[1,2,3]Triazolo[4,5-h]quinolones. A new class of potent antitubercular agents against multidrug resistant Mycobacterium tuberculosis strains

Article abstract of DOI:10.1016/j.bmcl.2007.06.064

In this preliminary study we report the activity of 3-methyl-9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazolo[4,5-h]quinolone-carboxylic acids and their esters as a new class of antiinfective agents against MDR Mycobacterium tuberculosis. In antitubercular screening against H37Rv and 11 clinically isolated strains of M. tuberculosis several derivatives (1o,3a,c,i,j,p) showed MIC₉₀ in the range 0.5-3.2 μg/mL. 3c showed no cytotoxicity and proved to be the most potent derivative exhibiting MIC₉₀ = 0.5 μg/mL against all M. tuberculosis strains and infected human macrophages (J774-A1) tested.

Full text of DOI:10.1016/j.bmcl.2007.06.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4791–4794
[1,2,3]Triazolo[4,5-h]quinolones. A new class of
potent antitubercular agents against multidrug resistant
Mycobacterium tuberculosis strains
Antonio Carta,a,* Michele Palomba,a Giuseppe Paglietti,a Paola Molicotti,b
Bianca Paglietti,b Sara Cannasb and Stefania Zanettib
a
`
Dipartimento Farmaco Chimico Tossicologico, Universita degli Studi di Sassari, Via Muroni 23/a, 07100 Sassari, Italy
b
`
Dipartimento di Scienze Biomediche, Universita degli Studi di Sassari, Viale S. Pietro, 07100 Sassari, Italy
Received 25 May 2007; revised 15 June 2007; accepted 19 June 2007
Available online 26 June 2007
Abstract—In this preliminary study we report the activity of 3-methyl-9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazolo
[4,5-h]quinolone-carboxylic acids and their esters as a new class of antiinfective agents against MDR Mycobacterium tuberculosis.
In antitubercular screening against H37Rv and 11 clinically isolated strains of M. tuberculosis several derivatives (1o,3a,c,i,j,p)
showed MIC90 in the range 0.5–3.2 lg/mL. 3c showed no cytotoxicity and proved to be the most potent derivative exhibiting
MIC90 = 0.5 lg/mL against all M. tuberculosis strains and infected human macrophages (J774-A1) tested.
Ó 2007 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (M. tuberculosis) is the etio-
logical agent for the Tuberculosis (TB). The incidence of
TB has steadily risen in the last years and TB is the
world’s second most common cause of death from infec-
tious diseases, after acquired immunodeficiency syn-
drome (AIDS).1 Furthermore, association of TB and
AIDS is wide-spread.2,3 This resurgence affects both
developed and developing countries with high rates of
human immunodeficiency virus (HIV) infection. The
World Health Organization (WHO) has estimated that
8 million of people develop active TB every year and
almost one-forth of these die.4
and successively, the addition of Isoniazid, Ethambutol,
Rifampin and Pyrazinamide, generally used in combina-
tion, made possible a high decrease in the mortality and
in the duration of treatment. Today the reemergence of
TB infection is further complicated by an increase of
M. tuberculosis strains resistant to drugs used in conven-
tional antitubercular therapy (XDR-TB) which are
becoming a threat to public health worldwide. The
emergence of XDR-TB shows that the development of
novel mechanism-based antitubercular agents is
necessary.7
It is known that multidrug resistant (MDR) bacilli to
both Isoniazid and Rifampin are sensitive to some fluo-
roquinolones (Ciprofloxacin, Ofloxacin, Levofloxacin,
etc.) which inhibit the topoisomerases II and IV as well
as the DNA gyrases, essential enzymes to maintain the
supercoils in bacterial DNA.8 Consequently a huge
effort has been made by scientists in order to discover
new quinolone derivatives endowed with antitubercular
activity.9–12
Prior to the advent of effective chemotherapy, 50% of
patients with active pulmonary TB died within 2 years.5
Since 1940s the introduction of the combination of
Streptomycin and para-aminosalicylic acid in therapy6
Abbreviations: TB, tuberculosis; AIDS, acquired immunodeficiency
syndrome; WHO, World Health Organization; MDR, multidrug
resistant; MIC90, inhibition constant; EMME, diethyl ethoxymethyl-
enemalonate; Dowtherm A, mixture of biphenyl and diphenyl ether
(26.5:73.5 ratio); SAR, structure activity relationships; XDR-TB,
extensive drug resistance tuberculosis.
In the past some of us have reported that new classes of
heterocycles are also endowed with antitubercular activ-
ity. In fact a certain number of quinoxaline 1,4-dioxides
and benzotriazole derivatives were described as in vitro
antitubercular agents at very low concentrations.13–16
Now, owing to our continuous interest in this field we
Keywords: Triazolo[4,5-h]quinolones; MDR M. tuberculosis; In vitro
antitubercular activity.
*
Corresponding author. Tel.: +39 079 228722; fax: +39 079 228720;
e-mail: acarta@uniss.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.064

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A. Carta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4791–4794
a) R=CH2CH3; R1=CH3;
have taken into account a previously described class of
angular triazolo[4,5-h] and [4,5-f]quinolone-carboxylic
acids and their esters (1a–d,f–h,j,k,l,n–s and 2d–h,j,k–o
of Fig. 1) as antiinfectives of the urinary tract17–19 as
possible antitubercular agents on the basis that they
are very closely related to fluoroquinolones but bear a
triazole ring which can influence either the lipophilicity
or the activity as whole. Furthermore, synthetic strate-
gies and antibacterial activity of known triazoloquino-
lone derivatives have been reviewed by Milata.20 In
this context we have submitted those compounds to
antitubercular test against M. tuberculosis H37Rv and
thus we have observed that none of the triazolo[4,5-
f]quinolone carboxylic derivatives were active, while in-
stead three triazolo[4,5-h]quinolone carboxylic acids
(1c,n,o,) exhibited interesting low MIC90 values (4.8,
5.0, and 1.6 lg/mL, respectively). In addition we have
also observed that the activity was related to the length
and position of the substituent at triazolo-nitrogen. In
fact an ethyl group in 1s, homologous of the most active
1o, lowered its activity (MIC90 value from 1.6 to >32 lg/
mL). Regarding the shift of the methyl group from
nitrogen 3–2 or 1 position it was observed that the activ-
ity disappears. This observation prompted us to prepare
the present new series of 3-methyl-9-substituted-6-oxo-
6,9-dihydro-3H-[1,2,3]-triazolo[4,5-h]quinolone-carbox-
ylic acids and their esters of structure 3a–p depicted in
Figure 2, where we considered a variety of substituents
on the quinolone nitrogen in order to improve activity
as such.
b) R=CH2CH3; R1=CH2CH2CH3;
c)R=CH2CH3; R1=CH2CH2CH2CH3;
d) R=CH2CH3; R1=CH2CH=CH2;
e) R=CH2CH3; R1=benzyl;
1
O
f
g
h
) R=CH2CH3; R =4-Me-benzyl;
1
COOR
) R=CH2CH3; R =4-Br-benzyl;
1
) R=CH2CH3; R =CH2CH2-phenyl;
i) R=H; R1=CH3;
N
R1
H3C
N
N
j) R=H; R1=CH2CH2CH3;
k) R=H; R1=CH2CH2CH2CH3;
l) R=H; R1=CH2CH=CH2;
N
3a-p
m) R=H; R1=benzyl;
1
n
o
) R=H; R =4-Me-benzyl;
1
) R=H; R =4-Br-benzyl;
p) R=H; R1=CH2CH2-phenyl;
Figure 2. 3-Methyl-9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazol-
o[4,5-h]quinoline-7-carboxylic acids and ethyl esters (3a–p).
processes. Alkylation with dimethyl sulfate of 5a yielded
the mixture of the three methyl isomers (6a–c) from
which after chromatography resolution 6a was obtained
in 20% yield. The isomers (5a,b) and (6a–c) were unam-
biguously identified according to procedures previously
reported.21 Hydrogenation of 6a under moderate pres-
sure (Parr apparatus) afforded the aminoderivative (7)
in quantitative yield, which according to Gould–Jacobs
reaction
with
diethyl
ethoxymethylenemalonate
(EMME) in Dowtherm A gave the aminomethylenemal-
onate (8) in 96% yield. Thermal ring closure of 8 at
250 °C in Dowtherm A gave the corresponding quino-
lone 9 in 75% yield. The latter underwent alkylation at
N-9 position with the appropriate bromide in the
presence of sodium hydride to give the corresponding
9-substituted-triazolo[4,5-h]quinolone esters (3a–h) in
10–45% yields accompanied by their corresponding acids
(3i–p) in 5–65%. All the acids (3i–p) were also obtained
in 90–98% yields on alkaline hydrolysis of the esters
(3a–h).
The general synthesis of 3a–p is outlined in Scheme 1.
The preparation of the known key intermediate (9)19
was further modified to improve the yield. The commer-
cially available benzotriazole (4) underwent nitration
with a mixture of nitric acid (96%) and sulfuric acid
(95%). A mixture of regioisomers (5a,b) in 3:1 ratio
was obtained and recrystallization from acetone afforded
5a in high yield (73%), avoiding tedious chromatography
All the new derivatives (3a–p) were tested against
H37Rv and further 11 clinically isolated strains of
M. tuberculosis (SS1-11) endowed with different drug-
resistance (Table 1). Comparison of the activity of our
compounds with four standard antitubercular drugs
such as Streptomycin, Isoniazid, Rifampin, and Ethambutol
(Table 2) performed according to international
protocols22,23 showed that three strains (H37Rv, SS 5,
and SS10) were sensitive to all drugs, whereas SS2
(Rifampin), SS3 (Ethambutol), SS6 (Rifampin), and
SS7 (Streptomycin) were resistant to a single drug (in
bracket). In addition other strains SS8 and SS11 were
resistant to two drugs (Streptomycin–Isoniazid and Eth-
ambutol–Isoniazid, respectively) and eventually SS9 to
three (Streptomycin, Isoniazid, and Ethambutol). Com-
pounds 3a,c,i,j,p and 1o showed MIC90 values in the
range 0.5–3.2 lg/mL, while compounds 3b,d,e–h,k–o
were inactive exhibiting MIC90 > 32 lg/mL. Derivative
(3c) was the most potent exhibiting MIC90 = 0.5 lg/
mL against all strains tested.
a) R1=R2=R3=H;
b) R1=N2-CH3, R2=R3=H;
O
1
2
3
c
d
) R =N3-CH3, R =R =H;
COOR1
1
3
2
) R =R =H, R =CH2CH3;
e) R1=N1-CH3, R2=CH2CH3, R3=H;
f) R1=N2-CH3, R2=CH2CH3, R3=H;
g) R1=N3-CH3, R2=CH2CH3, R3=H;
h) R1=H, R2=R3=CH2CH3;
3
N
R3
N
N
2
N
R1
1
1a-d,f-h, j,k,l,n-s
i) R1=N1-CH3, R2=R3=CH2CH3;
1
2
3
j
k
l
) R =N2-CH3, R =R =CH2CH3;
1
2
3
) R =N3-CH3, R =R =CH2CH3;
R1
2
N
1
2
3
1
) R =R =H, R =CH2CH3;
O
N
m) R1=N1-CH3, R2=H, R3=CH2CH3;
n) R1=N2-CH3, R2=H, R3=CH2CH3;
o) R1=N3-CH3, R2=H, R3=CH2CH3;
p) R1=N2-CH2CH3, R2=R3=CH2CH3;
q) R1=N3-CH2CH3, R2=R3=CH2CH3;
COOR2
N
3
N
R3
1
2
3
r
s
) R =N2-CH2CH3, R =H, R =CH2CH3;
2d-h,j,k-o
1
2
3
) R =N3-CH2CH3, R =H, R =CH2CH3;
Figure 1. 6-Oxo-6,9-dihydro-3H-[1,2,3]-triazolo[4,5-h]quinoline-7-car-
boxylic acids and ethyl esters (1a–d,f–h,j,k,l,n–s) and 6-oxo-6,9-dihydro-
3H-[1,2,3]-triazolo[4,5-f]quinoline-7- carboxylic acids and ethyl esters
(2d–h,j,k–o) tested.
The latter derivative (3c) was selected as lead com-
pound for further experiments. Human macrophages

A. Carta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4791–4794
4793
a
NO2
+
H
N
N
NO2
H
N
N
H
N
N
N
N
N
4
5a
5b
b
+
+
NO2
NO2
CH3
H
N
N
N
N
NO2
NO2
N
N
H3C
H3C
N
N
N
N
N
N
5a
6c
6b
6a
E
E
c
d
E = CO2C2H5
NO2
NH2
H3C
N
N
H3C
N
N
N
H
H3C
N
N
N
N
N
6a
7
8
e
O
O
O
CO2C2H5
CO2C2H5
CO2H
f
N
H
H3C
N
N
N
R
+
N
R
H3C N
H3C
N
N
3i-p
Br-R
N
N
N
3a-h
N
9
g
Scheme 1. Synthesis of 3-methyl-9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazolo[4,5-h]quinoline-7-carboxylic acids and ethyl esters (3a–p).
Reagents and conditions: (a) HNO3 96%, H2SO4 concd, 60 °C, 1 h; (b) (CH3)2SO4, NaOH 2N, rt, 30 min; (c) H2 45 psi, Pd/C 10%, rt, 3 h; (d)
EMME, Dowtherm, 150 °C, 7 h; (e) Dowtherm, 250 °C, 10 min; (f) NaH (50%), DMF, rt, 90 °C, 0.5–20 h; NaOH 2 N, 100 °C, 2 h.
Table 1. Sensitive concentration (lg/mL) of the most active triazoloquinolones (1o,3a,c,i,j,p) and reference drug (Rifampin) against SS1-11 and
H37Rv strains
M. Tuberculosis strains
1o
3a
3c
3i
3j
3p
Rifampin
SS1
SS2
1.6
0.8
3.2
3.2
1.6
1.6
3.2
1.6
1.6
1.6
1.6
1.6
0.5
0.5
0.5
2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
1.6
1.6
1.6
3.2
3.2
1.6
3.2
1.6
1.6
1.6
1.6
1.6
0.8
0.8
3.2
3.2
3.2
3.2
3.2
0.8
0.8
3.2
0.8
0.8
>4.0
>4.0
0.5
SS3
SS4
0.7
0.5
SS5
SS6
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
>4.0
0.8
SS7
SS8
0.5
0.5
SS9
SS10
SS11
H37Rv
0.8
0.5
0.7
J774-A1 were infected with H37Rv strain and succes-
sively grown in the absence of antitubercular agent or
in the presence of 3c at both the concentrations of
0.5 and 0.25 lg/mL. After 7 days the macrophages
were lysed and the growth of mycobacterial culture
was of 5000 and 8000 CFU/mL, respectively, while
the untreated culture grew regularly. Cytotoxicity of
3c was tested against both human macrophages and
Hep-2 cells and the corresponding CC50 values were
in both cases higher than 50 lg/mL.
Preliminary structure activity relationship (SAR) studies
suggest that in general the presence of an alkyl substitu-
ent at N-9 position (1o and 3a,c,i,j) was more favorable
than propenyl or benzyl groups, while a phenylethyl
group was tolerated (3p).

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A. Carta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4791–4794
Table 2. Breakpoint concentrations of Streptomycin, Isoniazid, Rifampin, and Ethambutol against SS1-11 and H37Rv strains
M. Tuberculosis strains
Streptomycin (1 lg/mL)
Isoniazid (0.1 lg/mL)
Rifampin (1 lg/mL)
Ethambutol (5.0 lg/mL)
SS1
SS2
R
S
S
S
R
R
S
S
S
R
S
S
S
S
S
S
S
S
SS3
SS4
S
S
R
R
S
R
S
R
S
SS5
SS6
S
S
S
SS7
SS8
R
R
R
S
S
S
R
R
S
S
SS9
R
S
SS10
SS11
H37Rv
S
R
S
R
S
S
R, resistant; S, sensitive.
10. Shandil, R. K.; Jayaram, R.; Kaur, P.; Gaonkar, S.;
Suresh, B. L.; Mahesh, B. N.; Jayashree, R.; Nandi, V.;
Bharath, S.; Balasubramanian, V. Antimicrob. Agents
Chemother. 2007, 51, 576.
11. Lalloo, U. G.; Naidoo, R.; Ambaram, A. Curr. Opin.
Pulm. Med. 2006, 12, 179.
12. Sriram, D.; Yogeeswari, P.; Basha, J. S.; Radha, D. R.;
Nagaraja, V. Biorg. Med. Chem. 2005, 13, 5774.
13. Sanna, P.; Carta, A.; Rahbar-Nikookar, M. E. Eur.
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In summary, our experiments demonstrated that 3-methyl-
9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazolo[4,5-
h]quinolone-7-carboxylic derivatives are endowed with
an excellent activity against MDR M. tuberculosis
strains associated with no cytotoxicity. Further in vivo
studies and SAR investigations will be reported in due
course.
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