Synthesis and anti-mycobacterial activities of triazoloquinolones

Article abstract of DOI:10.1016/j.ejmech.2010.11.020

A number of quinolone derivatives have been reported to possess anti-mycobacterial activity. Generally. Mycobacterium tuberculosis isolates expressing resistance to both isoniazid and rifampin are susceptible to fluoroquinolones. Benzotriazole is a hetero

Full text of DOI:10.1016/j.ejmech.2010.11.020

European Journal of Medicinal Chemistry 46 (2011) 320e326
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-mycobacterial activities of triazoloquinolones
,
a
a
a
a
b
Antonio Carta a , Michele Palomba , Irene Briguglio , Paola Corona , Sandra Piras , Daniela Jabes ,
*
Paola Guglierame b, Paola Molicotti c, Stefania Zanetti c
a Department of Medicinal and Toxicological Chemistry, University of Sassari, Via Muroni 23/a, 07100 Sassari, Italy
b NeED Pharmaceuticals, Via G. Fantoli 6/15, 20138 Milano, Italy
c Department of Biological and Medicinal Sciences, University of Sassari, Viale San Pietro, 07100 Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2010
Received in revised form
24 September 2010
Accepted 11 November 2010
Available online 19 November 2010
A number of quinolone derivatives have been reported to possess anti-mycobacterial activity. Generally.
Mycobacterium tuberculosis isolates expressing resistance to both isoniazid and rifampin are susceptible
to fluoroquinolones. Benzotriazole is a hetero-bicyclic aromatic ring endowed with interesting chemical
and biological properties and pharmacological activities. In a preliminary study we have recently
reported the activity of triazolo[4,5-h]quinolone-carboxylic acids, a new class of benzotriazole deriva-
tives active against multi-drug resistant M. tuberculosis (MDR-Mtb). In this study we confirm that this
novel class of quinolones is endowed with a selective anti-mycobacterial activity, coupled with absence
of cytotoxicity.
Keywords:
Triazoloquinolones
Benzotriazolo
M. tuberculosis
Anti-mycobacterial activity
The SAR analysis of the new derivatives in comparison with the previous series shows that the methyl
group is the most effective substituent in both N-3 and N-9 positions of the ring system.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
7,8-triazoloquinolin-5-arsonic acidby Slaterin1932 [25]and recently
obtained by Ferlin et al. through an alternative route [26].
Benzo[d][1,2,3]triazole, here named benzotriazole, has been
extensively investigated by Katritzky et al. as a synthetic auxiliary for
it behaves as good leaving group after reaction with a variety of
carbonyl group [1e3], whereas Sparatore and Pagani have studied the
biological properties [4,5] and described the interesting pharmaco-
logical activities of several benzotriazole derivatives bearing substit-
uents at positions 1 or 2 [6e14]. Furthermore, our group has
developed a separate chemistry and further investigated the biolog-
ical potential of benzotriazoles [15e19]. In particular, we described
several 3-aryl-2-benzotriazol-(2)-yl-acrylonitriles which were found
to be endowed with antitubercular activity [20e23], partly retained
by the corresponding amides and carboxylic acids [21], and 2-ben-
zotriazol-(1)-yl-3-phenylacrylonitriles, which possess an interesting
anticancer activity (Fig. 1) [22,23].
Furthermore, by connection of either f or h sides of the quinoline
ring and both the adjacent carbon atoms of the triazole the angular
N-tricyclic systems triazolo[4,5-f]quinoline (2) and triazolo[4,5-h]
quinoline (3), were also obtained (Fig. 2).
Triazoloquinoline (2) was first reported by German Authors in
1934 [24], but not mentioned in Chemical Abstracts, while the isomer
derivative 3 was reported as a side product during the preparation of
Several derivatives of 2 and 3 endowed with biological properties
have been reported in the literature over the last three decades.
Among them, carboxylic acid derivatives of triazolo[4,5-f]quinolines
(2a) and triazolo[4,5-h]quinolines (3a), depicted in Fig. 3, showed
antimicrobial activity [27,28], whereas some 9-aminoalkylamino-
triazolo[4,5-f]quinolines (2b) exhibited anticancer activity [29].
The linear derivative triazolo[4,5-g]quinoline and its 5-Cl-
derivative were obtained by our group in 2000 [30]. As summarized
in Fig. 4, further substituted triazolo[4,5-g]quinolines (4) were later
synthesized [31e33]. In particular, 9-dihydrotriazolo[4,5-g]quino-
line-1-oxide (4a) stood out as a new small molecule endowed with
a selective, promising activity in cell-based assays against HIV-1wt
and clinically relevant NNRTI resistant mutants [34].
Concerning angular triazolo[4,5-h]quinoline derivatives (3),
Fig. 5 summarized the structural formula of 3,9-disubstituted-6-
oxo-6,9-dihydrotriazolo[4,5-h]quinolone-carboxylic acids and their
esters (3b) which were recently reported by our group as a new class
of quinolones possessing a potent anti-mycobacterial activity. On
the contrary, triazolo[4,5-f]quinolines (2a) were completely inac-
tive. The triazolo[4,5-h]quinilines (3b) turned out to be particularly
interesting for their activity against MDR-Mtb, several derivatives
exhibiting MIC values in the range of 0.5e3.2 mg/mL [35,36].
Recently the derivative 3,9-dimethyl-6-oxo-6,9-dihydrotriazolo
[4,5-h]quinoline-7-carboxylic acid (3c) has been selected for further
* Corresponding author. Tel./fax: þ39 079 228720.
E-mail address: acarta@uniss.it (A. Carta).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.020

A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
321
Fig. 4. Triazolo[4,5-g]quinoline derivatives.
Fig. 1. Benzotriazolyl-phenylacrylonitriles endowed with antitubercular (1a) or anti-
proliferative (1b) activities.
selected 3c) were evaluated in cell-based assays for cytotoxicity in
human hematological tumors cell cultures (MT-4).
2. Chemistry
The synthetic route which led to 9-alkyl-3-benzyl-6-oxo-6,9-
dihydrotriazole[4,5-h]quinoline-7-carboxylic ethyl esters and to
the acids (10cej and 11cej respectively) is depicted in Schemes
1e3.
Fig. 2. Triazolo[4,5-f]quinoline (2) and triazolo[4,5-h]quinoline (3).
Alkylation of 4-nitrobenzotriazole in acetonitrile with benzyl
bromide using NaOH as base was previously described by Diehl and
Kendall [37]. In the cited patent, the Authors claimed that the
reaction of 4-nitrobenzotriazole with an unspecified benzyl halide
in the same conditions yielded only two of three possible isomers,
namely 1-benzyl-4-nitrobenzotriazole (6a) and 2-benzyl-4-nitro-
benzotriazole (6b), but not the 1-benzyl-7-nitrobenzotriazole (6c)
reported in Scheme 1. The two isomers were isolated by means of
their different solubility in acidic media. No data relating to struc-
ture elucidation and characterization of the two compounds were
reported with the exception of melting points.
Following the same procedure a routine thin layer chromatog-
raphy (Hexane/Ethyl acetate 7/3) conversely showed three different
spots, instead of the two expected, at Rf values of 0.68, 0.65, 0.63.
Consequently we were able to isolate via column chromatography
three isomeric benzyl benzotriazoles which were fully identified and
characterized by NMR spectroscopy using a previously reported
methodology applied for the corresponding alkyl derivatives [14,17].
By 13C NMR spectroscopy, we were able to assign structure 6b to
the isomer with Rf ¼ 0.65 which showed a characteristic signal at
61 ppm for the benzyl carbon while the other two isolated
compounds showed for the same carbon signals at higher fields (55
and 53 ppm). In fact when a side alkyl chain is bound to 2-nitrogen
of the benzotriazole ring, the atom electron withdrawing effect of
the two adjacent nitrogen atoms causes a carbon chemical shift at
lower fields compared to the corresponding 1- or 3- alkyl deriva-
tives. Furthermore, the differences in the methylene carbon
chemical shifts could also arise from the steric interactions
between the benzyl methylene and proximate aromatic proton in
6a and methylene proton and the nitro group in 6c which could
shift the chemical shift of methylene carbons in these compounds
relative to that in 6b.
experiments. When determined by the twofold broth or agar dilu-
tion method, the MIC against mycobacteria, including Mycobacte-
rium tuberculosis, was 0.5e2 mg/mL. To test the intracellular activity,
human macrophages J774-A1 were infected with M. tuberculosis
H37Rv and successively grown in the absence or presence of 3c at 2.0
and 1.0 mg/mL. After 7 days the macrophages were lysed and the
growth of mycobacteria determined by plate counting. In treated
cultures, the number of viable mycobacteria was 5 ꢀ 103 and
8 ꢀ 103 CFU/mL, at 1.0 and 2.0
mg/mL, respectively, while in the
untreated controls the number of mycobacteria was 1 ꢀ106 CFU/mL.
In the aim to extend the SAR analysis for this class of compounds,
we have now designed, synthesized and submitted to biological
evaluation two novel series of triazolo[4,5-f]quinolone carboxylic
acids (11cef and 11gej), and their ethyl ester parents as more
lipophilic pro-drugs (10cef and 10gej respectively). In the first
series we have kept the best subtituents of the previous series in N9
(methyl, ethyl, allyl and tert-butyl) [35] and replaced the methyl
with a benzyl in N3 (11cef) in order to evaluate the biological effect
of increased steric bulk of this substituent in this position.
In the second series we have kept the methyl group in N3 and
introduced on N9 a further series of substituents (2-methylallyl,
cyclopropylmethyl, cyclobutylmethyl and 2,4-difluorobenzyl)
(11gej) in order to evaluate the biological effect of both increased
steric bulk and lipophilia of these substituents in this position.
All the new triazolo[4,5-f]quinolone carboxylic acids (11cej) were
tested against M. tuberculosis H37Rv, M. tuberculosis H37Ra, Myco-
bacterium smegmatis and Mycobacterium bovis. Moreover, to investi-
gate the spectrum of antibacterial activity, all compounds were tested
against a panel of gram-positive bacteria including enterococci
(Enterococcus faecalis), staphylococci (Staphylococcus aureus, Staphy-
lococcus epidermidis), gram-negative bacteria including Acinetobacter
baumanii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumo-
oniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and
against Candida sp. The most promising compounds (11c,11g and the
The assignment of the correct structure to the compounds with
Rf ¼ 0.68 and 0.63 was achieved by NOE experiments. Compound
having Rf ¼ 0.63 was identified as 1-benzyl-4-nitrobenzotriazole (6a)
Fig. 3. Triazolo[4,5-f]quinolines (2) and triazolo[4,5-h]quinolines (3) endowed with antimicrobial and anticancer activities.

322
A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
Fig. 5. Triazolo[4,5-f]quinolone derivatives endowed with potent anti-mycobacterial activity.
Scheme 1. Synthesis of N-benzyl-4(7)-nitrobenzotriazoles (6aec).
in that it showed a positive NOE effect on H-7 when the singlet at
5.96 ppmwas irradiated. No positive NOE effect was instead observed
for compound with Rf ¼ 0.68 when the benzyl methylene at 6.27 ppm
was irradiated, according to the structure of compounds 6c.
As reported in Scheme 2, hydrogenation of 6a by hydrazine and
palladium/charcoal under refluxing ethanol afforded the amino-
derivative (7) in good yield, which according to GouldeJacobs
reaction with diethyl ethoxymethylenemalonate in Dowtherm gave
the aminomethylenemalonate derivative (8) in 56% yield. Thermal
ring closure of 8 at 250 ꢁC in Dowtherm gave the corresponding
quinolone (9a) in 55% yield.
The reactions reported in Scheme 3 describe the synthesis of the
desired 3,9-disubstituted-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
line-7-carboxylic acids (11cej).
Ethyl 3-benzyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-
carboxylate (9a) and ethyl 3-methyl-6-oxo-6,9-dihydro-triazolo
[4,5-h]quinoline-7-carboxylate (9b) underwent alkylation at N-9
position with the appropriate alkyl bromide in the presence of KOH
or NaH to give the corresponding 9-substituted-triazolo[4,5-h]
quinolone esters (10cej) in 8e76% yields accompanied by their
corresponding acids (11cej) in 7e75%. 3-Methyl-intermediate (9b)
was in turn prepared according to the pathway previous reported
[35,36].
All the acids (11cej) were also obtained in 90e98% yields by
alkaline hydrolysis of the esters (10cej).
3. Results and discussion
The activity (MIC: mg/mL) of all the new derivatives (esters 10cej
and acids 11cej) and 3c against M. tuberculosis H37Rv, M. tuberculosis
H37Ra and other mycobacteria (M. smegmatis and M. bovis), is
reported in Table 1
Comparison of the activity of our compounds with reference
drugs such as ethambutol, rifampin, streptomycin and cipro-
floxacin, showed that only compounds (11c and 11g) were active
with MICs in the range 4e32 mg/mL, whereas all other compounds
(acids and esters) displayed limited or no activity. Only compound
3c had activity comparable to reference drugs.
When MICs were determined against a panel of gram-positive
and gram-negative bacteria and against Candida sp. all compounds
(10cej, 11cej and 3c) were inactive (MIC > 64 mg/mL).
Scheme 2. Synthesis of the key intermediate 3-benzyl-6-oxo-6,9-dihydro-]triazolo[4,5-h]quinoline-7-carboxylate (9).

A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
323
Scheme 3. Synthesis of 3,9-disubstituted-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-carboxylic acids (11cej).
The spectrum of antibacterial activity demonstrated by this new
class of quinolones resulted very narrow, suggesting a specific anti-
mycobacterial potential. This characteristic is quite relevant in the
context of a new anti-mycobacterial drug for two main reasons.
First, the limited spectrum shall not disturb the existing normal
flora and, therefore, will likely be better tolerated as compared with
wide spectrum existing treatments such as rifamycins and quino-
lones. Second, the presence of a reduced number of susceptible
organisms will limit the selection of resistant mutants among
species that are not specifically targeted by the treatment, therefore
reducing the risk of vertical transmission of resistance across
species.
These interesting results, supported also by the good cytotox-
icity profile, suggest to further characterize compound 3c, currently
selected as lead compound, and to continue the chemical and
biological exploration of this new class of potential anti-mycobac-
terial agents.
Table 1
In vitro evaluation (MIC:
mycobacteria.
mg/mL) of compounds (10cej and 11cej) against
Comp
M. tuberculosis
M. tuberculosis
H37Ra
M. smegmatis
M. bovis
BCG
H37Rv
mc2155
10c
10d
10e
10f
10g
10h
10i
10j
11c
11d
11e
11f
11g
11h
11i
11j
64
64
64
64
32
64
64
64
8
64
64
64
16
64
32
32
0.5
5
>128
>128
>128
32
>128
>128
>128
>128
16
64
>128
64
64
8
4
>128
>128
>128
4
>128
>128
>128
32
>128
64
64
4
8
4
64
8
32
16
16
0.5
4
64
16
8
>128
>128
32
64
64
64
5
0.5
64
16
32
16
16
4
3c
5. Experimental
Ethambutol
Rifampin
Streptomycin
Ciprofloxacin
2
<0.5
1
0.5
ꢃ0.06
8
e
e
5.1. Chemistry
1
e
0.5
0.125
0.25
Melting points were measured with a Digital Electrothermal
IA9100 melting points apparatus and are uncorrected. 1H NMR
spectra were recorded on a Varian XL-200 (200 MHz) spectrometer.
Solutions were typically prepared in either deuterochloroform
(CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) with chemical
shifts referenced to tetramethylsilane (TMS) as an internal stan-
dard. Chemical shifts are reported in ppm. MS were recorded using
combined HP 5790-HP 5970 GC/MS apparatus. Column chroma-
tography was performed using 70e230 mesh (Merck silica gel 60).
The Rf values and the progress of the reactions and the product
purity were checked by TLC using Merck F-254 commercial plates.
Analyses indicated by the symbols of the elements were within
ꢂ0.4% of the theoretical values.
As far as the cytotoxicity is concerned, the tested compounds
(11c, 11g and 3c) showed CC50 > 100 g/mL against MT-4 cells.
m
Overall, the several favourable characteristics of the most
promising compound 3c, which include activity against mycobac-
teria (particularly M. tuberculosis) comparable to reference
compounds, good in vitro therapeutic index obtained comparing
the cytotoxicity [35,36] and the anti-mycobacterial activity, and the
very narrow spectrum of antibacterial activity, directed only against
mycobacteria, are supportive of further evaluation.
4. Conclusion
5.1.1. Starting materials
In summary, our experiments confirm that 3,9-disubstituted-6-
oxo-6,9-dihydrotriazolo[4,5-h]quinolone-carboxylic acids are in
general endowed with anti-mycobacterial activity, whereas their
ethyl ester parents are not active in vitro. The SAR analysis of the
new derivatives in comparison with the previous series shows that
the methyl group is the most effective substituent in N-3 position of
the ring system and, very interestingly, and conversely to known
quinolones, the methyl group is the best substituent also in the N-9
position.
Allchemicals wereobtained fromcommerciallyavailable sources
(Aldrich) and used without purification. 4-Nitrobenzotriazole (5)
and ethyl 3-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-7-
carboxylate were prepared according to literature [24,35,36].
5.1.2. Preparation of 1(2)-benzyl-4-nitrobenzotriazole (6a,b) and
1-benzyl-7-nitrobenzotriazole (6c)
To a solution of 4-nitrobenzotriazole (5) (89.5 mmol) in aceto-
nitrile (440 mL) and NaOH (pellets) (1.5 eq,) benzyl bromide

324
A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
(89.5 mmol) was slowly added. The resulting mixture was then
warmed to 85 ꢁC and stirred overnight. The crude precipitate was
then collected by filtration and purified by flash chromatography
(hexane/ethyl acetate 7/3) to separate the three isomers 6aec.
Yields, melting points, analytical and spectroscopical data are
reported below.
5.1.5. Preparation of ethyl 3-benzyl-6-oxo-6,9-dihydro-triazolo[4,5-
h]quinoline-7-carboxylate (9a)
A suspension of aminomethylenemalonate derivative 8 (2.74 g,
6.95 mmol) in 30 mL of hot Dowtherm (250 ꢁC) was vigorously
stirred for 15 min. The reaction mixture was then cooled at room
temperature and poured into 250 mL of hexane and stirred for 2 h.
The crude solid obtained collected by filtration and purified by
chromatography (CHCl3/MeOH 9.5:0.5) gave the title compound as
a solid.
5.1.2.1. 1-Benzyl-4-nitrobenzotriazole (6a). This compound was
obtained in 30% yield, TLC (Hexane/Ethyl acetate 7/3): Rf 0.63, mp
129e130 ꢁC (ethanol). 1H NMR (CDCl3):
d
8.24 (d, 1H, J ¼ 8.8, H-5),
Yield 55%, mp 255e257 ꢁC (ethanol). 1H NMR (CDCl3 þ DMSO):
7.73 (d, 1H, J ¼ 8.2 Hz, H-7), 7.55 (dd, 1H, J ¼ 8.8 and 8.4 Hz, H-6),
d
13.27 (s,1H, NH), 8.57 (s,1H, H-8), 8.33 (d,1H, J ¼ 8.8 Hz, H-5), 7.54
7.40e7.34 (m, 5 phenyl-H), 5.96 (s, 2H, CH2); 13C NMR (CDCl3):
(d, 1H, J ¼ 8.8 Hz, H-4), 7.34 (m, 5 phenyl-H), 5.98 (s, 2H, CH2ePh),
4.32 (q, 2H, J ¼ 7.4, OeCH2eCH3), 1.37 (t, 3H, J ¼ 7.4, OeCH2eCH3).
MS m/z 348 (Mþ). Anal. Calcd for C19H16N4O3: C, 65.51; H, 4.63; N,
16.08. Found C, 65.22; H, 4.70; N, 16.37.
d
141.47 (Cq), 138.77 (Cq), 134.97 (Cq), 133.61 (Cq), 129.17 (2CH),
128.89 (CH), 127.52 (2CH), 126.66 (CH), 121.20 (CH), 116.94 (CH),
52.92 (CH2). MS m/z 254 (Mþ). Anal. Calcd for C13H10N4O2: C, 61.41;
H, 3.96; N, 22.04. Found C, 61.73; H, 4.19; N, 21.87.
5.1.6. Preparation of 3,9-disubstituted-6-oxo-6,9-dihydro-triazolo
[4,5-h]quinoline-7-carboxylic esters and acids (10cej and 11cej)
To a suspension of 10 mmol of finely powered KOH (method v)
or of NaH (50% oil suspension) (method vi) in DMSO (6 mL),
3.0 mmol of ethyl 3-benzyl(or methyl)-quinoline-7-carboxylate
derivative 9a (or 9b) was added and left stirring at rt for 15 min.
Then the appropriate alkyl bromide (3.5 mmol) was added and the
stirring was continued at rt for 1 h (method v) or at 90 ꢁC for 8 h
(method vi). On cooling the reaction mixture was added of 40 mL of
water. The precipitate when formed was filtered while the mother
liquors were extracted with chloroform (3 ꢀ 50 mL) and the
combined extracts were dried on anhydrous sodium sulphate and
evaporated in vacuo. The crude solids obtained, corresponding to
the 9-alkylated-esters (10cej), were crystallized by ethanol. The
mother liquors acidified by 2N HCl afforded a further precipitate
corresponding to the acids: 11c (34%); 11d (75%); 11e (16%); 11f
(17%); 11g (7%); 11h (17%); 11i (14%); 11j (12%). All the acids (11cej)
were also obtained in 90e98% yields by hydrolysis of the esters
(10cej) in the presence of 2N NaOH heating at 100 ꢁC for 2 h
(method vii). Method of preparation, yields, melting points,
analytical and spectroscopical data are reported below.
5.1.2.2. 1-Benzyl-7-nitrobenzotriazole (6c). This compound was
obtained in 13% yield, TLC (Hexane/Ethyl acetate 7/3): Rf 0.68, mp
82e84 ꢁC (ethanol). 1H NMR (CDCl3):
d
8.44 (d, 1H, J ¼ 8.2, H-6),
8.23 (d, 1H, J ¼ 7.8 Hz, H-4), 7.50 (dd, 1H, J ¼ 8.2 and 7.8 Hz, H-5),
7.26e7.09 (m, 5 phenyl-H), 6.27 (s, 2H, CH2); 13C NMR (CDCl3):
d
149.50 (Cq), 135.34 (Cq), 135.10 (Cq), 133.65 (Cq), 128.84 (2CH),
128.21 (CH), 127.30 (CH), 127.15 (2CH), 125.27 (CH), 123.25 (CH),
55.26 (CH2). MS m/z 254 (Mþ). Anal. Calcd for C13H10N4O2: C, 61.41;
H, 3.96; N, 22.04. Found C, 61.73; H, 4.19; N, 21.87.
5.1.2.3. 2-Benzyl-4-nitrobenzotriazole (6b). This compound was
obtained in 30% yield, TLC (Hexane/Ethyl acetate 7/3): Rf 0.65, mp
101e102 ꢁC (ethanol). 1H NMR (CDCl3):
d
8.40 (d,1H, J ¼ 8.0 Hz, H-5),
8.26 (d,1H, J ¼ 8.2 Hz, H-7), 7.57e7.36 (m, 6H, 5 phenyl-H þ H-6), 6.01
(s, 2H, CH2); 13C NMR (CDCl3):
146.71 (Cq), 137.97 (Cq), 136.65 (Cq),
d
133.66 (Cq), 128.93 (3CH), 128.58 (2CH), 126.23 (CH), 124.91 (CH),
124.52(CH),61.01(CH2).MSm/z254(Mþ). Anal. CalcdforC13H10N4O2:
C, 61.41; H, 3.96; N, 22.04. Found C, 61.19; H, 4.03; N, 21.91.
5.1.3. Preparation of 1-benzyl-4-aminobenzotriazole (7)
A suspension of compound 6a (1.0 g, 3.9 mmol), 10% palla-
dium/charcoal (0.1 g) and hydrazine monohydrate (0.5 mL) in
ethanol (15 mL) was refluxed under stirring for 1 h. The reaction
mixture was then cooled at room temperature, the catalyst
filtered off, and the solvent and the excess of hydrazine removed
in vacuo. The crude product was used without any further
purification.
5.1.6.1. Ethyl 3-benzyl-9-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quin-
oline-7-carboxylate (10c). Method (v): yield 53%, mp 228e230 ꢁC. 1H
NMR (CDCl3 þ DMSO):
d
8.57 (s, 1H, H-8), 8.52 (d, 1H, J ¼ 9.0 Hz, H-5),
7.47 (d, 1H, J ¼ 9.0 Hz, H-4), 7.34 (m, 5 phenyl-H), 5.94 (s, 2H, CH2Ph),
4.65 (s, 3H, NeCH3), 4.38 (q, 2H, J ¼ 7.0 Hz, OeCH2CH3),
d 1.41 (t, 3H,
J ¼ 7.0 Hz, OCH2CH3). MS m/z 362 (Mþ). Anal. Calcd for C20H18N4O3: C,
Yield 88%, mp 92e94 ꢁC (diethyl ether).1H NMR (CDCl3 þ DMSO):
66.29; H, 5.01; N, 15.46. Found C, 65.92; H, 5.23; N, 15.29.
d
7.30 (m, 5 phenyl-H), 7.12 (dd,1H, J ¼ 8.0 and 7.8Hz, H-6), 6.73 (d,1H,
J ¼ 8.0 Hz, H-5), 6.40 (d,1H, J ¼ 7.8 Hz, H-7), 5.97 (s, 2H, NH2), 5.81 (s,
2H, CH2);MSm/z 224 (Mþ). Anal. Calcd forC13H12N4: C, 69.62;H, 5.39;
N, 24.98. Found C, 69.29; H, 5.60; N, 24.79.
5.1.6.2. 3-Benzyl-9-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
line-7-carboxylic acid (11c). Method (v): yield 34%, method (vii):
yield 94%, mp 265e267 ꢁC. 1H NMR (CDCl3 þ DMSO):
d 15.45 (s, 1H,
COOH), 9.12 (s, 1H, H-8), 8.43 (d, 1H, J ¼ 8.8 Hz, H-5), 8.05 (d, 1H,
J ¼ 8.8, H-4), 7.36 (m, 5 phenyl-H), 6.12 (s, 2H, CH2ePh), 4.72 (s, 3H,
NeCH3). MS m/z 334 (Mþ). Anal. Calcd for C18H14N4O3: C, 64.66; H,
4.22; N, 16.76. Found C, 64.40; H, 4.59; N, 16.51.
5.1.4. Preparation of diethyl 2-((1-benzyl-benzotriazol-4-ylamino)
methylene)malonate (8)
To a stirred solution of 7 (0.86 g, 3.8 mmol) in 10 mL of Dowtherm
warmed at 60 ꢁC, diethyl ethoxymethylenemalonate (1.27 g,
5.9 mmol) was added dropwise. After then, the mixture was heated
upto 150ꢁC and stirred for 7 h. Aftercooling at room temperature, the
mixture was poured over 90 mL of hexane and stirred for 1 h vigor-
ously. The solid obtained was used without any further purification.
Yield 56%, mp 133e135 ꢁC (diethyl ether). 1H NMR (CDCl3):
5.1.6.3. Ethyl 3-benzyl-9-ethyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
line-7-carboxylate (10d). Method (v): yield 8%, mp 161e163 ꢁC. 1H
NMR (DMSO):
d
8.61 (s, 1H, H-8), 8.51 (d, 1H, J ¼ 9.0 Hz, H-5), 7.64 (d,
1H, J ¼ 9.0 Hz, H-4), 7.40e7.20 (m, 5 phenyl-H), 5.98 (s, 2H, CH2Ph),
d
5.32 (q, 2H, J ¼ 7.2, NeCH2eCH3), 4.42 (q, 2H, J ¼ 7.0 Hz,
d
11.64 (d, 1H, J ¼ 13.8 Hz, NH), 9.51 (d, 1H, J ¼ 13.8 Hz, NHeCH]C),
OeCH2eCH3), 1.71e1.41 (m, 6H, 2CH3). MS m/z 376 (Mþ). Anal. Calcd
for C21H20N4O3: C, 67.01; H, 5.36; N, 14.88. Found C, 67.36; H, 5.22; N,
15.10.
7.37e7.20 (m, 6H, H-6 þ 5 phenyl-H), 7.07e6.97 (m, 2H, H-5 þ H-7),
5.85 (s, 2H, CH2ePh), 4.43e4.25 (m, 4H, 2OeCH2eCH3), 1.46e1.34
(m, 6H, 2OeCH2eCH3). MS m/z 394 (Mþ). Anal. Calcd for
C21H22N4O4: C, 63.95; H, 5.62; N, 14.20. Found C, 64.30; H, 5.41; N,
14.56.
5.1.6.4. 3-Benzyl-9-ethyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
line-7-carboxylic acid (11d). Method (v): yield 75%, method (vii):

A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
325
yield 98%, mp 210e212 ꢁC. 1H NMR (CDCl3 þ DMSO):
d
15.29 (s, 1H,
OeCH2eCH3), 1.93 (s, 3H, NeCH2C(CH3)]CH2), 1.37 (t, 3H, J ¼ 7.1 Hz,
OeCH2eCH3). MS m/z 326 (Mþ). Anal. Calcd for C17H18N4O3: C, 62.57;
H, 5.56; N, 17.17. Found C, 62.29; H, 5.71; N, 17.01.
COOH), 8.92 (s, 1H, H-8), 8.54 (d, 1H, J ¼ 8.8 Hz, H-5), 7.64 (d, 1H,
J ¼ 8.8, H-4), 7.36 (m, 5 phenyl-H), 5.98 (s, 2H, CH2ePh), 4.36 (q, 2H,
J ¼ 7.2 Hz, NeCH2eCH3), 1.705 (t, 3H, J ¼ 7.2 Hz, NeCH2eCH3). MS
m/z 348 (Mþ). Anal. Calcd for C19H16N4O3: C, 65.51; H, 4.63; N,
16.08. Found C, 65.85; H, 4.44; N, 16.31.
5.1.6.12. 3-Methyl-9-(2-methylallyl)-6-oxo-6,9-dihydro-triazolo[4,5-
h]quinoline-7-carboxylic acid (11h). Method (v): yield 17%, method
(vii): yield 91%, mp 167 ꢁC. 1H NMR (CDCl3):
d 15.10 (s, 1H, COOH),
5.1.6.5. Ethyl 9-allyl-3-benzyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
8.78 (s, 1H, H-8), 8.62 (d, 1H, J ¼ 8.7 Hz, H-5), 7.65 (d, 1H, J ¼ 8.7 Hz,
H-4), 5.80 (s, 2H, NeCH2C(CH3)]CH2), 4.90 (s, 2H, NeCH2C(CH3)]
CH2), 4.42 (s, 3H, NeCH3), 1.93 (s, 3H, NeCH2C(CH3)]CH2). MS m/z
298 (Mþ). Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78.
Found C, 60.11; H, 4.95; N, 18.49.
line-7-carboxylate (10e). Method (vi): yield 25%, mp 183e185 ꢁC. 1H
NMR (CDCl3):
d
9.02 (s, 1H, H-8), 7.78 (d, 1H, J ¼ 9.0 Hz, H-5), 7.47 (d,
1H, J ¼ 9.0 Hz, H-4), 7.40e7.20 (m, 5 phenyl-H), 5.98 (s, 2H, CH2Ph),
5.80 (m, 1H, CH2eCH]CH2), 5.20 (d, 2H, J ¼ 9.0 Hz, CH2eCH]CH2),
5.00 (d, 2H, J ¼ 9.0 Hz, CH2eCH]CH2), 4.20 (q, 2H, J ¼ 7.0 Hz,
OeCH2eCH3), 1.29 (t, 3H, J ¼ 7.0 Hz, OCH2CH3). MS m/z 388 (Mþ).
Anal. Calcd for C22H20N4O3: C, 68.03; H, 5.19; N, 14.42. Found C, 68.31;
H, 5.01; N, 14.22.
5.1.6.13. Ethyl 9-(cyclopropylmethyl)-3-methyl-6-oxo-6,9-dihydro-
triazolo[4,5-h]quinoline-7-carboxylate (10i). Method (v), yield 19%,
mp 183e85 ꢁC. 1H NMR (CDCl3):
d 9.08 (s, 1H, H-8), 8.53 (d, 1H,
J ¼ 9.2 Hz, H-5), 7.89 (d, 1H, J ¼ 9.2 Hz, H-4), 5.20 (d, 2H, J ¼ 7.8 Hz,
NeCH2eCHe(CH2)2), 4.47 (s, 3H, NeCH3), 4.34 (q, 2H, J ¼ 7.2 Hz,
OeCH2eCH3), 1.57 (m, 1H, NeCH2eCHe(CH2)2), 1.31 (t, 3H,
J ¼ 7.2 Hz, OeCH2eCH3), 0.65e0.62 (m, 4H, NeCH2eCHe(CH2)2).
MS m/z 326 (Mþ). Anal. Calcd for C17H18N4O3: C, 62.57; H, 5.56; N,
17.17. Found C, 62.88; H, 5.41; N, 17.40.
5.1.6.6. 9-Allyl-3-benzyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-
7-carboxylic acid (11e). Method (vi): yield 16%, method (vii): yield
90%, mp 216e218 ꢁC. 1H NMR (DMSO):
d
15.48 (s,1H, COOH), 9.12 (s,
1H, H-8), 8.45 (d, 1H, J ¼ 9.2 Hz, H-5), 8.13 (d, 1H, J ¼ 8.6 Hz, H-4),
7.38 (m, phenyl-H), 6.40e6.05 (m, 3H, NeCH2eCH]
5
CH2 þ NeCH2eCH]CH2), 5.96 (s, 2H, CH2Ph), 5.23e5.08 (m, 2H,
CH2eCH]CH2). MS m/z 360 (Mþ). Anal. Calcd for C20H16N4O3: C,
66.66; H, 4.48; N, 15.55. Found C, 66.31; H, 4.61; N, 15.83.
5.1.6.14. 9-(Cyclopropylmethyl)-3-methyl-6-oxo-6,9-dihydro-triazolo
[4,5-h]quinoline-7-carboxylic acid (11i). Method (v), yield 14%, method
(vii): yield 97%, mp 293e95ꢁC.1HNMR(DMSOþ CDCl3):
d15.41(s,1H,
5.1.6.7. Ethyl 3-benzyl-9-butyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quino-
COOH), 9.08 (s, 1H, H-8), 8.53 (d, 1H, J ¼ 9.2 Hz, H-5), 7.89 (d, 1H,
J¼ 9.2Hz, H-4), 5.20(d,2H, J¼ 7.8 Hz, NeCH2eCHe(CH2)2), 4.47 (s, 3H,
NeCH3), 2.62 (m, 1H, NeCH2eCHe(CH2)2), 0.65e0.62 (m, 4H,
NeCH2eCHe(CH2)2). MS m/z 298 (Mþ). Anal. Calcd for C15H14N4O3: C,
60.40; H, 4.73; N, 18.78. Found C, 60.11; H, 4.95; N, 18.49.
line-7-carboxylate (10f). Method (vi): yield 76%, mp 142e144 ꢁC. 1H
NMR (CDCl3):
d
8.58 (d,1H, J ¼ 8.8 Hz, H-5), 8.52 (s,1H, H-8), 7.45e7.30
(m, 6H, H-4 þ 5 phenyl-H), 5.89 (s, 2H, CH2Ph), 5.08 (d, 2H, J ¼ 7.2 Hz,
NeCH2eCH2eCH2eCH3), 4.41 (q, 2H, J ¼ 7.0 Hz, OeCH2eCH3),1.98 (m,
2H, NeCH2eCH2eCH2eCH3), 1.68e1.24 (m, 8H, NeCH2eCH2eCH2e
CH3 þ OCH2CH3). MS m/z 404 (Mþ). Anal. Calcd for C23H24N4O3: C,
68.30; H, 5.98; N, 13.85. Found C, 67.94; H, 6.17; N, 14.09.
5.1.6.15. Ethyl 9-(cyclobutylmethyl)-3-methyl-6-oxo-6,9-dihydro-3H-
[1e3]triazolo[4,5-h]quinoline-7-carboxylate (10j). Method (v), yield
31%, mp 179e81 ꢁC. 1H NMR (CDCl3):
d 9.15 (s, 1H, H-8), 8.47 (d, 1H,
5.1.6.8. 3-Benzyl-9-butyl-6-oxo-6,9-dihydro-triazolo[4,5-h]quinoline-
J ¼ 8.4 Hz, H-5), 8.08 (d, 1H, J ¼ 8.4 Hz, H-4), 5.36 (d, 2H, NeCH2eCHe
(CH2)3), 4.46 (s, 3H, NeCH3), 4.19 (q, 2H, J ¼ 7.0 Hz, OeCH2eCH3), 2.89
(m, 1H, NeCH2eCHe(CH2)3), 2.06e1.89 (m, 6H, NeCH2eCHe(CH2)3),
1.37 (t, 3H, J ¼ 7.0 Hz, OeCH2eCH3). MS m/z 340 (Mþ). Anal. Calcd for
C18H20N4O3: C, 63.52; H, 5.92; N, 16.46. Found C, 63.85; H, 6.12; N,
16.11.
7-carboxylic acid (11f). Method (vi): yield 17%, method (vii): yield 96%,
mp 235e237 ꢁC.1HNMR(CDCl3):
d15.13(s,1H, COOH), 9.14(s,1H,H-8),
8.44 (d,1H, J ¼ 8.8 Hz, H-5), 7.37 (m, 6H, H-4 þ 5 phenyl-H), 6.14 (s, 2H,
CH2Ph), 5.25 (d, 2H, J ¼ 7.2 Hz, NeCH2eCH2eCH2eCH3), 1.90 (m, 2H,
NeCH2eCH2eCH2eCH3), 1.55e1.26 (m, 5H, NeCH2eCH2eCH2eCH3).
MS m/z376 (Mþ). Anal. Calcd for C21H20N4O3: C, 67.01; H, 5.36; N,14.88.
Found C, 67.34; H, 5.30; N, 115.15.
5.1.6.16. 9-(Cyclobutylmethyl)-3-methyl-6-oxo-6,9-dihydro-triazolo
[4,5-h]quinoline-7-carboxylic acid (11j). Method (v), yield 12%,
method (vii): yield 95%, mp 222e228 ꢁC (dec). 1H NMR (DMSO):
5.1.6.9. Ethyl 9-tert-butyl-3-methyl-6-oxo-6,9-dihydro-triazolo[4,5-
h]quinoline-7-carboxylate (10g). Method (vi): yield 9%, mp
d
15.50 (s, 1H, COOH), 9.15 (s, 1H, H-8), 8.47 (d, 1H, J ¼ 8.4 Hz, H-5),
191e94 ꢁC.1H NMR (CDCl3):
d
8.68 (s,1H, H-8), 8.27 (d,1H, J ¼ 8.8 Hz,
8.08 (d, 1H, J ¼ 8.4 Hz, H-4), 5.36 (d, 2H, NeCH2eCHe(CH2)3), 4.46
(s, 3H, NeCH3), 2.89 (m,1H, NeCH2eCHe(CH2)3), 2.06e1.89 (m, 6H,
NeCH2eCHe(CH2)3). MS m/z 312 (Mþ). Anal. Calcd for C16H16N4O3:
C, 61.53; H, 5.16; N, 17.94. Found C, 61.08; H, 5.01; N, 17.56.
H-5), 7.91 (d, 1H, J ¼ 9.0 Hz, H-4), 4.29 (s, 3H, NeCH3), 4.25 (q, 2H,
J ¼ 7.0 Hz, OeCH2eCH3), 3.36 (s, 9H, Ce(CH3)3), 1.33 (t, 3H, J ¼ 7.0 Hz,
OeCH2eCH3). MS m/z 328 (Mþ). Anal. Calcd for C17H20N4O3: C, 62.18;
H, 6.14; N, 17.06. Found C, 62.51; H, 6.03; N, 17.40.
5.2. Microbiology
5.1.6.10. 9-Tert-butyl-3-methyl-6-oxo-6,9-dihydro-triazolo[4,5-h]
quinoline-7-carboxylic acid (11g). Method (vi): yield 7%, method
5.2.1. Anti-mycobacterial assays
(vii): yield 94%, mp > 300 ꢁC. 1H NMR (CDCl3):
d
14.43 (s, 1H,
Anti-mycobacterial activity was determined by the twofold agar
dilution method against M. tuberculosis H37Rv (ATCC 27294),
H37Ra, M. smegmatis mc2155 and M. bovis BCG. Strains were grown
in Middlebrook 7H11 medium supplemented with OADC. The
bacterial suspensions (at the standard turbidity of 1 MACFARLAND)
were diluted 1:10, 1:100 and 1:10,000 with Middlebrook 7H9 and
inoculated in duplicate Middlebrook 7H11 plates containing serial
concentrations of the test compounds. Antimycobacterial activity
against M. tuberculosis H37Ra, M. smegmatis mc2155 and M. bovis
BCG were performed by the broth microdilution methodology
according to NCCLS [38]. Stock cultures were prepared from isolated
COOH),
d
8.68 (s, 1H, H-8), 8.29 (d, 1H, J ¼ 8.8 Hz, H-5), 7.97 (d, 1H,
J ¼ 9.0 Hz, H-4), 4.45 (s, 3H, NeCH3), 3.29 (s, 9H, Ce(CH3)3). MS m/z
300 (Mþ). Anal. Calcd for C15H16N4O3: C, 59.99; H, 5.37; N, 18.66.
Found C, 60.36; H, 5.14; N, 18.98.
5.1.6.11. Ethyl 3-methyl-9-(2-methylallyl)-6-oxo-6,9-dihydro-triazolo
[4,5-h]quinoline-7-carboxylate (10h). Method (v): yield 22%, mp
148 ꢁC.1H NMR (CDCl3):
d
8.78(s,1H, H-8), 8.62(d,1H, J ¼ 8.7 Hz, H-5),
7.65 (d, 1H, J ¼ 8.7 Hz, H-4), 5.80 (s, 2H, NeCH2C(CH3)]CH2), 4.90 (s,
2H, NeCH2C(CH3)]CH2), 4.42 (s, 3H, NeCH3), 4.29 (q, 2H, J ¼ 7.1 Hz,

326
A. Carta et al. / European Journal of Medicinal Chemistry 46 (2011) 320e326
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1133e1140.
colonies selected on Middlebrook 7H11 agar plates and diluted in
Middlebrook 7H9. Final bacterial inocula were approximately
1e5 ꢀ 105 CFU/mL. Assays were performed in sterile 96-well
microtiter plates with round bottom wells, sealed in plastic bag.
Plates were incubated at 37 ꢁC and read after 72 h or 7 days for M.
smegmatis mc2155 and M. bovis BCG, respectively.
5.2.2. Anticandida assays
MICs against C. albicans and C. tropicalis were determined
according to NCCLS [39]. Fungal suspensions were obtained from
cultures incubated at 35 for 48 h in Sabouraud Dextran agar.
Colonies were collected and diluted to a density of 106 CFU/mL. Test
compounds were dissolved in dimethyl sulfoxide at an initial
concentration of 10 mg/mL and serially diluted in RPMI-1640. A
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1001e1019.
50 mL sample of the above serial dilutions of test compounds were
added, in, sterile 96-well microtiter plates with round bottom
wells, to equal volume of fungal suspensions and incubated at 35 ꢁC
for 48e72 h. MIC determination was performed in duplicate.
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5.2.3. Antibacterial assays
All Gram-positive and Gram-negative bacteria were clinical or
laboratorystrains,preparedfromisolatedcoloniesselectedonMueller
Hinton Agar (MHA). MIC assays were performed by broth micro-
dilution methodology in sterile 96-well microtiter plates, according to
CLSI procedure [40], using final inocula of 1e5 ꢀ 105 CFU/mL in
Mueller Hinton Broth. Test compounds were dissolved in dimethyl
sulfoxideataninitial concentrationof10mg/mLandseriallydiluted in
Mueller Hinton Broth. A 50 mL sample of each drug concentration was
distributed in the wells containing the same volume of the bacterial
inocula and the plates were incubated at 37 ꢁC for 18e24 h. MIC was
determined as the minimum concentration inhibiting visible growth.
MIC determination was performed in duplicate.
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(2003) 833e842.
5.2.4. Cytotoxicity assays
Exponentially growing cells derived from human hematological
tumors [CD4þ humanT-cells containing an integrated HTLV-1 genome
(MT-4)] were seeded at an initial density of 1 105 cells/mL in 96-well
plates in RPMI-1640 medium supplemented with 10% fetal calf serum
(FCS), 100 U/mL penicillin G, and 100 g/mL streptomycin. Cell cultures
were then incubated at 37 ꢁCinahumidified, 5% CO2 atmosphere in the
absence or presence of serial dilutions of test compounds. Cell viability
was determined after 96 h at 37 ꢁC by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) method [41].
[32] A. Carta, G. Paglietti, Arkivoc 5 (2004) 66e75.
[33] A. Carta, G. Paglietti, Triazolo[4,5-g]quinolines, imidazo[4,5-g]quinolines,
pyrido[2,3-g]quinoxalines and [4,7]phenantrolines. Easy pathway for the
syntheses of these N-Tricycles. in: T.S. Kaufman, E.L. Larghi (Eds.), Modern
Approaches to the Synthesis of O- and N-Heterocycles, vol. 1. Research
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(1978) 109512g.
Acknowledgement
[38] National Committee for Clinical Laboratory Standards: Susceptibility Testing
of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved
Standard 2006 e NCCLS document M24eA. NCCLS, Wayne, PA, 2006.
[39] National Committee for Clinical Laboratory Standards: Reference Methods for
Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard e
Second Edition e 2002 e NCCLS document M27eA2. NCCLS, Wayne, PA, 2006.
[40] National Committee for Clinical Laboratory Standards: Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically;
Approved Standard e Seventh Edition e 2006 e NCCLS document M7eA7.
NCCLS, Wayne, PA, 2006.
The Fondazione Banco di Sardegna is gratefully acknowledged
for financial support.
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