Microwave induced three-component synthesis and antimycobacterial activity of benzopyrazolo[3,4-b]quinolindiones

Article abstract of DOI:10.1016/j.ejmech.2013.12.037

Three series of novel 4-arylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 4, 7 and 9 have been efficiently obtained in good yields by three-component microwave assisted reaction between aminopyrazoles 1 or 6 (both 1-phenyl and 1-H substituted), 2-hydroxynaph

Full text of DOI:10.1016/j.ejmech.2013.12.037

Accepted Manuscript
Microwave induced three-component synthesis and antimycobacterial activity of
benzopyrazolo[3,4-b]quinolindiones
Jairo Quiroga, Ph.D. Yurina Diaz, Juan Bueno, Braulio Insuasty, Rodrigo Abonia,
Alejandro Ortiz, Manuel Nogueras, Justo Cobo
PII:
S0223-5234(13)00825-8
10.1016/j.ejmech.2013.12.037
EJMECH 6629
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 November 2012
Revised Date: 18 November 2013
Accepted Date: 24 December 2013
Please cite this article as: J. Quiroga, Y. Diaz, J. Bueno, B. Insuasty, R. Abonia, A. Ortiz, M.
Nogueras, J. Cobo, Microwave induced three-component synthesis and antimycobacterial activity of
benzopyrazolo[3,4-b]quinolindiones, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2013.12.037.
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ACCEPTED MANUSCRIPT
Graphical abstract
Microwave
induced
three-component
synthesis
and
antimycobacterial activity of benzopyrazolo[3,4-b]quinolindiones
1
1
2
1
1
Jairo Quiroga, Yurina Diaz, Juan Bueno, Braulio Insuasty, Rodrigo Abonia,
1
3
3
Alejandro Ortiz, Manuel Nogueras and Justo Cobo.
1
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad
del Valle, Cali, Colombia.
2
GrupoQ1 Micobacterias, Subdirección Red Nacional de Laboratorios, Instituto
Nacional de Salud, Bogotá, Centro Colombiano de Investigación en Tuberculosis
CCITB, Bogotá, D.C., Colombia.
3
Departamento de Química Inorgánica y Orgánica, Universidad de Jaén, Jaén,
España.
e-mail: jairo.quiroga@correounivalle.edu.co
1

ACCEPTED MANUSCRIPT
Microwave induced three-component synthesis and antimycobacterial
activity of benzopyrazolo[3,4-b]quinolindiones
Keywords: Aminopyrazoles; 2-Hydroxynaphthoquinones; Pyrazolo[3,4-b]quinolin-
5,6-diones; Microwave irradiation; Antimycobacterial activity; Three-component
reactions.
Corresponding Author: Jairo Quiroga, Ph.D.
Corresponding Author's Institution: Universidad del Valle
First Author: Jairo Quiroga, Ph.D.
Order of Authors: Yurina Diaz, Chemist; Juan Bueno, MD MSc; Braulio Insuasty,
Ph. D.; Rodrigo Abonia, Ph. D.; Alejandro Ortiz, Ph. D.; Manuel Nogueras, Ph. D.;
Justo Cobo, Ph. D.
Abstract: Three series of novel 4-arylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 4,
7
and 9 have been efficiently obtained in good yields by three-component
microwave assisted reaction between aminopyrazoles 1 or 6 (both 1-phenyl and 1-
H substituted), 2-hydroxynaphthoquinone 2 and benzaldehydes 3. Compounds 4, 7
and 9 have been evaluated against fifteen Mycobacterium spp strains, and six of
them have shown antimycobacterial activity. The highest inhibitory activity with MIC
≤
2 µg/mL for three of these compounds (4a, 4b and 4g) was related with their
highest lipophilicity and lesser polarity within these series.
2

ACCEPTED MANUSCRIPT
Microwave
induced
three-component
synthesis
and
antimycobacterial activity of benzopyrazolo[3,4-b]quinolindiones
1
1
2
1
1
Jairo Quiroga, Yurina Diaz, Juan Bueno, Braulio Insuasty, Rodrigo Abonia,
1
3
3
Alejandro Ortiz, Manuel Nogueras and Justo Cobo.
1
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad
del Valle, Cali, Colombia.
2
GrupoQ1 Micobacterias, Subdirección Red Nacional de Laboratorios, Instituto
Nacional de Salud, Bogotá, Centro Colombiano de Investigación en Tuberculosis
CCITB, Bogotá, D.C., Colombia.
3
Departamento de Química Inorgánica y Orgánica, Universidad de Jaén, Jaén,
España.
Abstract: Three series of novel 4-arylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 4,
7
and 9 have been efficiently obtained in good yields by three-component
microwave assisted reaction between aminopyrazoles 1 or 6 (both 1-phenyl and 1-
H substituted), 2-hydroxynaphthoquinone 2 and benzaldehydes 3. Compounds 4, 7
and 9 have been evaluated against fifteen Mycobacterium spp strains, and six of
them have shown antimycobacterial activity. The highest inhibitory activity with MIC
≤
2 µg/mL for three of these compounds (4a, 4b and 4g) was related with their
highest lipophilicity and lesser polarity within these series.
*
Corresponding Author: FAX: 57-2-33392440; e-mail: jairo.quiroga@correounivalle.edu.co
Keywords: Aminopyrazoles; 2-Hydroxynaphthoquinone; Pyrazolo[3,4-b]quinolin-
5,6-diones; Microwave irradiation; Antimycobacterial activity; Three-component
reactions.
3

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1. Introduction
Human tuberculosis (TB) is a contagious–infectious disease mainly caused by
Mycobacterium tuberculosis, which is an aerobic pathogenic bacterium that
establishes its infection usually in the lungs [1]. About one-third of the world’s
population is currently infected with M. tuberculosis; 10% of those infected will
develop clinical opportunistic disease in inmunocompromised patients, such those
infected by the human immunodeficiency virus (HIV) [1]. Although there are
medical regimens for treating TB, they are far from ideal [2]. In addition, there have
been reported an increasing number of multidrug-resistant strains (MDR-TB) and
extensively drug-resistant (XDR-TB) strains and HIV epidemics. These two factors
make necessary the development of new drug that could improve the
understanding of drug action molecular mechanisms and drug resistance, and
accordingly could provide significant insights for the development of new
generation of drug for the treatment of TB [2].
Numerous tri- and tetracyclic planar nitrogen-containing heterocycles, such as
amsacrine, the benzo[c]phenanthridines, the ellipticines, intoplicine and coralyne
are well known as topoisomerase inhibitors and have been investigated as
potential anticancer agents [3]. The benzoquinolines such as acridines or the
isomeric phenanthridine are important scaffolds in antitumour agents, as well as
their benzo-fused derivatives benzophenanthridines, representing an important
alkaloid family.
Between them, the benzoquinolinediones have exhibited the best overall activity
against both bacteria and fungi. Particularly noteworthy is the significant antifungal
activity, which is comparable to the activity of the standard antifungal antibiotic
amphotericin B [4]. These derivatives are a very important class of compounds for
the production of pharmaceuticals, herbicides, dyes, etc. [5]. There are several
classical methods for the synthesis of these compounds from various starting
4

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materials, but still need to improve the reaction efficiency employing readily
available starting materials.
We are interested in pyrazolo[3,4-b]quinoline derivatives, which are used as
pharmaceutical agents [6] and their derivates have shown antitubercular activity
[
7], as inhibitors of oncogenic Ras [8]. Additionally, pyrazolo[3,4-b]quinoline
derivatives exhibit a high fluorescence in both solution and solid state under
exposure to the white light, which make them appropriates in the design of
electroluminescent materials, like organic lightemitting diodes (OLEDs) and as a
dopant in the multiplayer OLED fabrication [9]. In the past several decades, three
general strategies for the synthesis of pyrazolo[3,4-b]quinolines have been
developed: i) by the Friedländer condensation reaction of 2-aminobenzophenones
and pyrazolin-5-ones [10], but the availability of 2-aminobenzophenones limits the
scope of this reaction; ii) by cyclocondensation of 4-arylidenepyrazolin-5-ones with
anilines [11] or 5-N-arylpyrazoles with aromatic aldehydes [12]; and iii) by a three-
component one-pot reaction of aromatic aldehydes, 5-amino-3-methyl-1-
phenylpyrazole and dimedone under thermal [13] or microwave irradiation
condition [14].
In this way, multicomponent reactions have shown great importance for its high
levels of brevity and diversity, allowing time-saving in one-pot operations, and the
combination of three or more reagents to get flexible building blocks [15]. The
structure of the reaction products can be diversified by systematic variation of each
starting material that could be either commercially available or easily prepared [16].
Microwave assisted organic synthesis (MAOS) has also emerged as a powerful
tool for high-throughput procedures. This can improve the yield and purity of the
final compounds in short reaction times through the precise control of parameters
such as power irradiation, pressure and temperature [17].
5

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We have concentrated much of our recent efforts in the preparation of
pyrazolo[3,4-b]quinolines as bioactive compounds, and have already described
simple and efficient procedures to prepare them in a three-component reaction
starting from 5-aminopyrazoles 1 [18]. In an extension to get benzo-fused
derivatives of those compounds, that can increase their biological potencial, we are
reporting here the synthesis of 4-arylphenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-
diones, which have been tested as antimycobacterial agents.
2
. Results and discussion
.1. Chemistry
2
For the preparation of the new tetracyclic pyrazolic derivatives 4, we have carried
out a microwave irradiation on an equimolar mixture of 5-amino-3-methyl-1-
phenylpyrazole 1, 2-hydroxynaphthoquinone 2 and benzaldehydes 3 during 3 min.
From this approach the new 4-aryl-3-methyl-1-phenylbenzo[h]pyrazolo[3,4-
b]quinolin-5,6-diones 4 were isolated (Scheme 1) in an easy way and in good
yields (80-85 %) as stable crystalline solids (see Experimental Section).
Scheme 1
In all cases this three-component reaction led regioselectively to non-linear
tetracyclic compounds 4 (but not 5) and their structures were characterized by
spectroscopic and analytical methods.
The NMR spectroscopic data were consistent with the proposed structures 4.
1
Regarding to the tetracyclic system, for example compound 4a exhibits a H NMR
spectrum with a singlet at 1.93 ppm corresponding to CH -group of pyrazolic ring
3
at position 3. At 7.60, 7.83, 8.15 and 8.81 ppm appear four signals, each one
integrating for one proton, assigned to H-7, H-8, H-9 and H-10 respectively,
corresponding to protons of the benzo-moiety.
6

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13
The whole carbon skeleton was assigned from C NMR spectra, combined with
DEPT and bidimensional experiments (COSY, HSQC and HMBC). Compound 4a
13
showed in its C NMR spectrum at 14.2 ppm the signal for the methyl group and at
79.9 and 180.5 ppm the signals for C-5 and C-6 carbonyl groups. The signals for
1
C11a and C-10b appeared at higher δ values, 150.6 and 153.7 ppm. In contrast,
carbon atoms C-3a and C-4a appeared unusually at low chemical shifts, 116.9 and
119.8 ppm respectively. Regarding to the mass spectra, all products 4 exhibited
similar behavior in their fragmentation pattern, showing the molecular ion peak
along with a typical loss of the aryl group at position 4 as the base peak.
The regioselective formation of the non-linear (angular) tetracyclic structure 4
instead of a linear one 5 was unambiguously confirmed by X-ray diffraction
experiments [19] (figure 1).
Figure 1. ORTEP drawing for 4a and 4b
It is worth mentioning that compounds 4a-c,e,g,h have previously been
synthesized elsewhere in solution but with reaction times between 10- 26 minutes
(
61-91 % [20]) and 60-90 minutes (87-91 % [21]). In order to verify the versatility of
the above described method, we carried out, the three-component reaction
between the 5-amino-1-NH-pyrazole 6, 2-hydroxynaphthoquinone and
benzaldehydes 3 under the same reaction conditions. In this case, the non-linear
,11-dihydrobenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 7 were also obtained, in a
2
4
regioselective manner, also in good isolated yields (Scheme 2). Although,
formation of the regioisomer 8 during the cyclization process was also expected
owing to the presence of an additional nucleophilic center (1-NH) in aminopyrazole
6
[10, 15]; the exclusive formation of compounds 7 was confirmed by spectroscopic
analysis. Possible formation of benzopyrazoloquinazolines 8 was finally excluded
based on NMR examination, mainly because of the absence of signals for H-3 (H-4
1
in precursors 6) in their H-nmr spectra.
7

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Scheme 2
1
Thus, H-NMR spectra of compounds 7a-f showed three singlets, which
corresponding to the protons H-4 (between 5.29- 5.70 ppm) and two deuterium
exchangeable protons 1-NH (between 12.02-12.90 ppm), 11-NH (around 10.07-
10.38 ppm) respectively, and signals corresponding to aromatic protons found
13
between 6.69 and 8.75 ppm. Type of carbon atoms was determined by C-NMR
DEPT-135 experiment) spectroscopy, and it was consistent with the proposed
(
structure 7. Structures of compounds 7 were thoroughly confirmed by NMR and
HR-MS.
It is important to remark that in some cases the reaction led to the formation of
aromatized
3,4-diaryl-1H-benzo[h]pyrazolo[3,4-b]quinoline-5,6-diones
9a-g,
depending on the type od substituent in the Ar-ring (Scheme 3). We assume that
the reaction proceeded through the initial formation of dihydroderivatives type 7,
which were then aromatized under the reaction conditions. Under inert atmosphere
this reaction also led to the formation of the aromatized products 9.
Scheme 3
The structure of benzopirazolo[3,4-b]quinolines 9a-g were confirmed by NMR and
HR-MS.
2.2. Antimycobacterial screening
Results of the antimycobacterial studies of thirteen selected pyrazolo[3,4-
b]quinolines derivates 4, 7, 9 are consigned in Table 1. Six of such compounds (i.e.
4a, 4b, 4g, 7d, 7f and 9b) exhibited antimycobacterial activity; three of them 4a, 4b
and 4g showed the highest antitubercular activity (MIC between 2-4 µg/mL for
8

ACCEPTED MANUSCRIPT
Mycobacterium tuberculosis species) and the remaining 7d, 7f and 9b showed
moderate activity (MIC between 8-16 µg/mL).
Table 1. The In vitro activity (MIC µg/mL) of pyrazolo[3,4-b]quinolinedione
derivatives against fifteen Mycobacterium spp.
The antimicrobial activity of our compounds might be associated with the fact, that
the pyrazolo[3,4-b]quinolines have previously shown to be bactericidal with an in
vitro significant inhibitory activity on bacterial serine/threonine protein kinases [22],
because these kinases are necessary for an adequate response to environmental
stimuli and functionality of these enzymes [23]. Owing to they are essential in
nutrition and cell division in mycobacteria, kinases have emerged as novel
therapeutic target eukaryotic-like [24], and their inhibitors become into important
candidates for new anti-bacterial drugs development, with the ability to avoid
resistance mechanisms [23-25].
In order to correlate a qualitative structure–activity relationship described above,
with quantitative parameters, we calculated the logP for several benzopyrazolo[3,4-
b]quinolinediones (4, 7 and 9), and attempted to find a relationship between these
values and their MICs (in µg/mL). For this study was selected MTB2556, the
culture cell that was inhibited by a larger number of compounds (Table 2).
Table 2. The In vitro activity (MIC µg/mL) of compounds 4, 7 and 9 assayed
against the MTB2556 and their calculated Log P and dipole.
For the calculation of Log P and dipole we used quantum mechanical at
semiempirical level using Mopac, with the parametric method 3 (PM3). The
molecular modeling were prepared using CS Chem-Office Software version 9.0
(Cambridge software) [26]. The models were minimizedation until the root mean
square (RMS) gradient value reached a value smaller than 0.0001 kcal/mol. The
lowest energy structure was used for each molecule to calculate log P values and
dipole, as lipophilicity measures. Antimicrobial activity against Mycobacterium
9

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species depends on how efficiently solutes cross the cell wall that constitutes an
efficient permeability barrier. Hydrophobic cell wall, principally in the form of very
long-chain fatty acids (mycolic acids), obviously acts as a barrier to penetration of
hydrophilic solutes, this fact causes that mycobacteria would be often more
susceptible to less-polar compounds [25]. The derivates (4a, 4g and 9b) showed
appropriate values for Log P and dipole agree with their antimicrobial activity. In
contrast, the 7f derivative showed the lowest lipophilicity in comparison with the
other reported compounds. However the log P and dipole values for compound 7f
disagree with its antimicrobial activity, what can be attributed to the presence of the
nitro group in the molecule. On the other hand, the remaining compounds exhibited
low values of Log P and dipole, therefore less antimycobacterial activities (Figure
2).
Figure 2. LogP vs. MIC and dipole vs. MIC of benzopyrazolo[3,4-b]quinolindiones
derivates against MTB2556 culture cell.
3. Conclusion
We are herein describing the synthesis and antimycobacterial activity of novel
series of 4-arylbenzopyrazolo[3,4-b]quinolindiones 4, 7 and 9. These new
compounds were obtained in a straightforward manner via a three-component
approach assisted by microwave irradiation. These 4-arylbenzopyrazolo[3,4-
b]quinolindiones have been tested against fifteen Mycobacterium, and six of them
have shown antimycobacterial activity. The highest inhibitory activity with MIC ≤ 2
µg/mL for compounds 4a, 4b and 4g was related with their higher lipophilicity and
lesser polarity within these series.
4. Experimental
Melting points were determined on a Büchi melting point apparatus and are
uncorrected. IR spectra were recorded on Shimadzu FTIR 8400
spectrophotometer in KBr disks. H and C NMR spectra were recorded on a
a
1
13
1
0

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Bruker Avance 400 spectrophotometer operating at 400 MHz and 100 MHz
respectively, using DMSO-d and CDCl as solvents and tetramethylsilane as
6
3
internal standard. Mass spectra were run on a SHIMADZU-GCMS 2010-DI-2010
spectrometer (equipped with a direct inlet probe) operating at 70 eV. High
Resolution Mass Spectra (HRMS) by electron impact were recorded on a
Micromass AutoSpec-Ultima, magnetic sector mass spectrometer at 70 eV. The
elemental analyses were obtained using a LECO CHNS-900 elemental analyzer
and the values are within ± 0.4% of the theoretical values. Microwave experiments
were carried out on a focused microwave reactor (300W CEM Discover). Silica gel
aluminum plates (Merck 60 F254) were used for analytical TLC. All chemicals
including the starting 2-hydroxynaphthoquinone 2 and benzaldehydes 3 were
purchased from Aldrich, Fluka and Acros (analytical reagent grades) and were
used without further purification. The key amines 1 and 6 were obtained according
to a procedure previously described by us [27].
4.1. Chemistry
4.1.1.
General
procedure
for
the
synthesis
of
4-aryl-3-methyl-1-
phenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 4a-h, 3,4-diaryl-4,11-dihydro-1H-
benzo[h]pyrazolo[3,4-b]quinolin(1H,11H)-5,6-diones 7a-f and 3,4-diaryl-1H-
benzo[h]pyrazolo[3,4-b]quinolin-5,6-diones 9a-g.
A mixture containing 1.0 mmol of 5-amino-3-methyl-1-phenylpyrazole 1 or 5-amino-
3-arylpyrazole 6, 2-hydroxynaphthoquinone 2 (1.0 mmol) and the appropriate
o
aldehyde 3 (1.0 mmol) was exposed to microwave irradiation for 3 min (120 C and
200 W). The solids obtained were purified by recrystallization from ethanol.
4.1.1.1. 3-Methyl-1,4-diphenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-dione 4a: yellow
1
solid, mp 272-273 ºC (85%) (272-273 °C (83%) [20]). H NMR (400 MHz, CDCl
3
) δ
(
ppm): 1.93 (s, 3H, CH ), 7.25-730 (m, 2H, H Ar), 7.39 (t, 1H, H NPh, J = 8,1),
3 m p
7
.50-7.54 (m, 3H, H Ar, H Ar), 7.60 (m, 3H, H-8, H NPh), 7.83 (t, 1H, H-9, J =
p o m
1
1

ACCEPTED MANUSCRIPT
8
1
1
1
4
o
.1), 8.15 (d, 1H, H-7, J = 7.9 Hz), 8.31 (d, 2H, H NPh, J = 8.0 Hz), 8.81 (d, 1H, H-
13
0, J =8.1 Hz). C NMR (100 MHz, CDCl ) δ (ppm): 14.2 (CH ), 117.0 (C-3a),
3
3
19.8 (C-4a), 121.3 (C
o
NPh), 126.5 (C
p
NPh), 127.0 (C
m
NPh), 127.2 (C-10),
28.4 (C , C Ar), 129.0 (C-7), 129.1 (C Ar), 131.3 (C-8), 131.7 (C-6a), 132.6 (C-
o
p
m
), 135.9 (C-9), 137.4 (C-10a), 138.7 (C NPh), 146.5 (C-3), 150.6 (C-11a), 152.2
i
+
(C
i
Ar), 153.7 (C-10b), 179.9 (C-5), 180.5 (C-6). EI MS (70eV): m/z (%): 415 (M ,
16), 386 (100). Anal. Calcd for C H N O : C:76.40, H: 4.27, N: 9.90, found: C:
27 17 3 2
76.07, H: 3.96, N: 9.66.
4.1.1.2. 3-Methyl-1-phenyl-4-(4-tolyl)benzo[h]pyrazolo[3,4-b]quinolin-5,6-dione 4b:
1
red solid, mp 273-274 ºC (85%) (276-277 ºC (81-84 %) [20, 21]). H NMR (400
MHz, CDCl ) δ (ppm): 1.97 (s, 3H, CH ), 2.48 (s, 3H, CH Ar) 7.17 (d, 2H, H Ar, J
3
3
3
m
=
7.2 Hz), 7.32 (d, 2H, H
o
p
Ar, J = 7.2 Hz), 7.38 (t, 1H, H NPh, J = 8.0 Hz), 7.59 (m,
3
H, H-8, H NPh), 7.82 (t, 1H, H-9, J = 8.0 Hz), 8.13 (d, 1H, H-7, J = 8.1 Hz), 8.31
m
13
(
d, 2H, H NPh, J = 8.0 Hz), 8.79 (d, 1H, H-10 J = 8.1). C NMR (100 MHz, CDCl )
o
3
δ (ppm): 14.3 (CH
3
), 21.4 (CH
3
Ph), 117.1 (C-3a), 120.0 (C-4a) 120.2 (C
p
Ar), 121.3
(
C NPh), 126.5 (C NPh), 127.0 (C Ar), 127.2 (C-10), 128.9 (C-7), 129.0 (C Ar),
o
p
m
o
1
1
5
m
29.1 (C NPh), 131.2 (C-8), 131.6 (C-6a), 132.8 (C-4) 135.8 (C-9), 137.4 (C-10a),
38.7 (C NPh), 146.6 (C-3), 150.5 (C-11a), 152.6 (C Ar) 153.7 (C-10b), 179.9 (C-
i
i
+2
+
), 180.5 (C-6). EI MS (70eV): m/z (%): 431/429 (M /M , 63/24), 400 (100), 386
19 3 2
(95). Anal. Calcd for C28H N O : C: 77.23, H: 4.55, N: 9.65, found: C: 77.43, H:
4.55, N: 9.65.
4
.1.1.3.
,6-dione 4c: Red solid, mp 280-281 ºC (80%) (279-281 C, 83% [20]). H NMR
) δ (ppm): 2.00 (s, 3H, CH ), 3.91 (s, 3H, OCH ), 7.50 (d, 2H, H
Ar, J = 8.7 Hz), 7.22 (d, 2H, H Ar, J = 8.7 Hz), 7.38 (t, 1H, H NPh, J = 7.7 Hz),
3-Methyl-4-(4-methoxyphenyl)-1-phenylbenzo[h]pyrazolo[3,4-b]quinolin-
o
1
5
(400 MHz, CDCl
3
3
3
m
o
p
m
7.59 (m, 3H, H-8, H Nph), 7.82 (t, 1H, H-9 J = 8.3 Hz), 8.13 (d, 1H, H-7, J = 8.3
13
Hz), 8.30 (d, 2H, H NPh, J = 7.7 Hz), 8.78 (d, 1H, H-10, J = 8.3 Hz). C NMR
o
(
100 MHz, CDCl ) δ (ppm): 14.3 (CH ), 56.3 (OCH ), 113.9 (C Ar), 117.3 (C-3a),
3
3
3
m
1
20.2 (C-4a), 121.3 (C
o
NPh), 126.5 (C
p
o
NPh), 127.2 (C-10), 128.6 (C Ar), 128.9
1
2

ACCEPTED MANUSCRIPT
(
C-7), 129.1 (C
m
NPh), 131.2 (C-8), 129.3 (C-6a), 131.6 (C-4) 135.9 (C-9), 137.4
(
C-10a), 138.7 (C NPh), 146.6 (C-3), 150.5 (C-11a), 152.4 (C Ar) 153.7 (C-10b),
i
i
+
1
p
59.9 (C Ar), 179.9 (C-5), 180.5 (C-6). EI MS (70eV): m/z (%): 445 (M , 22), 416
(
100), 386 (26). HR-MS calcd for C H N O 445.1426, found 445.1425.
28 19 3 3
4.1.1.4.
3-Methyl-4-(3,4,5-trimethoxyphenyl)-1-phenylbenzo[h]pyrazolo[3,4-
1
b]quinolin-5,6-dione 4d: Yellow solid, mp 314-315 ºC (85%). H NMR (400 MHz,
CDCl
3 3
) δ (ppm): 2.07 (s, 3H, CH ), 3,86 (s, 6H, OCH3m Ar), 3.97 (s, 3H, OCH3p Ar),
6
7
8
.51 (s, 2H, H Ar), 7.40 (t, 1H, H NPh, J = 7.9 Hz), 7.60 (m, 3H, H-8, H NPh),
o
p
m
.83 (t, 1H, H-9, J = 7.8 Hz), 8.15 (d, 1H, H-7, J = 7.8 Hz), 8.31 (d, 2H, H NPh, J =
o
13
3
.0 Hz), 8.80 (d, 1H, H10, J = 7.8 Hz). C NMR (100 MHz, CDCl ) δ (ppm): 14.2
(
CH ), 56.3 (OCH ), 61.1 (OCH ), 104.6 (C Ar), 117.0 (C-3a), 120.2 (C-4a),
3
3m
3p
o
1
21.4 (C
o
p m
NPh), 126.6 (C NPh), 127.3 (C-10), 129.1 (C-7), 129.2 (C NPh), 131.2
(
C-6a), 131.4 (C-8), 131.6 (C-4) 136.0 (C-9), 137.4 (C-10a), 138.3 (C Ar), 138.6
p
(
C NPh) 146.6 (C-3), 150.6 (C-11a), 152.0 (C Ar), 153.5 (C Ar), 153.7 (C-10b),
i
i
m
+
1
79.6 (C-5), 180.4 (C-6). EI MS (70eV): m/z (%): 505 (M , 93), 476 (47.7), 462
(
57), 462 (76), 446 (100), 430 (36). HR-MS calcd for C H N O 505.1638, found
30 23 3 5
23 3 5
505.1634. Anal. Calcd for C30H N O : C: 70.03, H: 4.70, N: 8.17, found: C: 70.39,
H: 4.53, N: 8.18.
4.1.1.5. 4-(4-Chlorophenyl)-1-phenyl-3-methylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-
1
dione 4e: Brown solid, mp 328-329ºC (85%) (248-250ºC (85%) [21]). H NMR (400
MHz, CDCl
3 3
) δ (ppm): 1.91 (s, 3H, CH ), 7.40 (d, 2H, H
o
Ar, J = 8.3 Hz), 7.42 (t,
1
H, H NPh, J = 7.9 Hz), 7.57 (d, 2H, H Ar, J = 8.3 Hz), 7.63 (m, 2H, H NPh),
p
m
m
7.68 (t, 1H, H-8, J = 7.6 Hz), 7.92 (t, 1H, H-9, J = 7.6 Hz), 8.03 (d, 1H, H-7, J = 7.6
13
o
Hz), 8.27 (d, 2H, H NPh, J = 8.0 Hz), 8.73 (d, 1H, H-10, J = 7.5 Hz). C NMR
(
100 MHz, CDCl ) δ (ppm): 14.3 (CH ), 116.6 (C-3a), 120.9 (C-4a), 121.5 (C
3 3
o
NPh), 126.9 (C
p
NPh), 127.1 (C-10), 128.5 (C
m
Ar), 128.6 (C-7), 129.7 (C
m
NPh),
1
1
1
29.9 (C Ar), 131.8 (C-8), 132.4 (C Ar), 133.6 (C-6a), 135.5 (C-4) 136.8 (C-9),
o
p
37.2 (C-10a), 138.9 (C NPh), 145.9 (C-3), 150.0 (C-11a), 150.6 (C Ar) 153.8 (C-
i
i
+2
+
0b), 179.7 (C-5), 180.2 (C-6). EI MS (70eV): m/z: 451/449 (M /M , 8/26), 420
1
3

ACCEPTED MANUSCRIPT
3 2
(100), 386 (17). HR-MS calcd for C27H16ClN O 449.0931 found 449.0930. Anal.
Calcd for C H ClN O : C, 72.08; H, 3.58; N, 9.34. found: C, 71.60; H, 3.46; N,
27
16
3
2
8.86.
4.1.1.6. 4-(4-Bromophenyl)-3-methyl-1-phenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-
1
dione 4f: yellow solid, mp 263-264 ºC (80%). H NMR (400 MHz, CDCl
3
) δ (ppm):
1
.99 (s, 3H, CH ), 7.18 (d, 2H, H Ar, J = 8.3 Hz), 7.40 (t, 1H, H NPh, J = 7.8 Hz),
3
m
p
7
.60 (m, 3H, H-8, H
m
o
NPh), 7.66 (d, 2H, H Ar, J = 8.3 Hz), 7.83 (t, 1H, H-9, J = 7.9
Hz), 8.14 (d, 1H, H-7, J = 7.9 Hz), 8.30 (d, 2H, H NPh, J = 7.8 Hz), 8.78 (d, 1H, H-
o
13
1
0 J = 7.9 Hz). C NMR (100 MHz, CDCl ) δ (ppm): 14.5 (CH ), 116.7 (C-3a),
3
3
119.7 (C-4a), 121.4 (C
o
NPh), 122.7 (C
p
Ar), 126.7 (C
p
NPh), 127.2 (C-10), 128.8
(
C NPh), 129.1 (C-7), 129.2 (C Ar), 131.4 (C-8), 131.6 (C Ar), 133.3 (C-6a),
m
m
o
1
1
4
34.8 (C-4), 136.0 (C-9), 137.1 (C-10a), 138.6 (C
i
NPh), 146.2 (C-3), 150.5 (C-
1a), 150.7 (C Ar) 153.8 (C-10b), 179.9 (C-5), 180.3 (C-6). EI MS (70eV): m/z (%):
i
+2
+
95/493 (M /M , 32/30), 466 (100), 356 (52), 77 (15). Anal. Calcd for
: C: 64.43, H: 3.40, N: 8.35, found: C: 64.85, H: 3.13, N: 8.08.
27 3 2
C H16BrN O
4.1.1.7. 4-(4-Fluorophenyl)-3-methyl-1-phenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-
1
dione 4g: yellow solid, mp 289-290 ºC (85%) (283-284 ºC (85%) [20, 21]). H NMR
(
400 MHz, CDCl ) δ (ppm): 1.97 (s, 3H, CH ), 7.21 (d, 2H, H Ar, J = 9.5 Hz), 7.28
3
3
m
(
d, 2H, H
o
p m
Ar, J = 9.5 Hz), 7.40 (t, 1H, H Ar J = 7.6, Hz), 7.60 (m, 3H, H-8, H
NPh), 7.83 (t, 1H, H-9, J = 8.4 Hz), 8.14 (d, 1H, H-7, J = 8.4 Hz), 8.30 (d, 2H, Ho
13
NPh, J = 7.6 Hz), 8.79 (d, 1H, H-10, J = 8.4 Hz). C NMR (100 MHz, CDCl
3
) δ
(
ppm): 14.5 (CH ), 116.7 (C-3a), 119.7 (C-4a), 121.4 (C NPh), 122.7 (C Ar),
3
o
p
1
26.7 (C NPh), 127.2 (C-10), 128.8 (C NPh), 129.1 (C-7), 129.2 (C Ar), 131.4
p m m
o
(C-8), 131.6 (C Ar), 133.4 (C-6a), 134.8 (C-4), 136.0 (C-9), 137.1 (C-10a), 136.6
(
C NPh), 146.2 (C-3), 150.5 (C-11a), 150.7 (C Ar) 153.8 (C-10b), 179.9 (C-5),
i
i
+
1
80.3 (C-6). EI MS (70eV): m/z (%): 433 (M , 11), 405 (100). Anal. Calcd for
C H FN O : C: 74.82, H: 3.72, N: 9.69, found: C: 74.62, H: 3.69, N: 9.45.
27
16
3
2
1
4

ACCEPTED MANUSCRIPT
4.1.1.8.
3-Methyl-4-(4-nitrophenyl)-1-phenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-
1
dione 4h: yellow solid, mp 338-340 ºC (80%) (334-336 ºC (83%) [21]). H NMR
(400 MHz, CDCl
3 3 p
) δ (ppm): 1.94 (s, 3H, CH ), 7.42 (t, 1H, H NPh, J = 7.8 Hz),
7
8
2
.50 (d, 2H, H Ar, J = 8.1 Hz), 7.61 (m, 3H, H-8, H NPh), 7.86 (t, 1H, H-9, J =
m
m
.0 Hz), 7.15 (d, 1H, H-7, J = 8.0 Hz), 8.29 (d, 2H, H NPh, J = 7.8 Hz), 8.40 (d,
o
13
o 3
H, H Ar J = 8.1 Hz), 8.80 (d, 1H, H-10, J = 8.0 Hz). C NMR (100 MHz, CDCl ) δ
(
ppm): 14.5 (CH ), 116.7 (C-3a), 119.7 (C-4a), 121.4 (C NPh), 122.7 (C Ar),
3
o
p
126.7 (C
p
NPh), 127.2 (C-10), 128.8 (C
m
Ar), 129.1 (C-7), 129.2 (C
m
NPh), 131.4
(C-8), 131.6 (C Ar), 133.8 (C-6a), 134.8 (C-4), 136.0 (C-9), 136.6 (C NPh), 137.1
o i
(
C-10a), 146.2 (C-3), 150.5 (C-11a), 150.7 (C Ar) 153.8 (C-10b), 179.9 (C-5),
i
+
1
80.3 (C-6). EI MS (70eV): m/z %: 460 (M , 27), 431 (100), 415 (69), 385 (98), 77
(
22). Anal. Calcd for C H N O : C: 67.78, H: 3.79, N: 11.71, found: C: 67.99, H:
27 16 4 4
3.64, N: 11.46.
4.1.1.9.
3,4-Diphenyl-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-b]quinolin(1H,11H)-
1
5
,6-dione 7a: red solid, mp 315-317 ºC (80%). H NMR (400 MHz, CDCl
3
) δ
(
ppm): 5.70 (s, 1H, H-4), 7.00 (t, 1H, H Ar, J = 7.2 Hz), 7.12 (t, 2H, H Ar, J = 7.2
p
m
Hz), 7.19 (d, 2H, H
o
Ar, J = 7.2 Hz), 7.32 (t, 1H, H
p
R, J = 7.4 Hz), 7.41 (t, 2H, H
m
R, J = 7.4 Hz), 7.54 (d, 2H, H R, J = 7.4 Hz), 7.73 (t, 1H, H-8, J = 7.5 Hz), 7.79 (t,
o
1
1
3
1
1
1
4
H, H-9, J = 7.5 Hz), 7.90 (d, 1H, H-7, J = 7.5 Hz), 7.99 (d, 1H, H-10, J = 7.5 Hz),
1
3
0.38 (s, 1H, NH-11), 12.83 (s, 1H, NH-1). C NMR (100 MHz, CDCl
3
) δ (ppm):
5.4 (C-4), 113.4 (C-4a), 114.5 (C-3a), 125.6 (C-10), 125.7 (C-7), 126.2 (C R),
o
p m o p i m
27.8 (C R), 128.1 (C R), 128.8 (C Ar), 130.2 (C Ar), 130.6 (C R), 132.4 (C Ar),
32.7 (C-8), 133.0 (C Ar), 134.1 (C-6a), 134.8 (C-3), 136.0 (C-9), 137.2 (C-10a),
i
46.4 (C-11a), 157.4 (C-10b), 180.4 (C-5), 180.7 (C-6). EI MS (70eV): m/z (%):
+
03 (M , 24), 326 (100). HR-MS calcd for C26
17 3
H N O
2
403.1321, found 403.1319.
4.1.1.10.
3-Phenyl-4-(4-tolyl)-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin(1H,11H)-5,6-dione 7b: purple solid, mp 315-317 ºC (80%). H NMR
(
400 MHz, CDCl ) δ (ppm): 2.11 (s, 3H, CH ), 5.66 (s, 1H, H-4), 6.90-6.95 (m, 3H,
3
3
H
p
R, H
m
Ar), 7.08 (d, 2H, H
o
m
Ar, J = 7.2 Hz), 7.41 (t, 2H, H R, J = 7.4 Hz), 7.56 (d,
1
5

ACCEPTED MANUSCRIPT
2
7
1
1
1
1
1
1
7
H, H
o
R, J = 7.4 Hz), 7.72 (t, 1H, H-8, J = 7.5 Hz), 7.78 (t, 1H, H-9, J = 7.5 Hz),
.89 (d, 1H, H-7, J = 7.5 Hz), 7.98 (d, 1H, H-10, J = 7.5 Hz), 10,34 (s, 1H, NH-11),
1
3
2,90 (s, 1H, NH-1). C NMR (100 MHz, CDCl
3 3
) δ (ppm): 21.1 (CH ), 35.4 (C-4),
13.4 C-4a), 114.5 (C-3a), 125.6 (C-10), 125.7 (C-7), 126.2 (C R), 127.8 (C R),
o
p
28.1 (C R), 128.8 (C Ar), 130.1 (C Ar), 130.7 (C R), 132.4 (C Ar), 132.7 (C-8),
m
o
p
i
m
33.0 (C
i
Ar), 134.10 (C-6a), 134.7 (C-3), 136.0 (C-9), 137.2 (C-10a), 146.4 (C-
+
1a), 157.5 (C-10b), 180.4 (C-5), 180.7 (C-6). EI MS (70eV): m/z (%): 417 (M ,
5), 326 (100). Anal. Calcd for C27
19 3 2
H N O : C: 77.68, H: 4.59, N: 10.07, found: C:
7.59, H: 4.53, N: 10.15.
4.1.1.11.
4-(4-Methoxyphenyl)-3-phenyl-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin(1H,11H)-5,6-dione 7c: purple solid, mp 313 ºC (70%). H NMR (400
MHz, CDCl
3
) δ (ppm): 3.61 (s, 3H, CH
3 m
), 5.67 (s, 1H, H-4), 6.71 (d, 2H, H Ar, J =
8
.8 Hz), 7.12 (d, 2H, H Ar, J = 8.8 Hz), 7.33 (t, 1H, H R, J = 8.3 Hz), 7.43 (t, 2H,
o
p
H R, J = 8.3 Hz), 7.58 (d, 2H, H R, J = 8.3 Hz), 7.73 (t, 1H, H-8, J = 7.3 Hz), 7.79
m
o
(t, 1H, H-9 J = 7.3 Hz), 7.91 (d, 1H, H-7, J = 7.3 Hz), 7.99 (d, 1H, H-10, J = 7.3 Hz),
1
3
1
3
1
1
1
6
4
0.34 (s, 1H, NH-11), 12.85 (s, 1H, NH-1). C NMR (100 MHz, CDCl ) δ (ppm):
3
5.6 (C-4), 55.3 (OCH
3 m p
), 113.8 (C Ar), 114.2 (C-4a), 115.8 (C-3a), 125.9 (C R),
25.9 (C-7), 126.2 (C-10), 126.5 (C R), 128.7 (C Ar), 128.6 (C Ar), 129.3 (C R),
o
o
i
m
30.6 (C R), 132.9 (C-6a), 133.1 (C-8), 134.2 (C-3), 135.2 (C-10), 139.0 (C-9),
i
p
40.3 (C-10a), 146.8 (C-11a), 156.9 (C-10b), 157.9 (C Ar), 170.8 (C-5), 181.2 (C-
+
). EI MS (70eV): m/z (%): 433 (M , 58), 326 (100). HR-MS calcd for C H N O
27
19
3
3
33.1426, found 433.1424.
4.1.1.12.
4-Phenyl-3-(4-tolyl)-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin(1H,11H)-5,6-dione 7d: red solid, mp > 320 ºC (70%). H NMR (400
MHz, CDCl ) δ (ppm): 2.28 (s, 3H, CH ), 5.63 (s, 1H, H-4), 7.02 (t, 1H, H Ar, J =
3
3
p
7
.9 Hz), 7.09-7.15 (m, 4H, H
m
Ar, H
o
Ar), 7.20 (d, 2H, H
m
R, J = 7.9 Hz), 7.50 (d, 2H,
H R, J = 7.9 Hz), 7.66 (t, 1H, H-8, J = 7.5 Hz), 7.82 (t, 1H, H-9, J = 7.5 Hz), 8.00
o
(d, 1H, H-7, J = 7.5 Hz), 8.52 (d, 1H, H-10, J = 7.5 Hz), 10.01 ( broad s, 1H, NH-
13
1
3 3
1), 12.50 ( broad s, 1H, NH-1). C NMR (100 MHz, CDCl ) δ (ppm): 20.6 (CH ),
1
6

ACCEPTED MANUSCRIPT
p
35.0 (C-4), 115.3 (C-3a), 119.6 (C-4a), 125.5 (C-10), 125.7 (C Ar), 125.6 (C-7),
1
25.9 (C Ar), 128.8 (C Ar), 129.3 (C R), 130.2 (C-8) 132.5 (C R), 132.6 (C Ar),
o
m
m
p
i
i o
134.1 (C-3), 134.7 (C R), 137.5 (C-6a), 138.6 (C R), 139.8 (C-9), 140.0 (C-10a),
1
55.2 (C-11a), 158.3 (C-10b), 180.3 (C-5), 180.5 (C-6). Anal. calcd for C H N O :
27 19 3 2
C: 77.68, H: 4.59, N: 10.07, found: C: 77.53, H: 4.49, N: 10.21.
4.1.1.13.
3-(4-Chlorophenyl)-4-phenyl-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin(1H,11H)-5,6-dione 7e: purple solid, mp > 300 (d) ºC (75%). H NMR
(400 MHz DMSO-d ) δ (ppm): 5.70 (s, 1H, H-4), 7.00 (t, 1H, H R, J = 7.3 Hz), 7.12
6 p
(
t, 2H, H R, J = 7.3 Hz), 7.19 (d, 2H, H R, J = 7.3 Hz), 7.47 (d, 2H, H Ar, J = 8.4
m
o
o
Hz), 7.57 (d, 2H, H
m
Ar, J = 8.4 Hz), 7.71 (t, 1H, H-8, J = 7.5 Hz), 7.77 (t, 1H, H-9, J
=
1
4
7.5 Hz), 7.88 (d, 1H, H-7, J = 7.5 Hz), 7.98 (d, 1H, H-10, J = 7.5 Hz), 10.38 (s,
13
H, NH-11), 12.89 (s, 1H, NH-1). C NMR (100 MHz, DMSO-d
), 113.5 (C-3a), 126.7 (C-10), 127.7 (C Ar), 127.9 (C R), 128.6 (C-7), 130.3 (C
o
6
) δ (ppm): 35.4 (C-
m
m
Ar), 130.9 (C R), 131.7 (C-8), 133.0 (C-6a), 133.2 (C R), 135.3 (C-3), 136.0 (C-9),
o
p
1
37.1 (C-10a), 132.5 (C
i
Ar), 147.1 (C
i
R), 149.3 (C
p
Ar), 152.8 (C-11a), 153.3 (C-
+2
+
1
0b), 179.8 (C-5), 180.0 (C-6). EI MS (70eV): m/z: 439/437 (M /M , 14/38), 360
: C: 71.32, H: 3.68, N: 9.60, found: C:
(100), 210 (22). Anal. calcd for C26
H N O
16 3 2
77.49, H: 3.57, N: 9.72.
4.1.1.14.
3-(4-Nitrophenyl)-4-phenyl-4,11-dihydro-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin(1H,11H)-5,6-dione 7f: Red solid, mp 260-261 ºC (70%). H NMR (400
MHz, CDCl
3 m p o
) δ (ppm): 5,61 (s, 1H, H4), 7.01 (m, 3H, H Ar, H Ar), 7.09 (d, 2H, H
Ar J = 7.9 Hz), 7.17 (d, 2H, H R, J = 8.3 Hz), 7.67 (t, 1H, H-8, J = 8.0 Hz), 7.83 (d,
o
2
H, H R, J = 8.3 Hz), 8.14 (t, 1H, H-9, J = 8.0 Hz), 8.30 (d, 1H, H-7, J = 8.0 Hz),
m
8.75 (d, 1H, H-10, J = 8.1 Hz), 10.11 (broad s, 1H, NH-11), 12.50 (broad s, 1H,
NH-1). Compound 7f is barely soluble in dimethyl sulfoxide or any other solvent
normally used for nmr spectroscopy; thus, made the registration of a high
13
+
resolution C-nmr spectrum impossible. EI MS (70eV): m/z (%): 446 (M , 39), 417
99), 371 (100), 186 (59). HR-MS calcd for C H N O 446.1015, found 446.1011.
(
27
16
4
5
1
7

ACCEPTED MANUSCRIPT
4.1.1.15. 4-(4-Nitrophenyl)-3-(4-tolyl)-1H-benzo[h]pyrazolo[3,4-b]quinolin-5,6-dione
1
9
a: Yellow solid, mp >350 ºC (75%). H NMR (400 MHz, DMSO-d ) δ (ppm): 2.17
6
(
(
(
s, 3H, CH
3 m o
), 6.74 (d, 2H, H Ar, J = 7.8 Hz), 6.81 (d, 2H, H Ar, J = 7.8 Hz), 7.31
d, 2H, H R J = 8.0 Hz), 7.68 (t, 1H, H-8, J = 7.9 Hz), 7.91 (m, 3H, H-9, H R), 8.02
o
m
13
d, 1H, H-7, J = 7.9 Hz), 8.75 (d, 1H, H-10, J = 8.1 Hz), 14.34 (s, 1H, NH-1).
C
6 3
NMR (100 MHz, DMSO-d ) δ (ppm): 21.1 (CH ), 113.2 (C-3a), 120.0 (C-4a), 122.9
(
C Ar), 126.7 (C-10), 128.3 (C Ar), 128.6 (C-7), 129.2 (C R), 129.8 (C-6a), 129.9
m
o
m
(C
o
p i
R), 131.2 (C R), 131.8 (C-8), 132.3 (C R), 132.3 (C-4), 136.0 (C-9), 137.1 (C-
1
1
3
0a), 137.5 (C Ar), 144.0 (C Ar), 148.1 (C-11a), 152.7 (C Ar), 153.2 (C-10b),
p i p
+
79.4 (C-5), 179.6 (C-6). EI MS (70eV): m/z (%):460 (M , 11), 431 (32), 417 (100),
13 (61). HR-MS calcd for C27 460.1170, found 460.1170.
16 4 4
H N O
4.1.1.16.
3-(4-Chlorophenyl)-4-(4-methoxyphenyl)-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin-5,6-dione 9b: purple solid, mp 270 (d) ºC (70%). H NMR (400 MHz
DMSO-d ) δ (ppm): 3.74 (s, 3H, OCH ), 6.66 (d, 2H, H Ar, J = 8.8), 6.88 (d, 2H,
6
3
m
H
o
o m
R, J = 8.5), 6.97 (d, 2H, H Ar, J = 8.8), 7.08 (d, 2H, H R, J = 8.5), 7.67 (t, 1H,
H-8, J = 7.6), 7.92 (t, 1H, H-9, J = 7.6), 8.01 (d, 1H, H-7, J = 7.6), 8.75 (d, 1H, H-
13
1
0, J = 7.6), 14.35 (s, 1H, NH-1). C NMR (100 MHz, DMSO-d
6
) δ (ppm): 55.2
(
OCH ), 113.0 (C Ar), 113.3 (C-3a), 120.2 (C-4a), 126.3 (C-10), 127.2 (C Ar),
3
o
m
1
1
1
27.8 (C Ar), 128.0 (C-7), 128.1 (C-8), 129.6 (C R), 130.4 (C R) 131.2 (C Ar),
p
m
o
i
31.5 (C
p
R), 131.7 (C3), 132.2 (C-6a), 132.7 (C-4), 133.2 (C
i
R), 135.5 (C-9),
36.9 (C-10a), 152.9 (C-11a), 159.2 (C-10b), 179.7 (C-5), 179.9 (C-6). HR-MS
465.0880, found 465.0901.
3 2
calcd for C27H16ClN O
4.1.1.17. 3,4-di(4-Chlorophenyl)-1H-benzo[h]pyrazolo[3,4-b]quinolin-5,6-dione 9c:
1
Brown solid, mp >300 ºC (d) (70%). H NMR (400 MHz, DMSO-d
6
) δ (ppm): 6.90
(
d, 2H, H R, J = 7.5 Hz), 7.07 (d, 2H, H Ar, J = 7.9 Hz), 7.11 (d, 2H, H Ar, J = 7.9
m
m
o
Hz), 7.17 (d, 2H, H
o
R, J = 7.5 Hz), 7.67 (t, 1H, H-8, J = 7.5 Hz), 7.91 (t, 1H, H-9, J
=
1
1
7.5 Hz), 8.01 (d, 1H, H-7, J = 7.5 Hz), 8.74 (d, 1H, H-10, J = 7.5 Hz), 14.41 (s,
13
H, NH-1). C NMR (100 MHz, DMSO-d ) δ (ppm): 113.5 (C-3a), 120.4 (C-4a),
6
o o m m
26.7 (C-10), 127.7 (C Ar), 127.9 (C R), 128.6 (C-7), 130.3 (C Ar), 130.9 (C R),
1
8

ACCEPTED MANUSCRIPT
1
1
1
2
31.6 (C-4), 131.7 (C-8), 133.0 (C-6a), 133.2 (C
p i
R), 133.2 (C Ar), 135.3 (C-3),
36.0 (C-9), 137.2 (C-10a), 147.1 (C R), 149.3 (C Ar), 152.8 (C-11a), 153.4 (C-
i
p
+4
+2
+
0b), 179.8 (C-5), 180.0 (C-6). EI MS (70eV): m/z (%):473/471/469 (M / M /M ,
7/68/100), 434 (30). HR-MS calcd for C H N O 469.0385 found 469.0365.
26
13
3
2
4.1.1.18. 3-(4-Chlorophenyl)-4-(4-nitrophenyl)-1H-benzo[h]pyrazolo[3,4-b]quinolin-
1
5
,6-dione 9d: Yellow solid, mp >350 ºC (75%). H NMR (400 MHz, DMSO-d ) δ
6
o m
(ppm): 6.95 (d, 2H, H R, J = 8.3 Hz), 7.08 (d, 2H, H R, J = 8.3 Hz), 7.37 (d, 2H,
H Ar), 7.70 (t, 1H, H-8, J = 7.6 Hz), 7.90-7.98 (m, 3H, H-9, H Ar), 8.05 (d, 1H, H-
o
m
13
7
, J = 7.6 Hz), 8.80 (d, 1H, H-10, J = 7.7 Hz), 14.27 (s, 1H, NH-1). C NMR (100
MHz, DMSO-d ) δ (ppm): 114.5 (C-3a), 119.8 (C-4a), 122.5 (C Ar), 126.2 (C-10),
6
m
1
27.3 (C R), 128.1 (C-7), 129.5 (C Ar), 130.6 (C R), 131.1 (C8), 131.3 (C Ar),
m
o
o
i
1
32.8 (C3), 132.8 (C6a), 135.5 (C9), 136.5 (C10a), 138.2 (C-4) 143.5 (C
p
R), 146.4
(
C R), 146.7 (C Ar), 152.3 (C-11a), 152.9 (C-10b), 178.9 (C-5), 179.04 (C-6). EI
i p
+
MS (70eV): m/z (%):480 (M , 12), 451 (100), 417 (78), 370 (44). HR-MS calcd for
C H13ClN O
26 4 4
480.0625, found 480.0618.
4.1.1.19.
4-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-benzo[h]pyrazolo[3,4-
1
b]quinolin-5,6-dione 9e: Red solid, mp >300 ºC (80%). H NMR (400 MHz, DMSO-
d ) δ (ppm): 3.68 (s, 3H, OCH ), 6.66 (d, 2H, H Ar, J = 8.4), 6.99 (d, 2H, H Ar, J =
6
3
m
o
8
.4), 7.14 (d, 2H, H
o
R, J = 8.3), 7.69 (t, 1H, H-8, J = 8), 7.78 (d, 2H, H
m
R, J = 8.3),
7.92 (t, 1H, H-9, J = 8), 8.01 (d, 1H, H-7, J = 8), 8.76 (d, 1H, H-10, J = 8.1), 14.49
13
(
s, 1H, NH-1). C NMR (100 MHz, DMSO-d
6
3 m
) δ (ppm): 54.9 (OCH ), 112.9 (C Ar),
1
1
1
20.0 (C-4a), 113.6 (C-3a), 121.9 (C R), 126.2 (C-10), 127.8 (C Ar), 128.1 (C-3),
m
p
28.3 (C-7), 129.5 (C Ar), 129.7 (C R), 130.0 (C-6a), 131.0 (C-8), 131.5 (C-4),
o
o
35.4 (C-9), 136.7 (C
i
R), 137.5 (C-10a), 146.1 (C
p
R), 152.7 (C-11a), 153.0 (C
i
+
Ar), 159.3 (C-10b), 179.6 (C-5), 179.8 (C-6). EI MS (70eV): m/z (%):476 (M , 26),
16 4 5
447 (100), 401 (42). HR-MS calcd for C27H N O 476.1121, found 476.1205.
4.1.1.20. 4-(4-Chlorophenyl)-3-(4-nitrophenyl)-1H-benzo[h]pyrazolo[3,4-b]quinolin-
1
5
,6-dione 9f: Brown solid, mp >350 ºC (85%). H NMR (400 MHz, DMSO-d
6
) δ
1
9

ACCEPTED MANUSCRIPT
(
ppm): 7.11 (d, 2H, H
o
m o
Ar, J = 8.6 Hz), 7.15-7.21 (m, 4H, H Ar, H R), 7.71 (t, 1H,
H-8, J = 7.6 Hz), 7.91-7.98 (m, 3H, H-9, H R), 8.05 (d, 1H, H-7, J = 7.6 Hz), 8.77
m
13
(
d, 1H, H-10, J = 7.7 Hz), 14.65 (s, 1H, NH). C NMR (100 MHz, DMSO-d
6
) δ
(
ppm): 113.9 (C-3a), 120.7 (C-4a), 122.8 (C R), 126.8 (C-10), 128.0 (C Ar),
m
m
1
1
1
28.6 (C-7), 130.5 (C Ar), 130.5 (C R), 131.8 (C-8), 132.3 (C Ar), 133.4 (C-3),
o
o
i
35.6 (C-6a), 136.0 (C-9), 137.1 (C-10a), 139.7 (C-4) 146.2 (C
p
R), 146.8 (C
i
R),
49.0 (C Ar), 152.8 (C-11a), 153.6 (C-10b), 179.6 (C-5), 179.8 (C-6). EI MS
p
+
(
70eV): m/z (%):480 (M , 27), 451 (100), 435 (36), 405 (54), 370 (25). HR-MS
calcd for C H ClN O 480.0625, found 480.0625.
26
13
4
4
4.1.1.21. 3,4-di(4-Nitrophenyl)-1H-benzo[h]pyrazolo[3,4-b]quinolin-5,6-dione 9g:
1
Purple solid, mp >300 ºC (70%). H NMR (400 MHz, DMSO-d ) δ (ppm): ): 7.21 (d,
6
2
7
7
H, H
m o
R, J = 8.6 Hz), 7.40 (d, 2H, H Ar, J = 8.8 Hz), 7.71 (t, 1H, H-8, J = 7.7 Hz),
.87 (d, 2H, H Ar, J = 8.8 Hz), 7.91-7.98 (m, 3H, H-9, H R), 8.06 (d, 1H, H-7, J =
m
o
1
3
.7 Hz), 8.81 (d, 1H, H-10, J = 7.7 Hz), 14.41 (s, 1H, NH-1). C NMR (100 MHz,
) δ (ppm): 112.9 (C-3a), 120.0 (C-4a), 122.2 (C R), 122.6 (C Ar), 126.2
C-10), 128.1 (C-7), 129.5 (C R), 130.1 (C Ar), 131.4 (C-8), 131.9 (C-4), 135.5 (C-
DMSO-d
6
m
m
(
o
o
p p i
6a), 136.5 (C-9), 138.9 (C-10a), 143.5 (C Ar), 145.5 (C R), 146.5 (C R), 146.8
(
C-3), 147.2 (C-11a), 152.3 (C Ar), 153.1 (C-10b), 178.8 (C-5), 178.9 (C-6). EI MS
i
+
(
70eV): m/z (%):491 (M , 13), 462 (92), 446 (78), 416 (100), 370 (56). HR-MS
13 5 6
calcd for C26H N O 491.0866, found 491.1022.
4.2. Antimycobacterial evaluation
Mycobacterium species were obtained from the Laboratorio Micobacterias, Instituto
Nacional de Salud, Bogotá, Colombia, M. tuberculosis H37Rv (ATCC 27294), their
resistant variants (ATCC 35837 ethambutol resistant, ATCC 35838 rifampin
resistant, ATCC 35822 isoniazid resistant and ATCC 35820 streptomycin
resistant), five strains of M. tuberculosis Beijing genotype belong to Colombia
National Study of Drug resistance and five clinical isolates from humans with
mesotherapy associated outbreak caused by nontuberculous mycobacteria (NTM):
2
0

ACCEPTED MANUSCRIPT
M. chelonae, M. fortuitum, M. intracellulare, M. scrofulaceum and M. szulgai, were
used. Antimycobacterial activity of the derivates dissolved in DMSO (Sigma, New
Jersey, USA) was evaluated following the MTT colorimetric microdilution protocol,
described by Caviedes et al. with modifications [28, 29]. For standard tests, the
MIC values of rifampin and isoniazid, (Sigma, New Jersey, USA) were determined
each time. The minimum inhibitory concentration (MIC) of each molecule
corresponded to the lowest concentration at which the bacteria tested did not show
growth. Susceptibility testing was performed 3 times.
Acknowledgements
The authors are grateful to the Instituto Nacional de Salud, Bogotá, INS,
Universidad del Valle, Universidad de Jaén and COLCIENCIAS for the support of
this project.
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4

ACCEPTED MANUSCRIPT
1. Introduction
2
2
2
. Results and discussion
.1. Chemistry
.2. Antimycobacterial screening
. Conclusion
3
4
4
4
. Experimental
.1. Chemistry
.2. Antimycobacterial evaluation
Acknowledgements
References
2
5

ACCEPTED MANUSCRIPT
Highlights
ꢀ
ꢀ
New phenylbenzo[h]pyrazolo[3,4-b]quinolin-5,6-diones were synthesized.
Microwave-assisted synthesis gave product in good yield at short reactions
time.
ꢀ
ꢀ
Synthesized compounds were evaluated against 15 Mycobacterium spp.
The major inhibition in Mycobacterium spp was obtained with compounds
4a, 4b and 4g.
2
6

ACCEPTED MANUSCRIPT
Ar = 4a C
6
H
5
, 4b 4-CH
3
6
C H
4
, 4c 4-CH
3
OC
6
H
4
, 4d 3,4,5-tri-CH
h 4-O NC
3
OC
6
H
2
, 4e 4-ClC
6
H
4
, 4f 4-BrC
6
4 6 4
H , 4g 4-FC H ,
4
2
6 4
H
Scheme 1
Ar
O
R
O
3
O
4
5
6
HO
N 1
1
1
N
H
R 3
N
4
H
7
N 1
R 2
N
NH2
O 2
O
3
+
N
MW
H
6
3
min
1
NH
N
Ar
H
Ar
3
O
O
8
7
a R = Ar = C
6
H
5
; 7b R = C
6
H
5
; Ar = 4-CH
3
C
6
H
4
; 7c R = C
6
H
5
, Ar = 4-CH
NC
3
OC
6 4
H ;
7
d R = 4-CH
3
C
6
H
4
, Ar = C ; 7e R = 4-ClC
6
H
5
6
H
4
; Ar = C ; 7f R = 4-O
H
6 5
2
6
H
4
, Ar = C H
6 5
Scheme 2
2
7

ACCEPTED MANUSCRIPT
O
HO
+
R
Ar
O
Ar
O
R
R
3
O
O
4
5
N
O
O
2
6
-H2
NH2
N 1
N
N
H
6
MW
N
N
N
H
7
N
9
3 min
H
H
Ar
H
3
9
a R = 4-CH
3
C
6
H
4
, Ar = 4-O
NC ; 9e R = 4-O
g R = Ar = 4-O
2
NC
6
H
4
; 9b R = 4-ClC
NC , Ar = 4-CH
6 4
NC H
6
H
4
, Ar = 4-CH
3
OC
6
H
4; 9c R = Ar = 4-ClC
6 4
H ;
9
d R = 4-ClC ,Ar = 4-O
6
H
4
2
6
H
4
2
6
H
4
3
OC
6
H
4
; 9f R = 4-O
2
NC , Ar = 4-ClC H ;
6
H
4
6 4
9
2
Scheme 3
Figure 1. ORTEP drawing for 4a and 4b
2
8

ACCEPTED MANUSCRIPT
7
a
7e
4d
4b
4e
4d
9a 7c 7e
3
3
2
2
1
1
5
0
5
0
5
0
5
0
9
a
35
7
c
7b
7
7
b
a
3
0
5
2
7
d
20
15
10
5
7d
6
5
5
2
6
5
5
2
7f
9b
9b
7f
B
T
B
T
M
M
4
a
4g
4a
4g
4b
4b
0
2
3
4
5
6
7
2
3
4
5
6
7
8
9
10
11
Log P
Dipole (D)
Figure 2. LogP vs. MIC and dipole vs. MIC of benzopyrazolo[3,4-b]quinolindiones
derivates against MTB2556 culture cell.
2
9