Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones able to inhibit the M. tuberculosis DNA gyrase

Article abstract of DOI:10.1016/j.ejmech.2018.10.031

A number of new F-triazolequinolones (FTQs) and alkoxy-triazolequinolones (ATQs) were designed, synthesized and evaluated for their activity against Mycobacterium tuberculosis H37Rv. Five out of 21 compounds exhibited interesting minimum inhibitory concen

Full text of DOI:10.1016/j.ejmech.2018.10.031

Accepted Manuscript
Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones able to
inhibit the M. tuberculosis DNA gyrase
Antonio Carta, Alessandra Bua, Paola Corona, Sandra Piras, Irene Briguglio, Paola
Molicotti, Stefania Zanetti, Erik Laurini, Suzana Aulic, Maurizio Fermeglia, Sabrina
Pricl
PII:
S0223-5234(18)30899-7
10.1016/j.ejmech.2018.10.031
EJMECH 10817
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 August 2018
Revised Date: 9 October 2018
Accepted Date: 12 October 2018
Please cite this article as: A. Carta, A. Bua, P. Corona, S. Piras, I. Briguglio, P. Molicotti, S. Zanetti,
E. Laurini, S. Aulic, M. Fermeglia, S. Pricl, Design, synthesis and antitubercular activity of 4-alkoxy-
triazoloquinolones able to inhibit the M. tuberculosis DNA gyrase, European Journal of Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.031.
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Graphical abstract
Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones
able to inhibit the M. tuberculosis DNA gyrase.
Antonio Carta, Alessandra Bua, Paola Corona, Sandra Piras, Irene Briguglio, Paola Molicotti,
Stefania Zanetti, Erik Laurini, Maurizio Fermeglia, Sabrina Pricl
Two alkoxy-triazolequinolones, 21a and 30a, showed selective inhibition against Mycobacterium
tuberculosis H37Rv and a set of Mtb strains obtained from clinical isolates. They were assessed for
their ability to inhibit Mtb DNA gyrase in a DNA supercoiling activity assay, when compared to
ciprofloxacin they were found to be equally active.

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Design, synthesis and antitubercular activity of 4-alkoxy-triazoloquinolones able
to inhibit the M. tuberculosis DNA gyrase.
Antonio Cartaa,*,, Alessandra Buab,1, Paola Coronaa,1, Sandra Pirasa, Irene Briguglioa, Paola
Molicottib, Stefania Zanettib, Erik Laurinic, Suzana Aulicc, Maurizio Fermegliac, Sabrina Pricl c,*
a
Department of Chemistry and Pharmacy, University of Sassari, Via Muroni 23, 07100 Sassari,
Italy.
b
Department of Biomedical Sciences, University of Sassari, V.le San Pietro 43/C, 07100 Sassari,
Italy.
c
Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), DEA, University of
Trieste, Piazzale Europa 1,
34127 Trieste, Italy.
1Equal contribution.
*Corresponding authors.
E-mail addresses: acarta@uniss.it (A.Carta), Phone: +39-079-228722; sabrina.pricl@dia.units.it (S.
Pricl), Phone: +39-040.5583750
Abstract
A number of new F-triazolequinolones (FTQs) and alkoxy-triazolequinolones (ATQs) were
designed, synthesized and evaluated for their activity against Mycobacterium tuberculosis H37Rv.
Five out of 21 compounds exhibited interesting minimum inhibitory concentration (MIC) values
(6.6 – 57.9 µM), ATQs generally being more potent than FTQs. Two ATQs, 21a and 30a, were
endowed with the best anti-Mtb potency (MIC = 6.9 and 6.6 µM, respectively), and were not
cytotoxic in a Vero cell line. Tested for activity against M. tuberculosis DNA gyrase in a DNA
supercoiling activity assay, 21a and 30a showed IC50 values (27-28 µM) comparable to that of
ciprofloxacin (10.6 µM). 21a was next selected for screening against several Mtb strains obtained
from clinical isolates, including multi-drug-resistant (MDR) variants. Importantly, this compound
was effective in all cases, with very promising MIC values (4 µM) in the case of some
isoniazid/rifampicin-resistant Mtb strains. Finally, computer-based simulations revealed that the
binding mode of 21a in the Mtb gyrase cleavage core complexed with DNA and the relevant
network of intermolecular interactions are utterly similar to those described for ciprofloxacin,
yielding a molecular rationale for the comparable anti-mycobacterial and DNA gyrase inhibition
activity of this quinolone.
Keywords:
Triazoloquinolones; M. tuberculosis DNA gyrase; Antitubercular activity; M. tuberculosis clinical
strains, In vitro and in silico studies.
Highlights:
Triazole derivatives are of great interest in medicinal chemistry.
We have synthesized 4-alkoxy-triazoloquinolones endowed with antitubercular activity.
We have shown that 4-alkoxy-triazoloquinolones inhibit the M. tuberculosis DNA gyrase in a
different way compared to the classic quinolones.
We have shown that these derivatives are selective for M. tuberculosis and inactive against the other
bacteria.

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Acknowledgments
All the authors gratefully acknowledge the MIUR (Italy) for the generous financial support (grant
PRIN 2015).
E.L. and S.P. gratefully acknowledge the Associazione Italiana per la Ricerca sul Cancro (AIRC)
for the generous financial support (grant IG 2015 Id.17413 to S.P.).
1. INTRODUCTION
Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is one of the most
important pathogens of humans, second only to the HIV in the number of deaths caused annually
[1]. Mtb is estimated to latently infect one-third of the world’s population (≈ 2 billion people),
thereby creating a huge reservoir for future disease. In 2016 only, 10.4 million of the infected
people developed active TB and the death toll from this disease reached 1.7 million. At the same
time, 6.3 million new cases of TB were reported (up from 6.1 million in 2015), equivalent to 61%
of the estimated incidence of 10.4 million [1]. Control of Mtb is hindered by multidrug-resistant
(MDR)-TB, characterized by the presence of Mtb strains resistant to two primary anti-TB drugs,
rifampicin and isoniazid. Drug-resistant TB is a persistent threat, with 490000 cases of MDR-TB
emerging in 2016 and additional 110000 cases that were susceptible to isoniazid but resistant to
rifampicin (RR-TB), the most effective first-line anti-TB drug. Even more frightening is the
emergence of extensively drug resistant TB (XDR-TB) reported in all regions of the world. XDR-
TB involves resistance to isoniazid and rifampicin in addition to resistance to any fluoroquinolone
and one injectable second-line drug. The reasons why MDR-TB continues to emerge and spread are
mismanagement of TB treatment and person-to-person transmission [2]. Inappropriate or incorrect
use of antimicrobial drugs, use of ineffective drug formulations, and premature treatment
interruption can all lead to resistant form of TB, which can be further transmitted, particularly in
crowded or at-risk settings such as prisons and hospitals.
At present, Europe is facing TB recrudescence due to the human migration flux from TB high-
incidence countries [3]. This is a relevant phenomenon, increasing over time, considering that a
significant proportion (up to 70%) of TB cases notified in Europe are associated to foreign-born
populations. Irregular immigration represent one of the main problems, since the migratory journey,
often occurring under precarious conditions, increases the TB risk of migrants [4].
Under this glooming perspective, the discovery and development of new anti-TB drugs is very
pressing and challenging at the same. Indeed, to be highly effective any novel Mtb inhibitor should
be ideally endowed with acceptable tolerability profile (particularly in HIV-TB co-infected
patients), high efficacy to reduce treatment duration, be active against MDR/XDR and latent TB
forms.
Basically, the road to new anti-Mtb agents can be paved in different ways. One of these approaches
consists in the exploration of known and clinically validated bacterial targets using new chemical
entities or the modification of existing drug classes ultimately limiting/avoiding cross resistance
phenomena. Fluoroquinolones constitute a class of synthetic antibiotics that inhibit type-II
topoisomerases (i.e., DNA gyrase and topoisomerase IV) in most bacterial species [5-8]. DNA
gyrase is an ATP-dependent heterotetrameric (GyrA2GyrB2) enzyme that acts by creating a
transient double-stranded DNA break. It is unique in catalyzing the negative supercoiling of DNA
and is essential for efficient DNA replication, transcription, and recombination. Blocking the
activity of this protein thus results in pathogen cell death. Also, since DNA gyrase is the sole type II
topoisomerase in Mtb, this enzyme is likely the only target for fluoroquinolones in this

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microorganism [9-10]. Accordingly, a novel inhibitor of Mtb DNA gyrase would be effective
against MDR-Mtb, could be active against fluoroquinolone-resistant Mtb strains, and could be
active against non-replicating mycobacteria, another important aspect in the eradication of
persistent infections [11].
In the past several molecules active against Mtb were proposed by our group [12-15]. More
recently, [1,2,3]triazolo[4,5-h]quinolone (TQ) derivatives were also identified by us as new
chemical entities able to inhibit replication of MDR/XDR-Mtb strains [16-19]. Some of these
compounds (Figure 1) were highly potent against H37Rv, H37Ra and further 11 clinical isolates of
MDR/XDR-TB strains; concomitantly, they were devoid of cytotoxicity.
Figure 1. 9-Substituted-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (I)
With the aim of further enhancing the biological activity of the [1,2,3]triazolo[4,5-h]quinolone
scaffold, in the present effort we designed and synthesized a number of new F-triazolequinolones
(FTQs) and alkoxy-triazolequinolones (ATQs). Testing all TQ derivatives against Mtb H37Rv
highlighted 21a and 30a as the most active compounds in the full set. In vitro, we found that 21a
and 30a were both effective at inhibiting DNA supercoiling by Mtb gyrase, with IC50 values
comparable to that of ciprofloxacin (CPF), a quinolone-derivative antibiotic used in TB therapy.
Further testing carried out with 21a confirmed that this compound was quite active against a panel
of several other Mtb strains, including a set of multi-drug-resistant variants obtained from clinical
isolated. Finally, in silico modeling revealed that binding mode of 21a in the Mtb gyrase cleavage
core complexed with DNA and the relevant network of intermolecular interactions were utterly
similar to those described for ciprofloxacin, yielding a molecular rationale for the comparable
antibacterial and DNA gyrase inhibition activity of these two quinolones.
2. Chemistry
SAR considerations on previously synthesized TQs revealed that the most active derivatives
featured a small and lipophilic alkyl pendant on the triazole moiety. In particular, the presence of a
methyl group on the triazole N-3 atom was found to enhance the antitubercular activity of the TQ,
while the same substituent on the N-1 counterpart drastically reduced it. [6, 7, 9]. Starting from this
evidence, in this work we reasoned that, preserving the favorable –CH3 group on the triazole moiety
and introducing new substituents at different positions of the TQ scaffold we could obtain a new
series of efficient Mtb inhibitors (Figure 2).

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Figure 2. 4,9-Substituted-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (II)
Accordingly, we prepared two compounds bearing different lipophilic alkyl groups on the
quinolone N-9 atom (R1 = methyl and ethyl). Since fluoroquinolones are included in standard
regimens for the treatment of a variety of community and nosocomial infections, we also explored
the effect of the presence of a fluorine at position 4 of the TQ scaffold (R2). All new compounds
were prepared both in their free acid and ethyl ester form.
The synthesis of the intermediate 7-fluoro-1H-benzo[d][1,2,3]triazole (6) was already reported by
Kirk et al. in 1969 [20]; however, the process required a substantial adaptation to obtain the 2-
fluoro-6-nitroaniline (4) in good yield, as shown in Scheme 1.

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Scheme 1. Synthesis of the key intermediate 4-fluoro-1H-benzotriazole (6)
Methylation of 6 was carried out using dimethyl sulfate in NaOH 2M, as reported in Scheme 2.

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Scheme 2. Synthesis of the 4(7)-fluoro-1(2)-methyl-1H-benzotriazole (7a-c)
The reaction yielded a mixture of three isomers (7-fluoro-1-methyl-1H-benzotriazole (7a), 4-fluoro-
2-methyl-2H-benzotriazole (7b), and 4-fluoro-1-methyl-1H-benzotriazole (7c)) which were
separated by chromatography and identified by NMR [21]. Starting from compound 7a, the
synthesis of the intermediate ethyl-4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3] triazole [4,5-h]
quinolone (11) was performed as described in the Scheme 3.

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Scheme 3. Synthesis of the key intermediate 4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (11)
Alkylation of 11 at N-9 position with the CH3-I or CH3-CH2-I in the presence of potassium
hydroxide, followed by hydrolysis in NaOH 2M, gave the corresponding alkyl derivatives 11 and
15, respectively (Scheme 4).

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Scheme 4. Synthesis of 4-fluoro-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-
h]quinoline-7-carboxylic acid (13) and 4-fluoro-3-methyl-9-ethyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acid (15)

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Nucleophilic aromatic substitution of the electron-withdrawing F atom in compounds 7a-c with
alkoxy groups such as –OCH3 and –OCH2CH3 led us to obtain the new derivatives 21a,c, 23a-c,
30a and 32a via the chemical route depicted in Scheme 5. From the intermediate compounds 19a-c
only one (21a) of the three expected TQs was obtained. Indeed, the alkylation of 19c took place on
the oxygen atom at position 4, ultimately leading to the formation of the quinolinic derivatives 20c
and 22c. Non-alkylated derivatives on N-9 (24a,c) were also prepared to evaluate whether the
introduction of a methoxy group at position 4 still required the presence of an alkyl at the N-9
position for activity.
Of note, in the case of 4-ethoxy derivatives only compounds 30a and 32a were synthesized because
the corresponding derivative, without the methoxy group, of the series “b” and “c” resulted inactive
[16].
Scheme 5. Synthesis of 4-alkoxy-triazoloquinolones (21a,c 23a,b,c, 30a and 32a)
Finally, two other, unconventional TQs resulted from the reaction of the halogenated benzotriazoles
8a and 8c with strong nucleophiles such as ethanethiol or ammonia, respectively. Starting from
compound 8a, the final quinolone 37 was obtained via the 7-ethylthio intermediate derivative 33, as
shown in Scheme 6. On the contrary, starting from 8c the final quinolone 41 was synthesized
according to the pathway shown in Scheme 7. Based on what we saw for the previous series, also in
this case we prepared only quinolone derivatives with the methyl on N-3 [16]. Following the same
lead, we performed only a methylation on the quinolone moiety, since ethyl derivatives resulted less
potent than methylated ones.

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Scheme 6. Synthesis of 4-(ethylthio)-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-
h]quinoline-7-carboxylic acid (37).

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Scheme 7. Synthesis of 3,9-dimethyl-4-nitro-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-
7-carboxylic acid (41)
3. Results and Discussion
3.1. Minimum inhibitory concentration (MIC) determination of the new triazoquinolones against
Mtb H37Rv strain
All newly synthesized triazoloquinolones were initially tested against MTb H37Rv in a
conventional Resazurin Microtiter Assay (REMA) [22]. The results gathered in Table 1 show that
compounds 13, 20b, 21a, 23a, 30a, 32a, and 37 exhibits interesting minimum inhibitory
concentration (MIC) values in the range 6.6 to 57.9 µM. Taking 21a as a reference compound (MIC
= 6.9 µM), the biological data reveal that both the nature and position of the substituents exert an
effect on the antimycobacterial activity of the corresponding derivatives. Thus, while the
replacement of a methyl group with a longer (-CH2-CH3) chain on the O-4 atom of the quinolone
moiety does not result in an appreciable change of the corresponding MIC (30a, MIC = 6.6 µM),
the analogous substitution on the N-9 atom of the quinolone leads to a substantial decrease in
activity (23a, MIC = 52.9 µM). Finally, the double replacement of an ethyl group at both positions
of the quinolone moiety results in an intermediate MIC value for the corresponding compound (32a,
MIC = 25.3 µM). If a thio-ether is used as in compound (37), the antimycobacterial activity is seen
to drop (50.3 µM), as it is the case of compounds (41) and (13), featuring two electron withdrawing
groups at the same position (-NO2, MIC > 100 µM, and –F, MIC = 57.9 µM, respectively). The
position of the methyl substituent on the triazole ring is also found to have an effect on the activity
of these series of compounds. Indeed, with respect to (21a) the quinolone (20b) and the quinoiline
(21c), in which the –CH3 group is located on the N atom at position 2 or 1 of the triazole ring,
respectively, show MIC values of 27.8 µM and > 100 µM, respectively. Finally, compounds (20a
and 24a) were synthesized and tested to verify the fundamental role played by the –COOH
substitution at positions 7 (20a) and the –CH3 and on the N-9 (24a) of the quinolone ring in the
activity of these molecules. As expected, both compounds were devoid of antimycobacterial activity
(MIC > 100 µM).

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Table 1. In vitro antimycobacterial activity and cytotoxicity of compounds 13, 15, 20a,b,c, 21a,c,
23a,b,c, 24a,c, 29a, 30a, 31a, 32a, 37 and 41, against H37Rv.
MICa (µM)
% Verob cell viability at 100 µM
81.2 ± 4.4
95.1 ± 3.6
91.2 ± 5.9
89.5 ± 3.7
80.8 ± 4.9
90.4 ± 2.1
85.7 ± 4.2
84.4 ± 4.3
79.3 ± 6.1
85.4 ± 5.3
90.1 ± 5.9
94.2 ± 2.9
83.6 ± 5.0
90.2 ± 3.8
92.9 ± 4.8
92.0 ± 3.7
94.3 ± 4.5
95.1 ± 2.8
92.1 ± 6.0
87.7 ± 5.5
88.8 ± 3.4
95.2 ± 3.5
Compound
57.9
13
15
> 100
> 100
27.8
20a
20b
20c
21a
21c
22a
22b
22c
23a
23b
23c
24a
24c
29a
30a
31a
32a
37
> 100
6.9
> 100
> 100
> 100
> 100
52.9
> 100
> 100
> 100
> 100
> 100
6.6
> 100
25.3
50.3
> 100
1.5
41
CPFc
aMinimum inhibitory concentration; bAfrican kidney monkey cells (ATCC CCL-81);
cciprofloxacin.
All compounds were also examined for in vitro cytotoxicity in the mammalian VERO cell line by
the MTT colorimetric assay. The results, expressed as % cell viability in the presence of at 100 µM
of each compound, are summarized in the last column of Table 1. Cell viabilities ranged from 81.2

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to 94.3%, confirming that all molecules did not exhibit any significant toxicity effect on the selected
cell line.
3.2. In silico prediction of pharmacokinetic parameters for compounds 13, 20b, 21a, 23a, 24a, 30a,
32a, and 37
All compounds endowed with activity against Mtb H37Rv (compounds 13, 20b, 21a, 23a, 30a,
32a, and 37, Table 1) and 24a, as negative control, were subjected to in silico prediction of a
selection of pharmacokinetic parameters, i.e., molecular weight (MW), numbers of hydrogen bond
donors/acceptors (HBD/HBA), water-octanol partition coefficient (logP), water solubility (logS)
and polar surface area (PSA). For comparison purposes, ciprofloxacin was also analyzed as the
reference standard. All molecular properties were analyzed on the basis of the extended version of
the Lipinski rule of five [123,24], according to which an orally active drug should not violate more
than one of the following criteria: i) MW ≤ 500; ii) HBD and HBA (linked to compound membrane
permeability) ≤ 5 and ≤ 10, respectively; iii) logP and logS (helpful in predicting the intestinal
absorption and consequent efficiency of the trans-cellular transport of the drug) ≤ 5; iv) PSA < 140
Å2. As seen from Table 3, all compounds exhibited good drug likeness properties and did not
violated any of the pharmacokinetic parameters as proposed by Lipinski’s rule, suggesting these
molecules possess properties that would make them likely orally active drugs in humans.
Table 2. In silico predicted main pharmacokinetic parameters of compounds 13, 20b, 21a, 23a,
24a, 30a, 32a, and 37. Data for ciprofloxacin are reported for comparison.
Rule of 5
violation
Cpd
MWa
HBAb HBDc logPd logSe PSAf
Drug likeness
Rule
of 5
<500
≤10
≤5
≤5
≤5
-
≤1
276.22
288.26
288.26
302.29
274.23
302.29
316.31
318.35
5
6
6
6
6
6
6
6
4
1
1
1
1
2
1
1
1
2
0.27 -3.33 71.64
0.49 -2.65 78.82
0.09 -2.81 79.28
0.56 -3.80 79.28
0.00 -2.97 84.67
0.57 -3.18 78.85
1.04 -4.18 78.86
1.02 -3.92 71.64
1.29 -2.96 58.80
0
0
0
0
0
0
0
0
0
0.31
0.29
-0.02
0.17
-0.36
0.30
0.23
0.34
0.93
13
20b
21a
23a
24a
30a
32a
37
CPFg 331.35
aMolecular weight; bnumber of hydrogen bond acceptors; cnumber of hydrogen bond donors;
dlogarithm of n-octanol/water partition coefficient; elogarithm of water solubility; fpolar surface
area; gciprofloxacin.
3.3. MIC determination of 21a against clinical isolates of Mycobacterium tuberculosis

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Compounds 21a was also tested against 21 different Mtb strains obtained from clinical isolates.
Among these, 4 multi-drug-resistant (MDR) and 3 ciprofloxacin-resistant strains were included.
Mtb strains were defined as those that did not respond to least both isoniazid and rifampicin, the
two anti-Mtb drugs currently adopted in the clinics. Table 3 reports these results, together with the
MIC values for ciprofloxacin against the identified resistant. MIC values of 21a and ciprofloxacin
against Mtb strain H37Rv are also shown for comparison.
Table 3. MIC values (µM) of 21a against a set of Mtb strains obtained from clinical isolates. MDR
= multi-drug resistant strains. MIC values of ciprofloxacin against resistant Mtb strains are also
reported (in parenthesis) for comparison.
SMa INHb RIFc EMBd
Mtb strain
CPFe (µM)
21a (µM)
Sf
S
S
S
R
S
S
S
S
S
R
R
R
S
R
R
S
S
S
S
S
R
S
Rg
S
S
S
S
R
R
S
S
S
S
R
S
S
S
S
R
R
S
S
S
S
R
S
S
S
S
R
R
S
S
S
S
R
S
S
S
R
R
R
S
S
S
S
S
R
1.5
6.9
16
16
8
H37Rv
1358
S
R (16)
1670
R
R
S
R (8)
512 (MDR)
7 (MDR)
231
S
S
4
4
S
S
32
8
1574
S
S
1777
S
S
8
147
S
S
16
16
8
1726
S
S
203
S
S
204
S
S
8
157
R
R
R
S
S
8
368
S
16
4
571 (MDR)
1120 (MDR)
1657
S
S
4
600
S
R (4)
S
16
16
32
32
32
1535
S
S
S
1567
S
S
952
R
S
1097
aStreptomycin; bIsoniazid;, crifampicin; dethambutol; eciprofloxacin; fsensible; gresistant.

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While compound 21a was active on all identified Mtb resistant strains with MIC values in the range
4-32 µM), it is of particular interest to observe that this TQ derivative is remarkably active on 3
MDR strains, i.e., strains 7 and 1120 (MIC = 4 µM), and strain 512 (MIC = 8 µM).
3.4. Inhibition of Mtb DNA gyrase supercoiling activity by 21a and 30a
The two most active compounds 21a and 30a (MIC = 6.9 and 6.6 µM, Table 1) were next assessed
for their ability to inhibit Mtb DNA gyrase in a DNA supercoiling activity assay. A less active
compound 24a was also selected as negative control while ciprofloxacin was used as positive
control. When compared to ciprofloxacin (IC50 = 10.6 ± 0.60 µM), compounds 21a and 30a were
found to be equally active, with IC50 values of 27.6 ± 0.71 and 28.2 ± 1.56 µM, respectively
(Figure 3). As expected, compound 24a did not show comparable inhibition of the enzyme (IC50 =
91.3 ± 1.56 µM).
Figure 3. IC50 values for Mtb DNA gyrase inhibition by compounds 21a, 30a, and 24a. IC50 for
ciprofloxacin (CPF) is also shown for comparison.
Finally, in order to increase the knowledge of this new class of quinolones, derivatives 21a and 30a
were assesses for activity against a large panel of different microorganisms, including Gram
positive and Gram-negative bacteria (S. aureus, S. typhimurium, E. faecalis and E. coli) and yeasts
(C. albicans and C. tropicalis). Pleasingly, both compounds resulted devoid of antimicrobial
activity (MIC> 100 µM).
3.5. Molecular modeling study of the binding of compound 21a to the MTb DNA gyrase
The putative interactions of the most active compound 21a with the Mtb DNA gyrase were next
investigated by molecular dynamics (MD) simulations [25-27]. The available crystallographic
structure of the Mtb DNA gyrase (pdb code 5BTC) in complex with ciprofloxacin (CPF) [28] was
used as the starting point for these computational studies. As shown in Figure 4A, the predicted
binding mode of 21a in the Mtb DNA gyrase active site is very similar to that characterizing
ciprofloxacin and other related quinolones. Indeed, the keto-acid group of 21a coordinates a
magnesium ion while the triazole ring performs weak van der Waals interactions with the
neighboring GyrB residues Arg482, Thr500, and Glu501. In addition, other favorable contacts are
detected between 21a structure GyrA residues Asp94, Arg128, and Ptr129. These favorable
interaction network translates in a good affinity of 21a, the corresponding free energy of binding
(∆Gbind) value being equal to -6.88 ± 0.19 kcal/mol, in agreement with the experimental micromolar
inhibitory activity of the compound.

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•
Figure 4. (A) Putative binding mode of compound 21a onto the Mtb DNA gyrase. The
compound is shown as colored balls-and-sticks (gray, C; blue, N; red, O). The protein is
represented as transparent firebrick ribbons. The protein residues mainly involved in 21a
binding are evidenced and labeled (see also main text for more details). Gyrase residues
belonging to the subunit B (GyrB) are marked with an asterisk. Magnesium ion is depicted
as a chartreuse sphere while the adjacent DNA bases are represented in gold. Coordination
bonds are depicted as broken black lines. Hydrogen atoms, water molecules, ions and
counterions are omitted for clarity. (B) Comparison of the docked conformations of
compounds 21a (firebrick) and CPF (corn-flower blue) in the Mtb DNA gyrase binding site.
Figure 4B compares the conformations adopted by ciprofloxacin and compound 21a in the Mtb
DNA gyrase as extracted from the corresponding equilibrated MD simulation trajectories.. As seen
from this Figure, the two molecules can be largely superposed, the slightly more favorable ∆Gbind
value (-7.29 ± 0.21 kcal/mol) calculated for the CPF/gyrase complex being in line with a 4.5-fold
higher potency of ciprofloxacin compared to 21a (EC50 = 1.5 µM and 6.9 µM respectively, Table
1)..
4. Conclusion
In summary in this study we can assert that 4-methoxy (or ethoxy)-3,9-dimethyl-6-oxo-6,9-dihydro-
3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acids (21a and 30a) are endowed with anti-
mycobacterial activity. Surprisingly, on the contrary to what happens in the case of
fluoroquinolones, the presence of a fluorine in the classic 6 position of fluoroquinolones
(corresponding to 4 of the TQs) does not involve an increase in biological activity but a drastic
decrease. The same negative effect occurs with the substitution of the alkaloxy group with an
ethanethiol or with a nitro group. It is equally surprising that the most active substituent on the N-9
of the TQs (corresponding to the N-1 of the classic fluoroquinolones) is methyl and not ethyl.
Moreover, the activity, although not very high, of the compound 4-methoxy-2,9-dimethyl-6-oxo-
6,9-dihydro-2H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acid (20b) which differs from 21a by
the displacement of methyl from position 3 to 2 of the benzotriazole ring and of an ester group in
place of a carboxylic acid, reinforces the hypothesis of the different location of these molecules
within the M. tuberculosis DNA gyrase. The different mechanism of action of this new class of
quinolones is further demonstrated by their complete lack of activity against bacteria. This aspect is
very important because it avoids interference with the intestinal bacterial flora and reduces the risk
of vertical transmission of resistance across species. Interestingly, an analysis of different bacterial
DNA gyrases revealed that these enzymes share high sequence similarity and a highly conserved
active site [28]. As such, an hypothesis based on different binding modes of the present compounds
on their biological targets can be sensibly discarded as the molecular rationale underlying their
selective behavior towards the Mtb DNA gyrase, and further microbiological studies are needed to

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explain the specificity of action of these new triazolequinolones against Mycobacterium
tuberculosis.. Moreover, derivative 21a, chosen as lead compound, has generally shown to maintain
the same activity in clinical strains including MDRs. Although its ability to inhibit M. tuberculosis
DNA gyrase and its in vitro potency are slightly lower than that of ciprofloxacin, 21a do not
demonstrate any loss of potency against ciprofloxacin resistant strains. Finally, on the basis of
pharmacokinetic parameters and the drug solubility with PVP10, supported also by the good
cytotoxicity profile, suggest to us to submit the compound 21a further experiments in vivo.
5. Experimental section
5.1. Chemistry
Melting points were carried out with a Köfler hot stage or Digital Electrothermal melting point
apparatus. UV analysis was carried out using a UV–Vis Shimadzu UV-1800 spectrophotometer
(Shimadzu Corporation, Kyoto, Japan). Nuclear magnetic resonance (1H-NMR and 13C-NMR)
spectra were determined in CDCl3 or DMSO-δ6 and were recorded with a Bruker Avance III 200
and 400 NanoBay. Chemical shifts are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS) used as internal standard. Splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; br s, broad singlet; dd, double doublet.
Mass spectra (MS) were performed on combined Liquid Chromatograph-Agilent 1100 series Mass
Selective Detector (MSD). Analytical thin-layer chromatography (TLC) was performed on Merck
silica gel F-254 plates. Pure compounds showed a single spot in TLC. For flash chromatography,
Merck silica gel 60 was used with a particle size 0.040-0.063 mm (230-400 mesh ASTM).
Elemental analysis were performed on a Perkin-Elmer 2400 instrument and the results were within
± 0.4% of theoretical values.
5.1.1. Starting material and known intermediates
Polyvinylpyrrolidone with molecular weight 10 kDa (PVP10) was obtained from Sigma Aldrich
(USA) and PVP K-30 (Kollidon®, molecular weight 40 kDa) was supplied by BASF (Germany).
Hydroxypropyl methylcellulose K4M (HPMC) was obtained from Dow Chemicals Company
(USA). Methyl-β-cyclodextrin (Cavasol® W7 M Pharma) was purchased from Wacker-Chemie
GmbH (Germany). Eudragit® (L100–55) was generously donated by Degussa® (Germany). All
other chemicals were of analytical grade.
Key intermediates N-(2-fluorophenyl)acetamide (2) and N-(2-fluoro-6-nitrophenyl)acetamide (3a)
were prepared according to the procedures described in the literature [10]. 2-Fluoroaniline (1) was
commercially available. Details of synthesis of each compound are following provided.
5.1.2. 2-fluoro-6-nitroaniline (4). 3 g (0.0151 mol) of N-(2-fluoro-6-nitrophenyl)acetamide (3a) are
dissolved in 30 ml of concentrated H2SO4 and brought to 50 °C for 2 hours. After cooling, the
solution is poured into an ice bath and an abundant precipitate is observed. After filtration, the
acidic aqueus solution is extracted with diethyl ether. The pure compound is obtained through flash
chromatography. Yield 74%. M.P. 47-48 °C. 1H-NMR (DMSO-d6): δ 7.82 (d, 1H, J = 8.8MHz, H-
5), 7.42 (m, 1H, H-3), 7.25 (s, 2H, NH2), 6.63 (m, 1H, H-4). GC/MS: 157 (M+H). Anal. Calcd for
(C6H5FN2O2): C, 46.16; H, 3.23; F, 12.17; N, 17.94. Found: C, 46.18; H, 3.27; F, 12.15; N, 17.98.
5.1.3. 3-fluorobenzene-1,2-diamine (5). 4.6 g (0.029 mol) of 2-fluoro-6-nitroaniline (4) are
dissolved in 100 ml of ethanol and added with 0.46 g of 10% C/Pd. It is set to reduce by means of a
hydrogenator; the theoretical amount of H2 is rapidly absorbed at room temperature and at
atmospheric pressure. By filtration the catalyst is removed, while the solvent is removed by
evaporation: in this way a red oil is obtained which crystallizes in cold. Trough flash
chromatography 3.2 g of clean diamine are collected as a dark violet oil. Yield 97%. Liquid oil. 1H-

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NMR (DMSO-d6): δ 6.33 (m, 3H, H-4, H-5, H-6), 4.77 (s, 2H, NH2), 4.33 (s, 2H, NH2). GC/MS:
127 (M+H). Anal. Calcd for (C6H7FN2): C, 57.13; H, 5.59; F, 15.06; N, 22.21. Found: C, 57.09; H,
5.61; F, 15.02; N, 22.24.
5.1.4. 7-fluoro-1H-benzo[d][1,2,3]triazole (6). 3.66 g (0.029 mol) of 3-fluorobenzene-1,2-diamine
(5) are dissolved in 200 ml of HCl 2M and the solution is placed in an ice bath, bringing the
temperature to 0 °C. A solution obtained by dissolving 4.08 g of NaNO2 in 10 ml of H2O is added
drop by drop to over a period of ten minutes. After 12 h the formed precipitate is collected by
filtration. The acidic solution was extracted with ethyl acetate, and the rough obtained compound is
reunited to the precipitate. Trough flash chromatography we obtained the pure benzotriazole
1
derivative as a beige solid compound. Yield 83%. M.p. 134-135 °C. H-NMR (DMSO-d6): δ 7.68
13
(d, J=8.6 Hz 1H, H-4) ); 7.54 (m, 1H, H-5); 7.22 (m, 1H, H-6). C-NMR (CDCl3) δ: 168.50 (C),
144.79 (C), 142.26 (C), 125.80 (CH), 115.93 (CH), 108.70 (CH). GC/MS: 138. (M+H). Anal. Calcd
for (C6H4FN3): C, 52.56; H, 2.94; F, 13.86; N, 30.65. Found: C, 52.60; H, 2.99; F, 13.83; N, 30.60.
5.1.2. General procedure for the preparation of fluoro-(1,2,3)-methyl-benzotriazoles (7a-c). 1.5 g
(0.0109 mol) of 7- fluoro-1H-benzo[d][1,2,3]triazole (6) are dissolved in 21.7 ml of NaOH 2 M
(0.0434 mol). 1.61 ml (0.017 mol) 2.15 g of (CH3O)2SO2 are added at room temperature. The
solution is left to react for 18 hours. An oil is formed which separates from the alkaline solution.
The solution is extracted as it was first with ether, then with diethyl acetate, thus obtaining an oil
(1.4 g) which contains the 3 isomers. Isomers were separated and purified by flash chromatography.
1
5.1.2.1. 4-fluoro-1-methyl-1H-benzo[d][1,2,3]triazole (7a). Yield 38%. M.P. 79-80 °C. H-NMR:
(DMSO) δ: 8.53 (d, J=8.4 Hz, H-4); 7.68 (m, 1H, H-5); 7.22 (m, 1H, H-6); 4.33 (s, 3H, CH3). 13C-
NMR (CDCl3): 153.35 (C-3a), 150.82 (C-F), 135.02 (C-7a), 128.08 (CH), 108.21 (CH), 107.16
(CH), 34.35 (CH3). GC/MS: 152 (M+H). Anal. Calcd for (C7H6FN3): C, 55.63; H, 4.00; F, 12.57;
N, 27.80. Found: C, 55.58; H, 4.03; F, 12.53; N, 27.87.
1
5.1.2.2. 4-fluoro-2-methyl-2H-benzo[d][1,2,3]triazole (7b). Yield 25%. Liquid oil. H-NMR
(DMSO-d6), δ: 7.65 (d, J=8.4 Hz 1H, H-7); 7.33 (m, 1H, H-6) 7.13 (m, 1H, H-5); 4.51 (s, 3H, N-
CH3). 13C-NMR (DMSO-d6): 154.78 (C); 146.51 (C); 134.52 (C); 126.31 (CH); 113.87 (CH);
109.39 (CH); 43.27 (CH3). GC/MS:152 (M+H). Anal. Calcd for (C7H6FN3): C, 55.63; H, 4.00; F,
12.57; N, 27.80. Found: C, 55.59; H, 4.06; F, 12.61; N, 27.75.
1
5.1.2.3. 7-fluoro-1-methyl-1H-benzo[d][1,2,3]triazole (7c). Yield 33%. M.P. 65-66 °C. H -NMR:
(CDCl3) δ 7.81 (d, J=8.4 Hz, 1H, H-4); 7.27 (m, 1H, H-5); 7.12 (t, J=9.2 Hz, 1H, H-6); 4.45 (s, 3H,
N-CH3). 13C-NMR (CDCl3): 150.29 (C-F); 149.12 (C-3a), 145.32 (C-7a); 124.23 (CH); 115.70
(CH); 111.59 (CH); 36.27 (CH3). GC/MS: 152 (M+H). Anal. Calcd for (C7H6FN3): C, 55.63; H,
4.00; F, 12.57; N, 27.80. Found: C, 55.65; H, 4.01; F, 12.53; N, 27.82.
5.1.3. Synthesis of fluorine-methyl-triazolo derivatives (8a-15).
5.1.3.1. 7-fluoro-1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (8a). 4.2 g (0.0277 mol) 7-fluoro-1-
methyl-1H-benzotriazole (7c) are dissolved in 54.6 ml of H2SO4 conc. To the acidic solution a
mixture consisting of 10.3 g of KNO3 in 54.6 ml of H2SO4 conc. is added drop by drop at room
temperature. The temperature is then brought to 50 °C and left under stirring. After an hour we let
the solution return to room temperature and then poured into an ice bath. A light yellow precipitate
1
is formed and subsequently purified by chromatography. Yield 65%. M.P. 115-116 °C. H-NMR:
13
(CDCl3) δ: 8.31 (dd, 1H, H-5); 7.26 (t, J = 9.2 Hz, 1H, H-6); 4.56 (s, 3H, N-CH3). C-NMR:
(CDCl3) δ: 160.22 (C), 153.87 (C), 133.21 (C), 129.75 (C), 126.39 (CH), 107.86 (CH), 41.01
(CH3), GC/MS: 197 (M+H). Anal. Calcd for (C7H5FN4O2): C, 42.87; H, 2.57; F, 9.69; N, 28.57.
Found: C, 42.83; H, 2.61; F, 9.72; N, 28.55.

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5.1.3.2. 4-fluoro-2-methyl-7-nitro-2H-benzo[d][1,2,3]triazole (8b). 2.06 g (0.0136 mol) of 4-
fluoro-2-methyl-2H-benzo[d][1,2,3]triazole (7b) are dissolved in 5 ml of glacial CH3COOH and
brought to reflux. To the acidic solution were added 1.85 ml (0.053 mol) 2.50 g of fuming HNO3
drop by drop. The solution was left stirring for 24 h. Finally, the solution is cooled with an ice bath:
a solid mass is formed. 2.15 g of a pale yellow solid is filtered and subsequently purified by
chromatography. Yield: 51%. M.P. 52-53 °C. 1H-NMR (CDCl3), δ: 8.53 (dd, 1H, H-6); 7.52 (t, J =
13
9.2 Hz, 1H, H-5); 4.66 (s, 3H, N-CH3). C-NMR: (CDCl3) δ: 159.41 (C), 154.00 (C), 140.10 (C),
134.32 (C), 126.48 (CH), 109.20 (CH), 44.38 (CH3). GC/MS: 197 (M+H). Anal. Calcd for
(C7H5FN4O2): C, 42.87; H, 2.57; F, 9.69; N, 28.57. Found: C, 42.91; H, 2.55; F, 9.71; N, 28.53.
5.1.3.3. 7-fluoro-1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (8c). 2.00 g (0.013· mol) of 4-fluoro-
1-methyl-1H-benzo[d][1,2,3]triazole (7c) are dissolved in 26 ml of H2SO4 conc. To the acidic
solution a mixture consisting of 4.92 g of KNO3 in 26 ml of H2SO4 conc. is added drop by drop at
room temperature. The temperature is then brought to 50 °C and left under stirring. After an hour
we let the solution return to room temperature and then poured into an ice bath. A light yellow
precipitate is formed and subsequently purified by chromatography. Yield 53%. M.P. 83-84 °C. 1H-
13
NMR (DMSO-d6), δ: 8.42 (dd, 1H, H-6); 7.43 (t, J = 9.2 Hz, 1H, H-5); 4.83 (s, 3H, N-CH3). C-
NMR: (CDCl3) δ: 159.98 (C), 154.58 (C), 131.09 (C), 129.05 (C), 127.53 (CH), 108.48 (CH), 40.09
(CH3). GC/MS: 197 (M+H). Anal. Calcd for (C7H5FN4O2): C, 42.87; H, 2.57; F, 9.69; N, 28.57.
Found: C, 42.90; H, 2.55; F, 9.71; N, 28.54.
5.1.3.4. 7-fluoro-1-methyl-1H-benzo[d][1,2,3]triazol-4-amine (9). 1.71 g (8.72·10-3 mol) of 7-
fluoro-1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (8) are dissolved in 105 ml of 37% HCl. Then
19.5 g (8.38 · 10-2 mol, ≈ 10 equivalents) of SnCl2 H2O are added. The reaction temperature is then
brought to reflux and from the moment the solution becomes clear, one hour is expected. After
solution returns to room temperature, it is neutralized with 50% NaOH. The solution obtained now
contains abundant hydroxide which is filtered. The aqueous solution , neutralized and violet, are
further basified until yellowish coloration. The mothers are extracted with ethyl ether, and the solid
is also thoroughly washed with the same solvent. By evaporation of the organic phase we get pure
amine (9). Yield 70%. M.P. 118-119 °C. 1H-NMR: (CDCl3, δ ppm): 7.265 (s, 2H, NH2), 6.922 (dd,
1H, J = 10.6, J = 10.4, H-5), 6.325 (dd, 1H, J = 3.2, J = 8.4, H-6), 4.397 (s, 3H, CH3). ). 13C-NMR:
(CDCl3) δ: 155.68 (C), 150.18 (C), 133.79 (C),. 130.07 (C), 117.38 (CH), 115.88 (CH), 35.12
(CH3). GC/MS: 167 (M+H). Anal. Calcd for (C7H7FN4): C, 50.60; H, 4.25; F, 11.43; N, 33.72.
Found: C, 50.63; H, 4.21; F, 11.47; N, 33.76.
5.1.3.5. Diethyl 2-(((7-fluoro-1-methyl-1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate
(10). 1.63 g (9.81·10-3 mol) of 7-fluoro-1-methyl-1H-benzo[d][1,2,3]triazol-4-amine (9) are
suspended in 35 ml of Dowtherm. 2.33 g (1.08 · 10-2 mol) 2.16 ml of diethyl
(ethoxymethylene)malonate are then added. The reaction temperature is then brought up to 150 °C
and allowed to react for 17 hours. After returning the solution to room temperature, it is diluted with
hexane, obtaining copious precipitation. The product is then filtered to obtain a gray solid. The solid
1
is abundantly washed with ethyl ether, obtaining the pure product. Yield 90%. M.P. 76-77 °C. H-
NMR: (CDCl3) δ: 11.623 (d, J = 13, 1H, NH), 9.322 (d, 1H, J = 13, CH), 6.922 (m, 2H, H-6, H-5),
13
4.307 (m, 4H, 2 CH2), 1.404 (m, 6H, 2 CH3). C-NMR: (CDCl3) δ: 171.22 (2 C), 155.12 (C),
151.23 (CH), 149.73 (C), 134.34 (C),. 130.31 (C), 117.09 (CH), 115.74 (CH), 97.36 (C), 59.82 (2
CH2), 35.36 (CH3), 14.25 (2 CH3).GC/MS: 337 (M+H). Anal. Calcd for (C15H17FN4O4): C, 53.57;
H, 5.10; F, 5.65; N, 16.66. Found: C, 53.60; H, 5.07; F, 5.68; N, 16.70.
5.1.3.6.
Ethyl
4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (11). 1.70 g (5.05·10-3 mol) of 2-(((7-fluoro-1-methyl-1H-benzo[d][1,2,3]triazol-4-
yl)amino)methylene)malonate (10) are poured into 40 ml of boiling Dowtherm. It is left to react for
15 minutes, after which the solution is returned to rt. It is diluted with 40 ml of hexane, obtaining
copious precipitation. The obtained gelatinous product is then shelled with abundant ethyl ether,
obtaining pure compound as a dusty brown solid. Yield 84%. M.P. 254-256 °C. 1H-NMR: (CDCl3)

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δ: 8.48 (s, 2H, NH2), 8.21 (s, 1H, H-9), 7.87 (d, 1H, J = 11.4, H-6), 4.50 (s, 3H, CH3), 4.28 (q, 2H,
CH2-CH3), 1.33 (t, 3H, CH2-CH3). 13C-NMR: (CDCl3) δ: 172.17 (C), 165.31 (C), 153.89 (C),
146.39 (CH), 143.11 (C), 134.36 (C), 130.49 (C), 129.22 (C), 112.07 (C), 102.46 (CH), 61.21
(CH2), 35.60 (CH3), 14.12 (CH3). GC/MS: 291 (M+H). Anal. Calcd for (C13H11FN4O3): C, 53.80;
H, 3.82; F, 6.55; N, 19.30. Found: C, 53.77; H, 3.85; F, 6.36; N, 19.27.
5.1.3.7.
Ethyl
4-fluoro-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (12). 0.83 g (2.8·10-3 mol) of ethyl 4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (11) are added to a suspension of 0.63 g (1.12·10-2
mol) of KOH in 20 ml of DMSO at room temperature. After 30 minutes, 1.58 g (1.12·10-2 mol) 0.7
ml of CH3I are added and the reaction is stopped for 1 hour. The solution is then diluted with 3 ml
of water, obtaining complete precipitation. The solid is filtered, washed with water and dried, thus
obtaining 0.65 g of a pure compound. Yield: 75%. M.P.: 217-219 °C. 1H-NMR: (CDCl3) δ: 8.50 (s,
1H, H-8), 8.27 (d, 1H, J = 11.4, H-5), 4.59 (s, 3H, N3-CH3), 4.54 (s, 3H, N9-CH3), 4.42 (q, 2H,
CH2-CH3), 1.43 (t, 3H, CH3-CH2). 13C-NMR: (CDCl3) δ: 176.07 (C), 164.88 (C), 153.17 (C),
147.03 (CH), 143.86 (C), 134.36 (C), 131.02 (C), 129.62 (C), 112.41 (C), 103.21 (CH), 61.34
(CH2), 45.60 (CH3), 35.88 (CH3), 14.36 (CH3). GC/MS: 305 (M+H). Anal. Calcd for
(C14H13FN4O3): C, 55.26; H, 4.31; F, 6.24; N, 18.41. Found: C, 55.22; H, 4.37; F, 6.19; N, 18.46.
5.1.3.8. 4-fluoro-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
acid (13). 0.30 g (9.86·10-4 mol) of Ethyl 4-fluoro-3,9-dimethyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (12) are dissolved in 20 ml of NaOH 2M. The
reaction temperature is brought to 100 °C and allowed to react for one hour. Returned to room
temperature, the solution is acidified with HCl 6M, obtaining complete precipitation. The gelatinous
solid is filtered, washed with plenty of water, and dried, obtaining the pure product. Yield: 82%.
M.P. > 300 °C. 1H-NMR: (Acetone) δ: 8.95 (s, 1H, H-8), 8.13 (d, 1H, H-5), 4.83 (s, 3H, CH3), 4.64
13
(s, 3H, CH3). C-NMR: (CDCl3) δ: 175.44 (C), 166.27 (C), 154.01 (C), 151.83 (CH), 144.51 (C),
134.92 (C), 131.42 (C), 129.88 (C), 112.90 (C), 102.89 (CH), 45.38 (CH3), 35.95 (CH3). GC/MS:
277 (M+H). Anal. Calcd for (C12H9FN4O3): C, 52.18; H, 3.28; F, 6.88; N, 20.28. Found: C, 52.15;
H, 3.33; F, 6.83; N, 20.31.
5.1.3.9. Ethyl 9-ethyl-4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (14). 0.82 g (2.8·10-3 mol) of Ethyl 4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (11) are dissolved in 10 ml of DMFa and brought to 0
°C. 0.135 g (5.63·10-3 mol) of NaH are added and allowed to react for 10 minutes. 0.5 ml (6.25·10-3
mol) 0.975 g of CH3CH2I are then dripped. It is left to react for 3 hours, after which the solution is
poured into ice. The aqueous solution is extracted with ethyl acetate. The solid product obtained is
purified by flash chromatography with CHCl3 to eliminate traces of the starting product. M.P.: 196-
197 °C. Yield: 51%. 1H-NMR: (CDCl3) δ: 8.53 (s, 1H, H-8), 8.28 (d, 1H, J = 11, H-5), 5.11 (q, 2H,
N9-CH2-CH3), 4.54 (s, 3H, N3-CH3), 4.43 (q, 2H, O-CH2-CH3), 1.61 (t, 3H, N9-CH2-CH3), 1.44 (t,
13
3H, O-CH2-CH3). C-NMR: (CDCl3) δ: 175.87 (C), 164.59 (C), 152.84 (C), 147.36 (CH), 144.09
(C), 134.83 (C), 131.27 (C), 129.47 (C), 111.32 (C), 105.19 (CH), 61.47 (CH2), 52.06 (CH2), 35.88
(CH3), 14.41 (CH3), 14,21 (CH3).GC/MS: 318 (M+H). Anal. Calcd for (C15H15FN4O3) C, 56.60; H,
4.75; F, 5.97; N, 17.60. Found: C, 56.57; H, 4.78; F, 6.01; N, 17.3.
5.1.3.10.
9-ethyl-4-fluoro-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (15). 0.09 g (2.83 10-4 mol) of . Ethyl 9-ethyl-4-fluoro-3-methyl-6-oxo-6,9-dihydro-
3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (14) are solubilized in 5 ml of DMSO and then
0.048 g (4.24 10-4 mol) of Potassium tert-butoxide are added. It is refluxed and allowed to react for
3 hours. After one hour the reaction was switched off and let to return at room temperature. 5 ml of
water are added and the solution is acidified with HCl 2 M, obtaining complete precipitation. The
solid is filtered, abundantly washed with water, washed with ethyl ether and finally dried to obtain a
1
pure gray compound. M.P.: 266-267 °C. Yield: 73%. H-NMR: (CDCl3) δ: 9.12 (s, 1H, H8), 8.03
(s, 1H, H5), 5.22 (q, 2H, CH2), 4.52 (s, 3H, CH3), 1.49 (t, 3H, CH3). 13C-NMR: (CDCl3) δ: 176.27

ACCEPTED MANUSCRIPT
(C), 167.19 (C), 155.12 (C), 150.39 (CH), 144.16 (C), 135.31 (C), 132.42 (C), 129.67 (C), 113.22
(C), 103.80 (CH), 51.89 (CH2), 35.68 (CH3), 14.80 (CH3). GC/MS: 291 (M+H). Anal. Calcd for
(C13H11FN4O3): C, 53.80; H, 3.82; F, 6.55; N, 19.30. Found: C, 53.83; H, 3.79; F, 6.51; N, 19.36.
5.1.4. General procedure for the preparation of the intermediate methoxy-methyl-nitro-
benzotriazoles (16a-c) and ethoxy-methyl-nitro-benzotriazole (25a). 0.5 g of the adequate fluoro-
methyl-nitro-benzotriazole (8a-c) are suspended in 20 ml of methanol or ethanol with 1 ml of Et3N.
The solution is refluxed for from 4 to 20 hours. A solid precipitate from the solution. After cooling
down the solution, the precipitate is filtered and then abundantly washed with ethyl ether, obtaining
the pure product.
5.1.4.1. 7-methoxy-1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (16a). Solvent: methanol. Time
1
reaction: 4 h. M.P.:116-117 °C. Yield: 53%. H-NMR: (CDCl3) δ: 8.28 (d, 1H, H-6); 6.82 (d, 1H,
H-5); 4.54 (s, 3H, NCH3); 4.14 (s, 3H, OCH3). GC/MS: 209 (M+H). Anal. Calcd for (C8H8N4O3) C,
46.16; H, 3.87; N, 26.91. Found: C, 46.20; H, 3.85; N, 26.88.
5.1.4.2. 4-methoxy-2-methyl-7-nitro-2H-benzo[d][1,2,3]triazole (16b). Solvent: methanol. Time
1
reaction: 20 h. M.P.:215-216 °C. Yield: 75%. H-NMR: (CDCl3) δ: 8.45 (d, J = 8, 1H, H-6); 6.75
(d, J = 8.4, 1H, H-5); 4.64 (s, 3H, NCH3); 4.19 (s, 3H, OCH3). GC/MS: 209 (M+H). Anal. Calcd
for (C8H8N4O3) C, 46.16; H, 3.87; N, 26.91. Found: C, 46.18; H, 3.90; N, 26.93.
5.1.4.3. 4-methoxy-1-methyl-7-nitro-1H-benzo[d][1,2,3]triazole (16c). Solvent: methanol. Time
1
reaction: 10 h. M.P.:72-73 °C. Yield: 81%. H-NMR: (CDCl3) δ: 8.40 (d, J = 9.2 Hz, 1H, H6); ),
6.77 (d, J = 8.6 Hz, 1H, H5); 4.62 (s, 3H, NCH3); 4.24 (s, 3H, OCH3). GC/MS: 209 (M+H). Anal.
Calcd for (C8H8N4O3) C, 46.16; H, 3.87; N, 26.91. Found: C, 46.17; H, 3.92; N, 26.87.
5.1.4.4. 4-ethoxy-1-methyl-7-nitro-1H-benzo[d][1,2,3]triazole (25a). Solvent: ethanol. Time
1
reaction: 18 h. M.P.:78-79 °C. Yield: 56%. H-NMR: (CDCl3) δ: 8.27 (d, 1H, H5); 6.78 (d, 1H,
H6); 4.55 (s, 3H, N-CH3); 4.36 (q, 2H, CH2CH3); 1.61 (t, 3H, CH3CH2). ). GC/MS: 223 (M+H).
Anal. Calcd for (C9H10N4O3) C, 48.65; H, 4.54; N, 25.21. Found: C, 48.67; H, 4.56; N, 25.19.
5.1.5. General procedure for the preparation of the intermediate methoxy-methyl-benzotriazol-
amines (17a-c) and ethoxy-methyl-nitro-benzotriazole (26a). 1 g of the adequate methoxy-methyl-
nitro-benzotriazoles (16a-c) or ethoxy-methyl-nitro-benzotriazole (25a) is dissolved in 100 ml of
THF or CHCl3 and added with 0.1 g of 10% C/Pd. It is set to reduce by means of a hydrogenator;
the theoretical amount of H2 is rapidly absorbed at room temperature and at atmospheric pressure.
By filtration the catalyst is removed, while the solvent is removed by evaporation: in this way a
crystal solid is obtained, which is crystalized in ethanol.
5.1.5.1. 7-methoxy-1-methyl-1H-benzo[d][1,2,3]triazol-4-amine (17a). Solvent: CHCl3. Time
reaction: 3 h. M.P.:119-120 °C. Yield: 77%. 1H-NMR: (CDCl3) δ: 6.57 (d, J = 8, 1H, H-6); 6.37 (d,
J = 8.2, 1H, H-5); 4.43 (s, 3H, NCH3); 3.89 (s, 3H, OCH3). GC/MS: 179 (M+H). Anal. Calcd for
(C8H10N4O) C, 53.92; H, 5.66; N, 31.44. Found C, 53.94; H, 5.68; N, 31.40.
5.1.4.2. 7-methoxy-2-methyl-2H-benzo[d][1,2,3]triazol-4-amine (17b). Solvent: THF. Time
reaction: 4 h. M.P.:217-218 °C. Yield: 75%. 1H-NMR: (CDCl3) δ: 8.45 (d, J = 8, 1H, H-6); 6.75 (d,
J = 8.4, 1H, H-5); 4.64 (s, 3H, NCH3); 4.19 (s, 3H, OCH3). GC/MS: 179 (M+H). Anal. Calcd for
(C8H10N4O) C, 53.92; H, 5.66; N, 31.44. Found C, 53.90; H, 5.67; N, 31.39.
5.1.4.3. 4-methoxy-1-methyl-1H-benzo[d][1,2,3]triazol-7-amine (17c). Solvent: THF. Time
1
reaction: 4 h. M.P.: 56-57 °C. Yield: 88%. H-NMR: (CDCl3) δ: 6.75 (s, 2H, NH2); 6.61 (d, 1H,
H6); 6.43 (d, 1H, H5); 4.51 (s, 3H, NCH3); 3.98 (s, 3H, OCH3). GC/MS: 179 (M+H). Anal. Calcd
for (C8H10N4O) C, 53.92; H, 5.66; N, 31.44. Found C, 53.89; H, 5.65; N, 31.45.
5.1.4.4. 7-ethoxy-1-methyl-1H-benzo[d][1,2,3]triazol-4-amine (26a). Solvent: CHCl3. Time
1
reaction: 2 h. M.P.: 132-133 °C. Yield: 69%. H-NMR: (CDCl3) δ: 6.57 (d, 1H, H5); 6.35 (d, 1H,
H6); 4.44 (s, 3H, N-CH3), 4.27 (s, 2H, NH2); 4.09 (q, 2H, CH2CH3); 1.47 (t, 3H, CH3CH2). GC/MS:

ACCEPTED MANUSCRIPT
193 (M+H). Anal. Calcd for (C9H12N4O) C, 56.24; H, 6.29; N, 29.15. Found C, 56.20; H, 6.32; N,
29.14.
5.1.5. General procedure for the preparation of the intermediate diethyl 2-[(methoxy-methyl-
benzotriazol-yl)amino)methylene]malonate derivatives (18a-c) and diethyl 2-(((7-ethoxy-1-methyl-
1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate (27a). 1 g of methoxy-methyl-
benzotriazol-amines (17a-c) or ethoxy-methyl-nitro-benzotriazole (26a). are suspended in 35 ml of
Dowtherm. Diethyl (ethoxymethylene)malonate is added to the solution in a molar ratio (1:1,1). The
reaction temperature is then brought up to 150 °C and allowed to react for 17 hours. After returning
the solution to room temperature, it is diluted with hexane, obtaining copious precipitation. The
product is then filtered to obtain a solid. The solid is abundantly washed with ethyl ether, obtaining
the pure product.
5.1.5.1.
Diethyl
2-(((7-methoxy-1-methyl-1H-benzo[d][1,2,3]triazol-4-
yl)amino)methylene)malonate (18a). M.P.: 130-131 °C. Yield: 63%. 1H-NMR: (CDCl3) δ: 11.55 (d,
J = 14, 1H, NH), 9.72 (d, J = 14, 1H, CH), 6.90 (d, J= 8, 1H, H6), 6.70 (d, J =9, 1 H, H6), 4.48 (s,
1H, NCH3), 4.32 (q, 4H, 2 CH2-CH3), 3.98 (s, 3H, OCH3), 1.39 (q, 6H, 2 CH3-CH2). GC/MS: 349
(M+H). Anal. Calcd for (C16H20N4O5): C, 55.17; H, 5.79; N, 16.08. Found: C, 55.19; H, 5.82; N,
16.10.
5.1.5.2.
Diethyl
2-(((7-methoxy-2-methyl-2H-benzo[d][1,2,3]triazol-4-
yl)amino)methylene)malonate (18b). M.P.: 118-119 °C. Yield: 66%. 1H-NMR: (CDCl3) δ: 11.44 (d,
1H, NH), 9.24 (d, 1H, CH), 6.95 (d, 1H, H5), 6.59 (d, 1H, H6), 4.53 (s, 3H, OCH3), 4.29 (m, 4H, 2
CH2CH3), 4.03 (s, 3H, CH3), 1.41 (q, 6H, 2 CH3CH2). GC/MS: 349 (M+H). Anal. Calcd for
(C16H20N4O5): C, 55.17; H, 5.79; N, 16.08. Found: C, 55.15; H, 5.74; N, 16.11.
5.1.5.3.
Diethyl
2-(((4-methoxy-1-methyl-1H-benzo[d][1,2,3]triazol-7-
yl)amino)methylene)malonate (18c). M.P.: 115-116 °C. Yield: 70%. 1H-NMR: (CDCl3) δ: 11.42 (d,
1H, J = 12.8 Hz, NH), 8.35 (d, 1H, J = 12.6 Hz, CH=), 7.17 (d, 1H, J = 8 Hz, H5), 6.46 (d, 1H, J = 8
Hz, H6), 4.51 (s, 3H, OCH3), 4.38-4.17 (m, 4H, 2 CH2CH3), 4.09 (s, 3H, CH3), 1.43-1.28 (m, 6H, 2
CH3CH2). GC/MS: 349 (M+H). Anal. Calcd for (C16H20N4O5): C, 55.17; H, 5.79; N, 16.08. Found:
C, 55.14; H, 5.77; N, 16.09.
5.1.5.4. Diethyl 2-(((7-ethoxy-1-methyl-1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate
1
(27a). M.P.: 141-142 °C. Yield: 57%. H-NMR: (CDCl3) δ: 11.54 (d, 1H, NH), 9.35 (d, 1H, CH),
6.88 (d, 1H, H5), 6.68 (d, 1H, H6), 4.49 (s, 3H, N-CH3), 4.14-4.38 (m, 6H, CH2CH3), 1.33-1.58 (m,
9H, CH3CH2). GC/MS: 363 (M+H). Anal. Calcd for (C17H22N4O5): C, 56.35; H, 6.12; N, 15.46.
Found: C, 56.38; H, 6.09; N, 15.51.
5.1.6. General procedure for the preparation of the intermediate ethyl-methoxy-methyl-6-oxo-6,9-
dihydro-triazolo[4,5-h]quinoline-7-carboxylate derivatives (19a-c) and ethyl 4-ethoxy-3-methyl-6-
oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (28a). 1 g of the appropriate
diethyl 2-[(methoxy-methyl-benzotriazol-yl)amino)methylene]malonate derivatives (18a-c) or
diethyl 2-(((7-ethoxy-1-methyl-1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate (27a)
are poured into 40 ml of boiling Dowtherm. It is left to react for 15 minutes, after which the
solution is returned to rt. The solution is diluted with 40 ml of hexane, obtaining copious
precipitation. The obtained product is then shelled with abundant ethyl ether. Finally, the obtained
compound is crystallized from ethanol.
5.1.6.1.
Ethyl
4-methoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (19a). M.P.: 263-264 °C. Yield: 88%. H-NMR: (CDCl3) δ: 4 12.32 (s, 1H, NH), 9.19
(s, 1H, Hquinolone), 7.38 (s, 1H, H6), 4.56 (m, 5H, NCH3 + CH2-CH3), .123 (s, 3H, OCH3), 1.49 (q,
3H, CH3-CH2). GC/MS: 303 (M+H). Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67; N, 18.53.
Found: C, 55.66; H, 4.70; N, 18.51.

ACCEPTED MANUSCRIPT
5.1.6.2.
Ethyl
4-methoxy-2-methyl-6-oxo-6,9-dihydro-2H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (19b). M.P.: 228-229 °C. Yield: 83%. H-NMR: (CDCl3) δ: 9.12 (s, 1H, H8), 7.29 (s,
1H, H5), 4.59 (s, 3H, OCH3), 4.51 (m, 2H, CH2CH3), 4.16 (s, 3H, CH3), 1.50 (t, 3H, CH3CH2).
GC/MS: 303 (M+H). Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67; N, 18.53. Found: C, 55.63;
H, 4.65; N, 18.55.
5.1.6.3. Ethyl 4-methoxy-1-methyl-6-oxo-6,9-dihydro-1H-[1,2,3]triazolo [4,5-h]quinoline-7-
carboxylate (19c). M.P.: 204-205 °C. Yield: 73%. 1H-NMR: (CDCl3) δ: 12.25 (s, 1H, NH), 9.07 (s,
1H, H8), 7,26 (s, 1H, H5), 4.83 (s, 3H, OCH3), 4.54 (m, 2H, CH2CH3), 4.18 (s, 3H, CH3), 1.49 (t,
3H, CH3CH2). Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67; N, 18.53. Found: C, 55.60; H,
4.71; N, 18.50.
5.1.6.4.
Ethyl
4-ethoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (28a). M.P.: 280-281 °C. Yield: 97%. 1H-NMR: (CDCl3) δ: 12.28 (s, 1H, NH), 9.19 (s,
1H, H8), 7.38 (s, 1H, H5), 4.60 (s, 3H, N-CH3), 4.30-4.55 (m, 4H, CH2CH3), 1.46-1.64 (m, 6H,
CH3CH2). GC/MS: 317 (M+H). Anal. Calcd for (C15H16N4O4): 56.96; H, 5.10; N, 17.71. Found:
56.99; H, 5.08; N, 17.73.
5.1.7. General procedure for the preparation of methoxy-dimethyl-6-oxo-6,9-dihydro-3H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (20a), 4-methoxy-2,9-dimethyl-6-oxo-6,9-dihydro-
2H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acid (20b), ethyl 4,6-dimethoxy-1-methyl-1H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (20c) and ethyl 4-methoxy-3,9-dimethyl-6-oxo-6,9-
dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (29a). 1.00 g of the appropriate ethyl-
methoxy-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-carboxylate derivatives (19a-c) or
ethyl 4-ethoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (28a).
are added to a suspension of 0.76 g of KOH in 20 ml of DMSO at room temperature. After 30
minutes, 1.90 g (1.33·10-2 mol) 0.83 ml of CH3I are added and the reaction is stopped for 1 hour.
The solution is then diluted with 3 ml of water, obtaining complete precipitation. The solid is
filtered, washed with water and dried. Pure compounds were crystalized from ethanol.
5.1.7.1. Ethyl 4-methoxy-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (20a). M.P.: 256-257 °C. Yield: 59%. H-NMR: (DMSO-d6) δ: 8.56 (s, 1H, H9), 7.53
(s, 1H, H6), 4.47 (s, 3H, N3-CH31)3, 4.30 (s, 3H, OCH3), 4.25 (q, 2H, CH2), 4.03 (s, 3H, N9-CH3),
1.31 (t, 3H, J = 7.4, CH3-CH2). C-NMR: (DMSO-d6) δ: 171.10 (C), 164.55 (C), 148.40 (CH),
143.50 (C), 139.23 (C), 128.64 (C), 127.48 (C), 125.65 (C), 111.18 (C), 101.77 (CH), 59.82 (CH2),
56.38 (CH3), 51.32 (CH3), 33.05 (CH3), 14.25 (CH3). GC/MS: 317 (M+H). Anal. Calcd for
(C15H16N4O4): C, 56.96; H, 5.10; N, 17.71. Found: C, 57.00; H, 5.12; N, 17.69.
5.1.7.2.
4-methoxy-2,9-dimethyl-6-oxo-6,9-dihydro-2H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylic acid (20b). M.P.> 300 °C. Yield: 41%. H-NMR: (CDCl3) δ: 7.91 (s, 1H, H8), 7.22 (s,
1H, H5), 4.64 (s, 3H, OCH3), 4.59 (s, 3H, CH3), 4.17 (s, 3H, CH3). GC/MS: 289 (M+H). Anal.
Calcd for (C13H12N4O4): C, 54.17; H, 4.20; N, 19.44. Found: C, 54.19; H, 4.18; N, 19.46.
5.1.7.3. Ethyl 4,6-dimethoxy-1-methyl-1H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate (20c).
M.P.: 146-147 °C. Yield: 80%. 1H-NMR: (DMSO-d6) δ: 8.98 (s, 1H, H8), 7.23 (s, 1H, H5), 4.72 (s,
3H, CH3), 4.33 (q, 2H, J = 6.8 Hz, CH2), 4.12 (s, 3H, CH3), 4.10 (s, 3H, CH3), 1.39 (t, 3H, J = 7.4,
13
CH3). C-NMR: (DMSO-d6) δ: 164.68 (C), 162.13 (C), 149.77 (C), 148.33 (CH), 139.99 (C),
136.25 (C), 130.45 (C), 123.15 (C), 115.77 (C), 94.51 (CH), 62.76 (CH3), 61.59 (CH2), 57.46
(CH3), 37.58 (CH3), 13.95 (CH3). GC/MS: 317 (M+H). Anal. Calcd for (C15H16N4O4): C, 56.96; H,
5.10; N, 17.71. Found: C, 56.98; H, 5.09; N, 17.74.
5.1.7.4. Ethyl 4-ethoxy-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (29a). M.P.:228-229 °C. Yield: 94%. H-NMR: (CDCl3) δ: 8.40 (s, 1H, H8), 7.45 (s,
1H, H5), 4.65 (s, 3H, CH3), 4.57 (s, 3H, CH3), 4.11-4.42 (m, 4H, CH2CH3), 1.54-1.61 (m, 6H,
CH3CH2). 13C-NMR: (DMSO-d6) δ: 175.18 (C), 164.97 (C), 148.37 (CH), 141.22 (C), 134.31 (C),

ACCEPTED MANUSCRIPT
129.93 (C), 127.66 (C), 120.88 (C), 109.82 (C), 107.83 (CH), 62.06 (CH2), 46.39 (CH3), 35.59
(CH3), 23.38 (CH2), 14.17 (CH3), 14.59 (CH3). GC/MS: 331 (M+H). Anal. Calcd for (C16H18N4O4):
C, 58.17; H, 5.49; N, 16.96. Found: C, 58.20; H, 5.46; N, 16.99.
5.1.8. General procedure for the synthesis of alkoxy-dimethyl-6-oxo-6,9-dihydro-triazoloquinoline-
carboxylic acid derivatives 21a,c and 30a. 0.50 g of the appropriate triazolo[4,5-h]quinoline-
carboxylate (20a,c or 29a) are dissolved in 20 ml of NaOH 2M. The reaction temperature is brought
to 100 °C and allowed to react for one hour. Returned to room temperature, the solution is acidified
with HCl 6M, obtaining complete precipitation. The gelatinous solid is filtered, washed with plenty
of water, and dried, obtaining the pure product.
5.1.8.1.
4-methoxy-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylic acid (21a). M.P.> 300 °C. Yield: 89%. H-NMR: (DMSO-d6) δ: 9.04 (s, 1H, H8), 7.68
(s, 1H, H5), 4.66 (s, 3H, O-CH3), 4.54 (s, 3H, N3-CH3), 4.14 (s, 3H, N9-CH3). 13C-NMR: (DMSO-
d6) δ: 175.16 (C), 166.23 (C), 150.98 (CH), 143.88 (C), 136.91 (C), 133.57 (C), 130.41 (C), 127.82
(C), 109.57 (C), 103.66 (CH), 55.89 (CH3), 47.17 (CH3), 35.22 (CH3). GC/MS: 289 (M+H). Anal.
Calcd for (C13H12N4O4): C, 54.17; H, 4.20; N, 19.44. Found: C, 54.15; H, 4.22; N, 19.46.
5.1.8.2. 4,6-dimethoxy-1-methyl-1H-[1,2,3]triazolo[4,5-h]quinoline-7- carboxylic acid (21c). M.P.>
300 °C. Yield: 42%. 1H-NMR: (CDCl3) δ: 8.89 (s, 1H, H8), 7.83 (s, 1H, H5), 4.61 (s, 3H, O-CH3),
4.42 (s, 3H, N3-CH3), 4.01 (s, 3H, -CH3). 13C-NMR: (DMSO-d6) δ: 175.77 (C), 165.95 (C), 147.27
(CH), 144.96 (C), 138.12 (C), 129.48 (C), 127.66 (C), 123.63 (C), 109.22 (C), 100.66 (CH), 59.87
(CH2), 56.77 (CH3), 52.63 (CH2), 37.39 (CH3), 15.73 (CH3). GC/MS: 288 (M+H). Anal. Calcd for
(C13H12N4O4): C, 54.17; H, 4.20; N, 19.44. Found: C, 54.12; H, 4.23; N, 19.42.
5.1.8.3. 4-ethoxy-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
1
acid (30a). M.P.> 300 °C. Yield: 82%. H-NMR: (CDCl3) δ: 15.62 (s, 1H, OH), 8.90 (s, 1H, H8),
7.73 (s, 1H, H5), 4.73 (s, 3H, CH3), 4.61 (s, 3H, OCH3), 4.40 (Q, 2H, CH2CH3), 1.61 (t, 3H,
CH3CH2). 13C-NMR: (DMSO-d6) δ: 176.39 (C), 165.74 (C), 151.66 (CH), 140.23 (C), 136.17 (C),
133.98 (C), 130.52 (C), 127.82 (C), 110.32 (C), 102.84 (CH), 64.31 (CH2), 46.37 (CH3), 35.89
(CH3), 15.03 (CH3). GC/MS: 303 (M+H). Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67; N,
18.53. Found: C, 55.66; H, 4.70; N, 18.50.
5.1.9. General procedure for the synthesis of methoxy-methyl-6-oxo-6,9-dihydro-triazolo[4,5-
h]quinoline-7-carboxylate derivatives (22a-c, 31a). 0.5 g of the adequate ethyl-alkoxy-methyl-6-
oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-carboxylate derivatives (19a-c or 28a) are added to a
suspension of 0.35 g of KOH in 15 ml of DMSO at room temperature. After 30 minutes, 0.55 ml of
CH3CH2I are added. After 30 minutes from the addition an abundant precipitate is formed. The
stirring is removed and the solution is diluted with 40 ml of water, obtaining a solid which is
filtered and dried.
5.1.9.1. Ethyl 9-ethyl-4-methoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (22a). M.P.: 225-226 °C. Yield: 90%. H-NMR: (DMSO-d6) δ: 8.63 (s, 1H, H8), 7.59
(s, 1H, H5), 5.02 (q, 2H, O- CH2-), 4.48 (s, 3H, NCH3), 4,26 (q, 2H, N-CH2-),4.04 (s, 3H, OCH3),
1.42 (t, 3H, CH3 etile), 1.32 (t, 3H, CH3). 13C-NMR: (DMSO-d6) δ: 171.18 (C), 164.60 (C), 147.25
(CH), 143.58 (C), 138.37 (C), 128.67 (C), 126.34 (C), 126.05 (C), 111.80 (C), 102.05 (CH), 59.87
(CH2), 56.41 (CH3), 51.34 (CH2), 37.17 (CH3), 15.76 (CH3), 14.26 (CH3). GC/MS: 331 (M+H).
Anal. Calcd for (C16H18N4O4): C, 58.17; H, 5.49; N, 16.96. Found: C, 58.19; H, 5.46; N, 16.99.
5.1.9.2. Ethyl 9-ethyl-4-methoxy-2-methyl-6-oxo-6,9-dihydro-2H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (22b). M.P.: 239-240 °C. Yield: 66%. H-NMR: (DMSO-d6) δ: 8.64 (s, 1H, H8), 7.52
(s, 1H, H5), 4.90 (q, 2H, CH2CH3), 4.58 (s, 3H, OCH3), 4.25 (q, 2H, CH2CH3), 4.03 (s, 3H, CH3),
13
1.43 (t, 3H, CH3CH2), 1.31 (t, 3H, CH3CH2). C-NMR: (DMSO-d6) δ: 171.27 (C), 164.60 (C),
147.10 (C), 146.63 (CH), 139.66 (C), 136.91 (C), 127.40 (C), 126.11 (C), 112.20 (C), 99.38 (CH),
59.89 (CH2), 55.88 (CH3), 51.02 (CH2), 40.42 (CH3), 15.80 (CH3), 14.25 (CH3). GC/MS: 331

ACCEPTED MANUSCRIPT
(M+H). Anal. Calcd for (C16H18N4O4): C, 58.17; H, 5.49; N, 16.96. Found: C, 58.15; H, 5.52; N,
16.93.
5.1.9.3. Ethyl 6-ethoxy-4-methoxy-1-methyl-1H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylate
1
(22c). M.P.: 105-106 °C. Yield: 84%. H-NMR: (DMSO-d6) δ: 8.97 (s, 1H, H8), 7.16 (s, 1H, H5),
4.71 (s, 3H, CH3), 4.42 (q, 2H, CH2CH3), 4.27 (q, 2H, CH2CH3), 4.11 (s, 3H, CH3), 1.47 (t, 3H,
13
CH3CH2), 1.39 (t, 3H, CH3CH2). C-NMR: (DMSO-d6) δ: 164.60 (C), 161.34 (C), 149.74 (C),
148.39 (CH), 140.02 (C), 136.38 (C), 130.47 (C), 123.74 (C), 116.46 (C), 94.60 (CH), 71.82 (CH2),
61.48 (CH2), 56.01 (CH3), 37.57 (CH3), 15.33 (CH3), 13.96 (CH3). GC/MS: 331 (M+H). Anal.
Calcd for (C16H18N4O4): C, 58.17; H, 5.49; N, 16.96. Found: C, 58.16; H, 5.50; N, 16.94.
5.1.9.4. Ethyl 4-ethoxy-9-ethyl-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylate (31a). M.P.: 192-193 °C. Yield: 73%. H-NMR: (CDCl3) δ: 8.49 (s, 1H, H8), 7.88 (s,
1H, H5), 5.10 (q, 2H, N- CH2CH3), 4.56 (s, 3H, CH3), 4.28-4.48 (m, 4H, 2 CH2CH3), 1.41-1.67 (m,
9H, 3 CH3CH2). 13C-NMR: (DMSO-d6) δ: 174.32 (C), 164.09 (C), 146.20 (CH), 142.98 (C), 138.66
(C), 128.03 (C), 126.49 (C), 126.05 (C), 110.83 (C), 102.57 (CH), 64.31 (CH2), 61.40 (CH2), 51.86
(CH2), 36.22 (CH3), 14.41 (CH3), 14.32 (CH3), 14.26 (CH3). GC/MS: 344 (M+H). ). Anal. Calcd
for (C17H20N4O4): C, 59.29; H, 5.85; N, 16.27. Found: C, 59.31; H, 5.88; N, 16.26.
5.1.10. General procedure for the synthesis of alkoxy-methyl-6-oxo-6,9-dihydro-triazolo[4,5-
h]quinoline-7-carboxylic acid (23a-c, 32a). 0.5 g of the adequate alkoxy-methyl-6-oxo-6,9-
dihydro-triazolo[4,5-h]quinoline-7-carboxylate derivatives (22a-c or 31a) are dissolved in 22 ml of
2 M NaOH. The mixture is brought to 100 ° C for 2 hours under magnetic stirring. The basic, clear
solution is returned to room temperature and subsequently acidified with 2 M HCl; a very fine
gelatinous precipitate is formed which is filtered (albeit with difficulty) and dried, obtaining pure
carboxylic acid.
5.1.10.1.
9-ethyl-4-methoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (23a). M.P.> 300 °C. Yield: 86%. 1H-NMR: (DMSO-d6) δ: 15.11 (s, 1H, OH), 9.08
(s, 1H, H8), 7.68 (s, 1H, H5), 5.25 (q, 2H, CH2-CH3), 4,54 (s, 3H, NCH3), 4.13 (s, 3H, OCH3), 1.50
13
(t, 3H, CH3-CH2). C-NMR: (DMSO-d6) δ: 175.77 (C), 165.95 (C), 147.27 (CH), 144.96 (C),
138.12 (C), 129.48 (C), 127.66 (C), 123.63 (C), 109.22 (C), 100.66 (CH), 59.87 (CH2), 56.77
(CH3), 52.63 (CH2), 37.39 (CH3), 15.73 (CH3). GC/MS: 303 (M+H). Anal. Calcd for (C14H14N4O4):
C, 55.63; H, 4.67; N, 18.53. Found: C, 55.60; H, 4.64; N, 18.54.
5.1.10.2.
9-ethyl-4-methoxy-2-methyl-6-oxo-6,9-dihydro-2H-[1,2,3]triazolo[4,5-h]quinoline-7-
1
carboxylic acid (23b). M.P.: 273-274 °C. Yield: 95%. H-NMR: (DMSO-d6) δ: 15.83 (s, 1H, OH),
9.03 (s, 1H, H8), 7.47 (s, 1H, H5), 5.05 (m, 2H, CH2CH3), 4.62 (s, 3H, OCH3), 4.08 (s, 3H, CH3),
13
1.47 (t, 3H, CH3CH2). C-NMR: (DMSO-d6) δ: 175.71 (C), 166.00 (C), 148.31 (C), 146.46 (CH),
142.27 (C), 140.14 (C), 127.56 (C), 125.03 (C), 111.10 (C), 99.05 (CH), 56.18 (CH3), 52.30 (CH2),
43.99 (CH3), 15.69 (CH3). GC/MS: 303 (M+H). Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67;
N, 18.53. Found: C, 55.66; H, 4.70; N, 18.51.
5.1.10.3. 6-ethoxy-4-methoxy-1-methyl-1H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acid (23c).
M.P.: 194-195 °C. Yield: 76%. 1H-NMR: (DMSO-d6) δ: 8.98 (s, 1H, H8), 7.17 (s, 1H, H5), 4.71 (s,
13
3H, OCH3), 4.32 (q, 2H, CH2), 4.11 (s, 3H, CH3), 1.46 (t, 3H, CH3). C-NMR: (DMSO-d6) δ:
166.16 (C), 161.25 (C), 149.58 (C), 148.66 (CH), 139.90 (C), 136.19 (C), 130.45 (C), 123.78 (C),
117.25 (C), 94.58 (CH), 71.55 (CH2), 57.21 (CH3), 37.55 (CH3), 15.57 (CH3). GC/MS: 303 (M+H).
Anal. Calcd for (C14H14N4O4): C, 55.63; H, 4.67; N, 18.53. Found: C, 55.62; H, 4.68; N, 18.55.
5.1.10.4.
4-ethoxy-9-ethyl-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (32a). M.P.: 263-264 °C. Yield: 57%. 1H-NMR: (CDCl3) δ: 15.43 (s, 1H, OH), 8.75
(s, 1H, H8), 7.75 (s, 1H, H5), 5.21 (q, 2H, OCH2CH3), 4.40 (q, 2H, OCH2CH3), 1.60 (m, 6H, 2
CH3CH2). 13C-NMR: (DMSO-d6) δ: 175.29 (C), 166.37 (C), 149.09 (CH), 140.33 (C), 138.01 (C),
128.47 (C), 126.39 (C), 126.37 (C), 110.77 (C), 102.67 (CH), 64.11 (CH2), 51.32 (CH2), 35.41

ACCEPTED MANUSCRIPT
(CH3), 14.87 (CH3), 14.26 (CH3). GC/MS: 316 (M+H). Anal. Calcd for (C15H16N4O4): C, 56.96; H,
5.10; N, 17.71. Found: C, 56.99; H, 5.08; N, 17.74.
5.1.11. General procedure for the synthesis of 4-methoxy-1/3-methyl-6-oxo-6,9-dihydro-1H-
[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic acid (24a,c). 0.5 g of the adequate mehoxy-methyl-6-
oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-carboxylate derivatives (19a,c) are dissolved in 22 ml
of 2 M NaOH. The mixture is brought to 100 ° C for 2 hours under magnetic stirring. The basic,
clear solution is returned to room temperature and subsequently acidified with 2 M HCl; a
precipitate is formed which is filtered and dried, obtaining pure carboxylic acid.
5.1.11.1. 4-methoxy-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
1
acid (24a). M.P.> 300 °C. Yield: 74%. H-NMR: (CDCl3) δ: 8.615 (s, 1H, H8), 7.507 (s, 1H, H5),
13
4.511 (s, 3H, N-CH3), 4.109 (s, 3H, O-CH3). C-NMR: (DMSO-d6) δ: 174.88 (C), 165.79 (C),
150.23 (CH), 144.21 (C), 139.97 (C), 132.09 (C), 130.56 (C), 127.37 (C), 111.68 (C), 103.17 (CH),
55.77 (CH3), 35.43 (CH3). GC/MS: 274 (M+H). Anal. Calcd for (C12H10N4O4): C, 52.56; H, 3.68;
N, 20.43. Found: C, 52.54; H, 3.71; N, 20.39.
5.1.11.2. 4-methoxy-1-methyl-6-oxo-6,9-dihydro-1H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
1
acid (24c). M.P.> 300 °C. Yield: 97%. H-NMR: (CDCl3) δ: 8.747 (s, 1H, H8), 7.514 (s, 1H, H5),
13
4.695 (s, 3H, N-CH3), 3.951 (s, 3H, O-CH3). C-NMR: (DMSO-d6) δ: 175.31 (C), 165.06 (C),
150.45 (CH), 144.19 (C), 140.22 (C), 132.35 (C), 130.25 (C), 127.56 (C), 110.41 (C), 102.89 (CH),
55.37 (CH3), 35.36 (CH3). GC/MS: 274 (M+H). Anal. Calcd for (C12H10N4O4): C, 52.56; H, 3.68;
N, 20.43. Found: C, 52.58; H, 3.66; N, 20.41.
5.1.12. Synthesis of ethylthio-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinolines (33-37)
5.1.12.1. 7-(ethylthio)-1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (33). 1 g (5.1·10-3 mol) of
benzotriazole intermediate 8a are dissolved in 5 ml (6·10-2 mol) 3.7 g of thioethane with 2 ml of
Et3N. The solution is refluxed for 4 hours, then brought to room temperature. After evaporation, a
pale yellow solid is collected. The raw compound is purified through flash chromatography. ). M.P.:
193-194 °C. Yield: 76%. 1H-NMR: (CDCl3) δ: 8.17 (d, 1H, J = 8.4 Hz, H6), 7.23 (d, 1H, J = 8.4 Hz,
H5), 4.67 (s, 3H, CH3), 3.23 (q, 2H, CH2), 1.50 (t, 3H, CH3). GC/MS: 239 (M+H). Anal. Calcd for
(C9H10N4O2S): C, 45.37; H, 4.23; N, 23.51; S, 13.46. Found C, 45.40; H, 4.20; N, 23.53; S, 13.49.
5.1.12.2. 7-(ethylthio)-1-methyl-1H-benzo[d][1,2,3]triazol-4-amine (34). 1.0 g (4.2 10-3 mol) of 7-
(ethylthio)-1-methyl-4-nitro-1H-benzotriazole (35) are dissolved in 50 ml of ethanol and added with
0.10 g of 10% C/Pd. It is set to reduce by means of a hydrogenator; the theoretical amount of H2 is
rapidly absorbed at room temperature and at atmospheric pressure. By filtration the catalyst is
removed, while the solvent is removed by evaporation. The pure amine is obtained as a violet oil.
1
Oil. Yield: 84%. H-NMR (CDCl3) δ: 7.36 (d, 2H, J =7.8, H6), 6.42 (d, 2H, J =7.8, H5), 4.58 (s,
13
3H, CH3), 2.72 (q, 2H, CH2), 1.19 (t, 3H, CH3). C-NMR (CDCl3) δ: 146.9 (C), 140.7 (C), 133.2
(C), 130.5 (C), 126.8 (CH), 121.1 (CH), 33.2 (CH3), 28.8 (CH2), 14.1 (CH3). GC/MS: 209 (M+H).
Anal. Calcd for (C9H12N4S): C, 51.90; H, 5.81; N, 26.90; S, 15.39. Found: C, 51.92; H, 5.79; N,
26.93; S, 15.41.
5.1.12.3. Ethyl 4-(ethylthio)-3-methyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (35). 1 g (5.2·10-3 mol) of 7-(ethylthio)-1-methyl-1H-benzotriazol-4-amine (34) is
suspended in 25 ml of Dowtherm, then 1.8 g (8.6·10-3 mol) 1.7 ml of diethyl
ethoxymethylenemalonate (EMME) are added. The solution is brought to 150 °C for 17 hours, but
reaction resulted incomplete. We brought temperature to 250 °C and we let it go for almost 30
minutes. Finally, we reported the reaction temperature at room temperature, and we added 30 ml of
hexane to obtain an abundant precipitate. The solid compound is filtered, washed with firstly with
hexane, then with diethyl ether and dried. Raw precipitate is purified through flash chromatography
1
obtaining pure quinolone derivative. M.P.: 225-226 °C. Yield: 66%. H-NMR (CDCl3) δ: 12.85 (s,
1 H, NH), 8.82 (s, 1H, H-5), 8.03 (s, 1H, H-8), 4.61 (s, 3H, N-CH3), 4.47 (q, 2H, O-CH2, J = 7.0),

ACCEPTED MANUSCRIPT
13
3.10 (q, 2H, S-CH2, J = 7.2), 1.51-1.37 (m, 6H, 2 CH3). C-NMR: (DMSO-d6) δ: 174.33 (C),
165.42 (C), 149.11 (CH), 141.64 (C), 134.25 (C), 129.34 (C), 126.89 (C), 121.34 (C), 111.02 (C),
115.22 (CH), 61.81 (CH2), 35.12 (CH3), 29.57 (CH2), 14.45 (CH3), 14.29 (CH3). GC/MS: 333
(M+H). Anal. Calcd for (C15H16N4O3S): C, 54.20; H, 4.85; N, 16.86; S, 9.65. Found: C, 54.22; H,
4.83; N, 16.89; S, 9.63.
5.1.12.4. Ethyl 4-(ethylthio)-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (36). 1 g (3.0·10-3 mol) of triazolo-quinoline-7-carboxylate (35) are added to a
suspension of 0.48 g of KOH in 20 ml of DMSO at room temperature. After 20 minutes, 0.5 ml
(8·10-3 mol), 1.15 g of CH3I are added and the reaction is left for 4 hours. The dark red solution is
then diluted with 25 ml of water, obtaining a brown solid, which is filtered and dried. We get an
1
impure solid which is purified through flash chromatography. M.P.: 214-215 °C. Yield: 83%. H-
NMR (CDCl3) δ: 9.08 (s, 1 H, H-5), 8.14 (s, 1H, H-8), 4.73 (s, 3H, N-CH3), 4.70 (s, 3H, N-CH3),
13
4.24 (q, 2H, O-CH2, J = 7.0), 3.23 (q, 2H, S-CH2, J = 7.2), 1.61-1.25 (m, 6H, 2 CH3). C-NMR:
(DMSO-d6) δ: 174.87 (C), 165.17 (C), 149.83 (CH), 141.01 (C), 134.52 (C), 129.39 (C), 127.77
(C), 121.17 (C), 111.25 (C), 115.32 (CH), 61.69 (CH2), 46.21 (CH3), 35.12 (CH3), 29.56 (CH2),
14.32 (CH3), 14.21 (CH3). GC/MS: 347 (M+H). Anal. Calcd for (C16H18N4O3S): C, 55.48; H, 5.24;
N, 16.17; S, 9.26. Found: C, 55.50; H, 5.22; N, 16.19; S, 9.23.
5.1.12.5.
4-(ethylthio)-3,9-dimethyl-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylic acid (37). 0.5 g (1.5·10-3 mol) of triazolo-quinoline-carboxylate (36) are dissolved in 22
ml of 2 M NaOH. The mixture is brought to 100 ° C for 2 hours under magnetic stirring. After
cooling, the solution is neutralized with 2 M HCl; a very fine gelatinous precipitate is formed,
which is filtered and dried giving the pure product. M.P.> 300 °C. Yield: 73%. 1H-NMR (CDCl3) δ:
15.12 (s, 1H, OH) 8.76 (s, 1 H, H-5), 8.30 (s, 1H, H-8), 4.76 (s, 3H, N-CH3), 4.68 (s, 3H, N-CH3),
3.27 (q, 2H, S-CH2, J = 7.2), 1.50-1.43 (m, 3H, CH3). 13C-NMR: (DMSO-d6) δ: 174.68 (C), 165.32
(C), 149.56 (CH), 141.39 (C), 134.27 (C), 129.81 (C), 127.83 (C), 121.143 (C), 111.46 (C), 115.07
(CH), 45.79 (CH3), 35.26 (CH3), 29.43 (CH2), 14.39 (CH3). GC/MS: 319 (M+H). Anal. Calcd for
(C14H14N4O3S): C, 52.82; H, 4.43; N, 17.60; S, 10.07. Found: C, 52.79; H, 4.45; N, 17.58; S, 10.10.
5.1.13. Synthesis of nitro-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinolines (38-41)
5.1.13.1. 1-methyl-7-nitro-1H-benzo[d][1,2,3]triazol-4-amine (38). 1 g (5.09·10-2 mol) of 7-fluoro-
1-methyl-4-nitro-1H-benzo[d][1,2,3]triazole (8c) are dissolved in 100 ml of ammonia propanol and
placed in a closed tube at 50 °C for 2 hours. The abundant precipitate obtained is cooled and
filtered, which is washed with ethyl ether and dried as a pure compound. M.P. 256-257 °C. Yield:
1
98%. H-NMR (CDCl3) δ: 8.18 (d, 1H, J = 8.8 Hz, H6), 6.46 (d, 1H, J = 8.8 , H-6), 4.47 (s, 3H,
CH3). GC/MS: 194 (M+H). Anal. Calcd for (C7H7N5O2): C, 43.53; H, 3.65; N, 36.26. Found: C,
43.50; H, 3.67; N, 36.23.
5.1.13.2. Diethyl 2-(((1-methyl-7-nitro-1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate
(39). 0.8 g (4.3·10-3 mol) of 1-methyl-7-nitro-1H-benzo[d][1,2,3]triazol-4-amine (38) are suspended
in 15 ml of Dowtherm. 1.03 g (4.73·10-3 mol) 0.96 ml of diethyl ethoxymethylenemalonate
(EMME) are added. The solution is brought to 150 °C and let go for 17 hours. Once the time has
expired, the mixture is brought to room temperature. Hexane is then added to obtain a precipitate
which is filtered, washed with hexane and dried. Pure compound is obtained through flash
1
chromatography. M.P.: 159-160 °C. Yield: 75%. H-NMR (CDCl3) δ: 11.973 (d, 1H, J = 11.8 Hz,
NH), 9.064 (d, 1H, =CH, J = 12), 8.405 (d, 1H, H6, J = 9 Hz), 7.076 (d, 1H, H5, J = 9 Hz), 4.655 (s,
3H, CH3), 4.463-4.263 (m, 4H, 2 CH2), 1.461-1.346 (m, 6H, 2 CH3). GC/MS: 364 (M+H). Anal.
Calcd for (C15H17N5O6): C, 49.59; H, 4.72; N, 19.28. Found: C, 49.62; H, 4.74; N, 19.25.
5.1.13.3.
Ethyl
3-methyl-4-nitro-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-
carboxylate (40). 20 ml of Dowtherm are brought to the boiling temperature, then 0.7 g (1.9·10-3
mol) of diethyl 2-(((1-methyl-7-nitro-1H-benzo[d][1,2,3]triazol-4-yl)amino)methylene)malonate
(39) are then rapidly added. After 35 minutes, the solution is allowed to cool down. A very fine

ACCEPTED MANUSCRIPT
precipitate is observed, which is filtered and washed with hexane and diethyl ether, obtaining the
pure compound. M.P.: 256-257 °C. Yield: 72%. 1H-NMR (CDCl3) δ: 13.720 (s, 1H, NH), 8.873 (s,
13
1 H, H-5), 8.460 (s, 1H, H-8), 4.569 (s, 3H, CH3), 4.262 (q, 2H, CH2), 1.306 (t, 3H, CH3). ). C-
NMR: (CDCl3) δ: 174.19 (C), 165.16 (C), 153.67 (C), 146.46 (CH), 142.71 (C), 134.47 (C), 130.21
(C), 129.45 (C), 112.29 (C), 112.06 (CH), 61.45 (CH2), 34.69 (CH3), 14.33 (CH3). GC/MS: 318
(M+H). Anal. Calcd for (C13H11N5O5): C, 49.22; H, 3.49; N, 22.07. Found: C, 49.19; H, 3.51; N,
22.10.
5.1.13.4. 3,9-dimethyl-4-nitro-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-h]quinoline-7-carboxylic
acid (41). 1.05 g (3.3·10-3) of ethyl 3-methyl-4-nitro-6-oxo-6,9-dihydro-3H-[1,2,3]triazolo[4,5-
h]quinoline-7-carboxylate (40) are added to a suspension of 0.73 g of KOH in 15 ml of DMSO at
room temperature. After 30 minutes, 0.85 ml of CH3I are added. After 20 minutes, an abundant
1
precipitate appears which is filtered and washed with chloroform. M.P.> 300 °C. Yield: 30%. H-
NMR (CDCl3) δ: 9.556 (s, 1 H, H5), 8.235 (s, 1H, H8), 4.309 (s, 3H, CH3), 3.55 (s, 3H, CH3). 13C-
NMR: (DMSO-d6) δ: 174.03 (C), 164.21 (C), 152.12 (C), 151.36 (CH), 143.66 (C), 135.26 (C),
131.79 (C), 130.22 (C), 112.90 (C), 112.37 (CH), 45.29 (CH3), 35.07 (CH3). GC/MS: 304 (M+H).
Anal. Calcd for (C12H9N5O5): C, 47.53; H, 2.99; N, 23.10. Found: C, 47.50; H, 3.02; N, 23.13.
5.2. Anti-mycobacterial activity assay
5.2.1. Resazurin Microtiter Assay (REMA)
100 µl 7H9 was dispensed in each well of a sterile 96-well plate, and serial twofold dilutions of
essential oil were prepared directly on the plate by adding 100 µl of the working solution of drug to
achieve the final concentration. For all mycobacteria strains the FBT concentration range used was
32-1 µg/mL. The inoculum was prepared from the 7H9 growth, adjusted to a McFarland tube scale
1. The suspension was diluted 1:10 and 100 µl was added to each well. The plates were covered,
sealed in plastic bags, and incubated for 7 days at 37ºC. After the final visual reading, 30 µl of
0.02% resazurin was added to each well and re-incubated overnight. A change in color from blue
(oxidized state) to pink (reduced) indicated bacterial growth and Minimal inhibitory concentration
(MIC) was defined as the lowest drug concentration that prevented the color change.
5.2.2. In vitro cytotoxicity assay
All compounds were tested for toxicity (EC50) in a mammalian VERO cell line (ATCC CCL-81,
ATCC, Manassas, VA, USA) at a concentration of 100 µM. The same concentration of
ciprofloxacin was used for comparison. After 72 hours of exposure, cell viability was assessed on
the basis of cellular conversion of MTT into a formazan product using the Promega CellTiter 96
non-radioactive cell proliferation assay (Promega, Madison, WI, USA).
5.2.3. Mycobacterium tuberculosis DNA gyrase inhibition assay
M. tuberculosis DNA gyrase supercoiling assays were performed in the absence or presence of
compounds (21a), (30a), (24a), and ciprofloxacin as control using the M. tuberculosis Gyrase
Supercoing Assay Kit according to the manufacturer’s recommendations (Inspiralis, Norwick, UK).
In brief, the assay (30 µL) was carried out in gyrase buffer (50 mM HEPES, KOH (pH = 7.9), 6
mM magnesium acetate 4 mM DTT, 1 mM ATP, 2 mM spermidine, 100 mM potassium glutamate,
and 0.05 mg/mL of albumin) containing 0.5 µg of relaxed plasmid pBR322 DNA, using 1 U of M.
tuberculosis DNA gyrase x assay buffer. Reactions were also carried in the presence of
ciprofloxacin and test compounds (2 µL); their vehicles, water and DMSO served as negative
control, respectively. Reactions were incubated for a period of of 30 min at 37 °C, and subsequently
quenched by the addition of 30 µL of STEB stop buffer (40% w/v sucrose, 100 mM Tris-HCl (pH
8), 10 mM EDTA, 0.5 mg/mL Bromophenol Blue) and 30 µL of 24:1 chloroform:isoamyl alcohol.
Reaction products were then loaded onto 1% agarose TEA gels and allow to rest for 40 min prior to