting
in an increase in percent inhibition of mycobacterial
growth from 40.2% (at 20 µg/mL) to 88%, and the IC50
value for MAO A inhibition decreased from 28.2 to14.1
µM. In the context of the influence of the ortho effect
(o-chloro-substituted phenyl; i.e., pyridazinoindoles 5h,
5q, 5u), as a rule, the insertion of the o-chloro substitu-
ent decreases the value of the percent inhibition of
mycobacterial growth and increases the IC50 value for
MAO inhibition. This ortho effect is noticeable in the
case of the sterically hindered o-aryl-substituted py-
ridazinoindoles that also have N-alkyl substituents. For
example, compound 5a inhibits [3H]-uracil incorporation
by 40.2% (at 20 µg/mL) and has an IC50 value for MAO

Pyridazino[4,3-b]indoles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3459
A inhibition of 28.2 µM, whereas the comparable figures
for the Cl-containing analogue 5h are 3.1% and 1000
µM. However, there is an exception in that the corre-
sponding figures for the Cl-containing compound, 5u,
are 50.8% and 17.8 µM, respectively.
tives. Overall, members of the 4-(4-isoprenyl)phenyl
series with either an N(5)-methyl or an 8-bromo sub-
stituent exhibited the greatest effect on M. tuberculosis
based on inhibition of 5,6-[3H]-uracil incorporation. A
partial correlation between inhibition of bacterial growth
and monoamine oxidase activity suggests that the
pyrazinoindole may act through inhibition of mycobac-
terial redox reactions.
Accordingly, the hypothesis that the antituberculosis
activity of pyridazinoindoles is due to inhibition of
bacterial MAO must be treated with caution. Although
the presence of the gene for an MAO homologue has
been reported in the M. tuberculosis genome,25 a func-
tioning MAO requires substantiation. Such a mycobac-
terial MAO may participate in the biochemical synthesis
of hydrogen peroxide playing a role in the response of
mycobacteria to oxidative stress and thus be a potential
target for new drug development with the pyridazinoin-
doles described herein as the initial lead compounds.
However, the most promising compounds arising from
this work will first require extensive testing as inhibi-
tors of mycobacterial growth in vitro and in the mouse
model of tuberculosis, in conjunction with toxicity
testing prior to extensive study of structural-functional
relationships.
Experimental Section
Chemistry. Melting points are uncorrected. 1H NMR (400
MHz) data were measured in DMSO-d6, and chemical shifts
are reported in parts per million (δ) downfield from tetra-
methylsilane as internal standard. Mass spectra were mea-
sured on a VG-7070E spectrometer (EI, 70 eV). Elemental
analyses were performed at the laboratory for microanalysis
of A. N. Nesmeyanov Institute of Organoelement Compounds,
Moscow. TLC was performed on Silufol UV-254 plates. Anhy-
drous solvents were purified by standard procedures.
Key Intermediates: 2-Arylidene-1,2-dihydro-3H-indol-
3-ones (1). 2-Arylidene-1,2-dihydro-3H-indol-3-ones (1) were
prepared as described27 (Table S1 in Supporting Information).
A product sufficiently pure to be used as such for the next step
was generated. Recrystallization was achieved from methanol/
water (2:1), ethyl acetate, and acetonitrile.
Tuberculosis (TB) case notifications have been in-
creasing in the newly independent states of the former
Soviet Union and parts of Russia.34,35 Human immu-
nodeficiency virus (HIV) associated TB is widespread
in the sub-Saharan African countries hardest hit by
acquired immunodeficiency syndrome (AIDS),36 and
there has been a disturbing increase in the number of
TB cases caused by organisms resistant to the two most
important drugs, isoniazid and rifampicin; a survey of
72 countries suggested that the multidrug-resistant TB
(MDR-TB) problem is more widespread than previously
thought and is likely worsening.37 MDR-TB appears to
be especially serious in the Russian Federation, where
it has spread in prisons and throughout the entire
population.38 No novel compounds to treat TB have been
introduced into clinical practice in the past 30 years.
Incremental improvements have been made by modify-
ing the rifampin structure to generate long-lasting
rifamycins: rifapentine, rifabutin, and rifalazid.34 Sev-
eral quinolones are part of the second-line regimen
applied to the management of MDR-TB.34 However,
these are modest alternative regimens in light of the
dimensions of TB and HIV/TB and the threat of MDR-
TB. There is an urgent need for new chemotherapy that
will allow a shortening of the total duration of effective
treatment, improve the treatment of MDR-TB, and
provide an antidote to latent TB infection. Further
experiments will tell whether the pyrazinoindoles con-
form to some of these specifications.
Procedure for General Cyanation of 2-Arylidene-1,2-
dihydro-3H-indol-3-ones (1) To Give 2-(r-cyanoaryl-
methylene)-1,2-dihydro-3H-indol-3-ones (2) (Table S2 in
Supporting Information). A mixture of 1 (10 mmol) and
potassium cyanide (1.95 g, 30 mmol) in DMSO (15 mL) was
stirred at room temperature for 3-5 days. The course of the
reaction was followed to completion by TLC in 15% ethyl
acetate/hexane. The green reaction mixture was quenched with
a saturated NaCl solution (50 mL). A solution with an orange
color resulted, and the product precipitated within 10-12 h
to give an orange residue. This final product 2 was filtered
and washed with water. Recrystallization was possible from
ethanol, 2-propanol, or 2-propanol/water (2:1). MS, m/z: 2a,
246 [M]+; 2b, 264 [M]+; 2d, 288 [M]+; 2e, 276 [M]+; 2g, 264
[M]+.
Procedure for General N-Alkylation of 2-(r-cyanoaryl-
methylene)-1,2-dihydro-3H-indol-3-ones: 1-Alkyl-2-(r-cy-
anoarylmethylene)-1,2-dihydro-3H-indol-3-ones (3) (Table
S2 in Supporting Information). To a solution of sodium
ethanolate prepared from sodium (0.25 g, 11 mmol) and
ethanol (50 mL) was added the appropriate cyanoindogenide
2 (10 mmol) in one portion, and the mixture was stirred at
room temperature for 30 min. Then alkyl halide (25 mmol)
was added to the dark-blue solution. The resulting mixture
was stirred for 5-8 h. The reaction was determined to be
complete by TLC in 15% ethyl acetate/hexane. The reaction
mixture then was diluted with water (50 mL), and the residue
was filtered and washed with ethanol/water (2:1). A product
sufficiently pure to be used as such for the next step was
generated. Recrystallization is possible from ethanol, 2-pro-
panol, or 2-propanol/water (2:1). MS, m/z: 3a, 260 [M]+.
General Ring Closure Procedure: 3-Amino-4-aryl-5H-
(or 5-alkyl)pyridazino[4,3-b]indoles (4 and 5) (Table S3
in Supporting Information). Method A. To a hot solution
of hydrazine acetate prepared from 80% hydrazine hydrate
(224 mmol, 14 mL) and acetic acid (30 mL) was added the
appropriate cyanoindogenide 2 or 3 (3.5 mmol) in one portion,
and the reaction mixture was heated to gentle reflux for 5 h.
After cooling, the reaction mixture was poured into ice/water
(100 mL). The final product was filtered and washed with
water. Recrystallization is possible from ethanol, 2-propanol,
or N,N-dimethylformamide/methanol (1:5). Method A was used
for the preparation of the following compounds: 4a,d,e,j and
5a,h,l,o,q,r,t. MS, m/z: 4a, 260 [M]+; 4b, 278 [M]+; 4d, 302
[M]+; 4e, 290 [M]+; 4g, 278 [M]+; 5a, 274 [M]+.
Conclusions
Development of novel classes of compounds capable
of shortening the duration of effective treatment of
tuberculosis, treating isoniazid- and rifampin-resistant
tuberculosis (defined as MDR-TB), and addressing
latent tuberculosis is a major priority of global tuber-
culosis control programs. There was evidence in the
earlier Russian literature that cyclic derivatives of
hydrazine, such as fused pyridazines, could inhibit the
growth of M. tuberculosis. We chose to search for such
activity among the newer series of pyrazino[4,3-b]-
indoles (pyrazinoindoles), tricyclic hydrazine deriva-
3-Amino-4-aryl-5H-(or 5-alkyl)pyridazino[4,3-b]indoles
hydrochlorides (4 and 5) (Table S3 in Supporting Infor-
mation). Method B. The 3-amino-4-aryl-5H-(or 5-alkyl)-

3460 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Velezheva et al.
pyridazino[4,3-b]indoles were prepared in the same manner
as indicated above. After cooling, the reaction mixture was
poured into ice/water (100 mL) and finally rendered acidic with
20% HCl. The final product was filtered and washed with cold
water. Recrystallization is possible from ethanol or acetonitrile
or N,N-dimethylformamide/methanol (2:5). Method B was used
for the preparation of the following compounds: 4b,c,f-h,k,l
and 5c,d,g,j,k,m,n,p,r,u.
General Diazotization Procedure: 2,5-Dihydro-4-aryl-
3H-pyridazino[4,3-b]indol-3-ones (6) (Table S3 in Sup-
porting Information). Sodium nitrite (150 mg, 2.17 mmol)
was added at 0 °C with vigorous stirring to concentrated H2-
SO4 (2.5 mL) followed by addition of the suspension or solution
of the appropriate amine 4 or 5 (1.92 mmol) in acetic acid (10
mL). The reaction mixture was stirred for 1 h at 20 °C followed
by addition of water (75 mL) until precipitation ceased and
was allowed to stand 5 h at room temperature. The final
product was filtered and washed three times with water. The
analytical sample was prepared after recrystallization from
N,N-dimethylformamide/methanol (2:5).
3-N-Formylamino-5-methyl-4-phenylpyridazino[4,3-b]-
indole (7a) (Table S3 in Supporting Information). The
mixture of 1-methyl-3-amino-2-phenylpyridazino[4,3-b]indole
5a (137 mg, 0.5 mmol), 98% formic acid (10 mL), and acetic
anhydride (1 mL) was refluxed for 3-5 h. After removal of
the liquids under reduced pressure, the same amounts of
formic acid and acetic anhydride were added and the mixture
was refluxed and triturated as above. This procedure was
repeated three times. After removal of the liquids under
reduced pressure, the residue was purified by silica gel column
chromatography (chloroform/acetone (10:1)) to afford 7a (48
mg, 50%) as a white powder and recovered starting material
5a (50 mg). The analytical sample was prepared by recrystal-
lization from chloroform/pentane (2:5).
3-N,N-Diacetylamino-1-methyl-4-phenylpyridazino-
[4,3-b]indole (7b) (Table S3 in Supporting Information).
The mixture of 1-methyl-3-amino-2-phenylpyridazino[4,3-b]-
indole 5a (140 mg, 0.51 mmol) and sodium acetate (4.6 mg,
0.056 mmol) was refluxed in acetic anhydride (2 mL) for 10
min and after cooling was diluted with 10 mL of water. The
final product was filtered and washed three times with water.
The analytical sample was prepared by recrystallization from
2-propanol.
Detroit, MI), and the total number of microcolonies in spots
was calculated under an inverted microscope (CK-2, Olympus,
Osaka, Japan) after culturing for 3 days at 37 °C. The bulk of
filtered cultures were stored at +4 °C. Earlier, it was found
that no change in the CFU content occurred during this
period.31
Antimycobacterial activity of compounds was estimated by
growth inhibition of M. tuberculosis, as measured by selective
incorporation of radiolabeled 5,6-[3H]-uracil into bacterial cells
as described.29,30 Briefly, on the basis of CFU results, cultures
were diluted so that the number of M. tuberculosis was 50 ×
104, 25 × 104, 12.5 × 104, and 6.25 × 104 CFU in 100 µL of
RPMI 1640 medium with 5mM Hepes, 2mM L-glutamine, and
2% FBS. Different concentrations (100 µL) of M. tuberculosis
were added to each well of 96-well tissue culture microplates
(Costar-Corning, Badhoevedorp, The Netherlands). The com-
pounds in the three concentrations of 20, 2.0, and 0.2 µg/mL
(100 µL of each) were added. Each concentration of M.
tuberculosis and the compound was studied in triplicate. After
24 h, 1 µCi of 5,6-[3H]-uracil (Isotop, St. Petersburg, Russia)
was added to every well and incubated for 18 h in a CO2
incubator at 37 °C. The harvests were collected onto mi-
croporous filters (Skatron Filter Mat 11731, dimensions of 102
mm × 256 mm) using a cell harvester (Skatron, Oslo, Norway).
After numerous washings with water and drying in an oven
for 30 min at 80 °C, the filters were placed in vials with
scintillant and â-radiation was registered with the Wallac 1409
scintillant register (Wallac, Turku, Finland). Incorporation of
5,6-[3H]-uracil into growing M. tuberculosis was measured in
counts per minutes (cpm). Results are expressed as mean cpm
( SD for triplicate cultures. Parallel estimations of mycobac-
terial CFU counts and 5,6-[3H]-uracil uptake under identical
cultural conditions and dynamics provided coefficients of
correlation higher than 0.9, validating the usage of this less
laborious and variable technique.29 The activity of compounds
was estimated as percent inhibition, which was determined
on the basis of the percent difference in incorporation of 5,6-
[3H]-uracil into growing M. tuberculosis over 42 h in the
presence and the absence of the compounds. The MIC50 value
was defined as the concentration of compounds resulting in
50% reduction in the cpm on that plate compared to those on
a plate free of the drug at the same dilution of M. tuberculosis
suspension.
Methods of Evaluation of MAO Inhibitory Activity.
Rat liver mitochondria isolated by the conventional method
of differential centrifugation were used as a source of MAO-A
and MAO-B.32 Isolated mitochondria were washed and sus-
pended in 50 mM phosphate buffer, pH 7.4, and stored at -20
°C. MAO activity was assayed radiometrically by accumulation
of 14C-labeled aldehydes formed during enzymatic deamination
of 14C-labeled amines.32 [14C]-5-Hydroxytryptamine creatinine
sulfate (0.1 mM) and [14C]-phenylethylamine (10 µM) were
used as substrates of MAO-A and MAO-B, respectively. The
enzyme activities were determined after preincubation of
mitochondria with inhibitors for 60 min at 37 °C using
concentrations in the range 10-8-10-3 (or 10-4) M. Protein
content was determined by the Lowry method39 using bovine
serum albumin as the standard.
5-Methyl-3-phthaloylamino-4-(4-isopropylphenyl)py-
ridazino[4,3-b]indole (7c) (Table S3 in Supporting In-
formation). The finely ground mixture of hydrochloride 5d
(51 mg, 0.145 mmol) and phthalic anhydride (80 mg, 0.54
mmol) was heated in an open flask at 150-160 °C for 1 h.
Then the excess sublimated phthalic anhydride was removed
mechanically. The residue was dissolved in chloroform and was
purified by silica gel column chromatography (chloroform/
acetone (10:1)) to afford 7c (31 mg, 74%). The analytical sample
was prepared by recrystallization from chloroform/pentane (2:
5).
Methods for Evaluation of Inhibitory Activities of
Compounds. Portions (5 mg) of each compound were dis-
solved in 120 µL of DMSO and then diluted in complete
medium, RPMI 1640, with 5 mM HEPES, 2 mM L-glutamine,
and 2% fetal bovine serum (FBS) (Sigma) to obtain concentra-
tions of 800, 80, 8.0, and 0.8 µg/mL. The final compound
concentrations were 20, 2.0, and 0.2 µg/mL. M. tuberculosis
H37Rv (Pasteur) from the Central TB Research Institute
Culture Museum, Moscow, as frozen samples were thawed,
added to 5 mL of Dubos broth (Difco, Detroit, MI), and
incubated for 1 week at 37 °C. The mycobacterial suspension
(0.5 mL) was diluted in 20 mL of fresh, warm Dubos medium
and further cultured for 1 week. The resulting suspension was
washed three times at 3000g, 4 °C, with 0.02% EDTA-PBS
(Ca2+- and Mg2+-free) solution, resuspended in RPMI-1640
medium supplemented with 2% FBS, 5 mM HEPES, 2 mM
l-glutamine (Sigma), and filtered through a 4 µm pore size
filter (Millipore Corporation, Badford, MA) to remove clumps.
To estimate CFU content in the filtrate, 10 µL from each 5-fold
serial dilution was plated on Dubos agar (Difco Laboratories,
Statistics. To evaluate the statistical value of differences
between averages, the Student’s T-test was used and the
BIOSTAT program (Stenten A. Glanz, 1993; translation by
“Praktika”, Moscow, 1998) was applied.
Acknowledgment. This work was supported by
a grant (Grant RB 2-2032) from the U.S. Civilian
Research and Development Foundation (CRDF) for the
Independent States of the Former Soviet Union, Ar-
lington, VA. P.J.B. was supported by the NIH, NIAID,
NCDDG-OI Program (Grant AI-46393). We thank Prof.
Alexander S. Apt of Central TB Research Institute
Russian Academy of Medical Sciences (CNIIT) for the
idea of using the selective incorporation of radiolabeled
5,6-[3H]-uracil exclusively into viable M. tuberculosis as

Pyridazino[4,3-b]indoles
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Supporting Information Available: Chemical data on
the indogenides 1 (Table S1), cyanoindogenides 2 and 3 (Table
S2), and the pyridazinoindoles 4-7 (Table S3), including
1
yields, melting points, H NMR data, and elemental analysis
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