New 6-nitroquinolones: Synthesis and antimicrobial activities

Article abstract of DOI:10.1016/j.farmac.2004.01.014

Pursuing our searches on quinolonecarboxylic acids we used a simple three-step one pot procedure to synthesize novel 1,7-disubstituted-6- nitroquinolones. The new derivatives were tested against Mycobacterium tuberculosis and Mycobacterium avium complex (

Full text of DOI:10.1016/j.farmac.2004.01.014

IL FARMACO 59 (2004) 463–471
www.elsevier.com/locate/farmac
New 6-nitroquinolones: synthesis and antimicrobial activities
Gianluca Sbardella a, Antonello Mai b, Marino Artico b,*, Maria Giovanna Setzu c,
Graziella Poni c, Paolo La Colla c,*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento di Studi Farmaceutici, Università degli Studi di Roma “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy
c Dipartimento di Biologia Sperimentale, Università di Cagliari, S.P. Monserrato-Sestu, Km. 0,700 - 09042 Monserrato (CA), Italy
Received 10 October 2003; accepted 29 January 2004
Available online 02 April 2004
Abstract
Pursuing our searches on quinolonecarboxylic acids we used a simple three-step one pot procedure to synthesize novel 1,7-disubstituted-
6-nitroquinolones. The new derivatives were tested against Mycobacterium tuberculosis and Mycobacterium avium complex (MAC) as well
as against both gram-positive and gram-negative bacteria. In vitro assays showed some derivatives were endowed with good inhibiting
activities against tested mycobacteria. Some derivatives were also found more potent than ciprofloxacin and ofloxacin (used as reference
drugs) against gram-positive bacteria.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Nitroquinolones; Antimycobacterial agents; Antibacterial agents; Tuberculosis
1. Introduction
Therefore, because of the global health problems of TB,
the increasing rate of MDR-TB and the high rate of a co-
infection with HIV, the development of potent new antituber-
culous drugs without cross-resistance with known antimyco-
bacterial agents is still urgently needed [13,14].
Tuberculosis (TB) is a growing international health con-
cern, since it is the leading infectious cause of death in the
world today [1,2]. Moreover, the resurgence of TB in indus-
trialized countries and the worldwide increase in the preva-
lence of Mycobacterium avium complex (MAC) infections in
immunocompromised hosts have prompted the quest for new
antimycobacterial drugs [3-6].
Many groups have recently reported [15-29] the synthesis
and antimycobacterial activity of novel classes of com-
pounds, including isonicotinic acid hydrazides, pyrazinoic
acids, b-lactams, compounds containing a thiocarbonyl group
(thioamides, thioureas), compounds containing an alkylthio
group bound to an electrondeficient atom of carbon, oxazoli-
dinones, fluoroquinolones, nitroimidazoles, and others.
In particular, the appearance of multidrug-resistant
(MDR) strains of M. tuberculosis, which exhibit in vitro
resistance to at least two major antituberculous drug (usually
isoniazid and rifampin) and cause intractable TB, has greatly
contributed to the increased incidence of TB [7-11].
Taking account that fluoroquinolones exhibit fairly good
antimycobacterial activity [30] and having in mind the not
fully expressed antitubercular potential of nitro pharmacoph-
ore [31], in a preliminary report we recently described the
synthesis and evaluation of a few novel quinolones as poten-
tial antimycobacterial agents [18].
Although M. tuberculosis (two of its strains) was the first
bacterial species to have the entire genome sequenced, this
was of little help in identifying targets for the development of
novel antimycobacterial drugs, mainly due to the complexity
of this genoma (with at least 100 genes involved, e.g., in the
synthesis of mycobacterial cell wall) [12].
The compounds were evaluated against both gram-
positive and gram-negative bacteria and against various my-
cobacteria. The results of these studies revealed good anti-
bacterial activity of tested compounds against Streptococcus
and Staphylococcus but, above all, some of these derivatives
showed to be effective against M. tuberculosis and other
mycobacteria.
* Corresponding authors.
E-mail addresses: marino.artico@uniroma1.it (M. Artico),
placolla@unica.it (P. La Colla).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.014

464
G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
O
O
O
O
O
O
F
O2N
H2N
OH
OH
OH
R2
R2
R2
N
N
R
N
N
R
N
N
R
R1
R1
R1
1a-e
2a-r
3a,b
Fig. 1. Novel 6-Fluoro-, 6-nitro- and 6-aminoquinolones 1-3. Simmetrically substituted quinolones (1a-e, 2a-l, 3a,b): R = R1 = H, alkyl, cycloalkyl,
2,4-difluorophenyl; R2 = H. Unsimmetrically substituted quinolones (2m-r): R = R1 = tert-butyl, cyclopropyl; R2 = H or R = tert-butyl, cyclopropyl;
R1-R2 = morpholine, thiomorpholine.
In the present paper we wish to report the synthesis and the
antimicrobial activities (both against mycobacteria and
gram-positive and -negative bacteria) of a series of
6-nitroquinolone carboxylic acids together with a few
6-fluoro- and 6-amino-analogues (1a-e, 2a-r, and 3a,b;
Figure 1).
potassium carbonate to yield ethyl quinolonecarboxylates
4a-e and 5a-j, furtherly hydrolyzed (in acidic or basic condi-
tions) to the corresponding quinolonecarboxylic acids 1a-e
and 2a-l (Scheme 1). 6-Aminoquinolones 3a,b were ob-
tained by reduction of the corresponding 6-nitrosubstituted
derivatives 2g,h with stannous chloride in the presence of
concentrated hydrochloric acid.
Unsymmetrically substituted 6-nitroquinolones 2m-r
were obtained starting from ethyl 3-(2-chloro-4-fluoro-5-
nitrophenyl)-3-oxopropanoate 8 by treatment with the proper
amine (1.1 eq) in the presence of potassium carbonate, to
yield 3-(2-chloro-5-nitro-4-substituted phenyl)-3-oxopro-
pionic ethyl esters 6a-d (Scheme 2). These intermediates
were then converted into the ethyl quinolonecarboxylates
5k-p by a three-step one-pot procedure similar to that de-
scribed before. Ethyl esters were finally hydrolyzed (in
acidic or basic conditions) to the desired quinolonecarboxy-
lic acids 2m-r.
2. Chemistry
1,7-Disubstituted-6-fluoro-, 6-nitro- and 6-amino-4-oxo-
1,4-dihydro-quinoline-3-carboxylic acids 1-3 were synthe-
sized as outlined in Scheme 1 and in Scheme 2.
In particular, symmetrically substituted 6-fluoro- and
6-nitro-quinolones 1a-e and 2a-l were obtained starting from
ethyl 3-(2-chloro-4,5-difluorophenyl)-3-oxopropanoate [32]
or ethyl 3-(2-chloro-4-fluoro-5-nitrophenyl)-3-oxopro-
panoate 8 [synthesized by us following the procedure illus-
trated in reference 32], respectively, by a three-step one-pot
reaction with triethyl orthoformate in acetic anhydride, the
proper amine (2.2 eq) in methyl sulphoxide and, at last,
When the 1-tert-butyl-7-tert-butylamino-6-nitro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid ethyl ester 5e was
hydrolyzed in acidic conditions, unsubstituted 7-amino-6-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 2a was
O
O
O
O
O
O
X
F
X
O
COOH
b,c,d
Cl
2N
F
X
OC 2H5
e,f,g
OH
OC 2H5
h or i
Cl
HN
N
R
HN
N
R1
7
R1
R
X = F
X = NO
8
4a-e, 5a-j
1a-e, 2a-l
2
O
N
O
a
H2N
j
X = NO
OH
2
COOH
Cl
HN
R1
R
3a,b
F
Scheme 1. Synthesis of compounds 1a-e, 2a-l, and 3a,b. Reagents and conditions: a) HNO3, H2SO4; b) SOCl2, reflux; c) KOOCCH2COOEt, MgCl2, Et3N,
acetonitrile; d) H2O, HCl; e) HC(OEt)3, Ac2O, reflux; f) RNH2 (R = R1), 2.2 eq, DMSO, r.t.; g) K2CO3, 90 °C; h) NaOH, H2O, reflux; i) Acetic acid, H2O,
H2SO4, reflux; j) SnCl2, HCl 37%, ethanol, reflux.
O
O
O
O
O
O
O
O
O2
N
F
O2N
O2
R2
N
O2N
OC 2H5
a
OC 2H5
b,c,d
OH
OC 2H5
e or f
R2
R2
Cl
N
Cl
N
N
N
N
R1
R1
R
2m-r
R1
R
5k-p
8
6a-d
Scheme 2. Synthesis of compounds 2m-r. Reagents: a) HNR1R2, K2CO3, CH3CN, reflux; b) HC(OEt)3, Ac2O, reflux; c) RNH2, DMSO, r.t.; d) K2CO3, 90 °C;
e) NaOH, H2O, reflux; f) Acetic acid, H2O, H2SO4, reflux.

G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
465
Table 1
In vitro antimycobacterial activity of compounds 1-3
a
c
Compd
R
R1
R2
CC50
MIC50b/MIC90
M. tuberculosis
MT-4
M. avium
complex
>200
ATCC 27294 (wt)
>200
1ad
1b
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>200
>200
>200
>200
>200
>200
>200
>200
54
>200
>200
1c
>200
>200
1d
>200
>200
1ed
2a
126/>200
>200
>200
H
>200
2b
2c
2d
2e
ethyl
ethyl
>200
>200
2-F-ethyl
ethenyl
2-F-ethyl
77/>200
>200
26/100
>200
2-F-ethyl
iso-propyl
iso-amyl
iso-propyl
iso-amyl
80
2.0/22.6
>200
>200
2f
>200
140
>200
2gd
2hd
2i
tert-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
tert-butyl
tert-butyl
tert-butyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
tert-butyl
tert-butyl
1.4/6
1.2/12
>200
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
cyclopropyl
morpholine
thiomorpholine
tert-butyl
66.6
>100
>200
>200
118
>200
>200
75.5/>200
81/>200
>200
2j
2k
2l
2md
2n
>200
>200
>200
>200
>200
166
31/>200
0.2/75
1.6/100
35/>200
14.6/65
8/100
47/>200
13.5/50
9/46
2o
>200
192
2pd
2q
H
54/>200
2.3/8.9
1.1/5.8
129/>200
140/>200
1/2
morpholine
thiomorpholine
cyclopropyl
tert-butyl
104
2r
78
3ad
3bd
CIPe
OFXf
H
H
>200
>200
60
>200
168/>200
1/2.5
>200
3/5
1.5/3
a Compound dose (µM) required to reduce the viability of MT-4 cells by 50%, as determined by the MTT method
b Minimum inhibitory concentration (µM) required to reduce the number of viable Mycobacteria by 50%, as determined by the MTT method
c Minimum inhibitory concentration (µM) required to reduce the number of viable Mycobacteria by 90%, as determined by the MTT method
d Rif. 18.
e CIP: ciprofloxacin; fOFX: ofloxacin
obtained. The reaction of ethyl 3-(2-chloro-4-fluoro-5-nitro-
phenyl)-3-oxopropanoate with 2-fluoroethylamine accord-
ing to the described procedure furnished the expected 1-(2-
fluoroethyl)-7-(2-fluoroethylamino)-6-nitro-4-oxo-1,4-dihy-
dro-quinoline-3-carboxylic acid ethyl ester 5a together with
the 7-(2-fluoroethylamino)-6-nitro-4-oxo-1-vinyl-1,4-dihy-
droquinoline-3-carboxylic acid ethyl ester 5b resulting from
the dehydrohalogenation of the fluoroethyl side chain at
position 1 of quinolone ring. The hydrolysis of the two esters
gave the corresponding derivatives 2c and 2d. The compound
1-ethyl-7-ethylamino-6-nitro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid 2b was directly obtained from 3-(2-chloro-
4-fluoro-5-nitrophenyl)-3-oxopropionic acid ethyl ester
without isolating the ester intermediate since we use ethy-
lamine hydrochloride in the presence of a double amount of
potassium carbonate.
complex (MAC), and representative strains of gram-positive
(group D Streptococcus, Staphylococcus aureus) and gram-
negative (Salmonella spp., Shigella spp.) bacteria. Ciprof-
loxacin and ofloxacin were used as reference drugs.
4. Results and discussion
Results of the in vitro evaluation of antibacterial and
antimycobacterial activity of tested compounds are reported
in Tables 1,2.
When tested against mycobacteria, 1-tert-butyl-7-tert-
butylamino derivative 2g resulted the most active compound,
exhibiting a potency (MIC50 = 0.5-1.5 µM) comparable, if
not superior, to those of ciprofloxacin and ofloxacin
(MIC50 = 1-1.2 µM and MIC50 = 1.5-3 µM, respectively).
The introduction of a morpholine or a thiomorpholine group
in the C7 position of quinolone ring resulted in the very active
compounds 2n,o and 2q,r, 1-tert-butyl derivatives 2n,o being
more active against M. tuberculosis whereas 1-cyclopropyl
counterparts 2q,r exhibited fairly good activity against
3. Biology
The quinolonecarboxylic acids 1-3 were evaluated in vitro
against M. tuberculosis (ATCC 27294 strain), M. avium

466
G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
Table 2
In vitro antibacterial activity of compounds 1-3
a
Compd
R
R1
R2
CC50
MT-4
MICb/MBCc
S. aureus Salmonella spp. Shigella spp.
group D
Streptococcus
12/12
1ad
1b
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>200
>200
>200
>200
>200
>200
>200
>200
54
12/12
>200
12/12
>200
>200
>200
7.4/7.4
>200
>200
1/1
12/25
>200
>200
>200
7.4/7.4
>200
>200
1/3
>200
1c
>200
>200
1d
>200
>200
1ed
2a
22/22
7.4/22
>200
H
>200
2b
2c
2d
2e
ethyl
ethyl
>200
>200
2-F-ethyl
ethenyl
2-F-ethyl
6/6
6/6
2-F-ethyl
50/50
25/25
66/200
>200
1/3
2/3
iso-propyl
iso-amyl
iso-propyl
iso-amyl
80
66/200
>200
>200
>200
3/6
>200
>200
6/6
2f
>200
140
2gd
2hd
2i
tert-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
tert-butyl
tert-butyl
tert-butyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
tert-butyl
tert-butyl
0.05/0.05
0.8/0.8
2.4/2.4
22/22
0.05/0.05
0.8/1.5
2.4/2.4
7.4/7.4
>200
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
cyclopropyl
morpholine
thiomorpholine
tert-butyl
66.6
>100
>200
>200
118
1.5/1.5
22/22
66/66
>200
>200
25/25
22/22
66/66
25/25
0.8/0.8
0.8/2.4
>200
>125
0.01/0.01
0.4/0.4
1.5/1.5
22/22
66/200
>200
>200
25/25
22/66
66/66
25/25
0.8/0.8
2.4/22
>200
>125
0.01/0.01
0.4/0.4
2j
2k
2l
2md
2n
>200
22/200
5/10
22/>200
2.5/10
22/22
22/22
0.6/1.2
0.8/2.4
0.2/0.8
25/25
>125
>200
166
22/22
2o
>200
192
22/22
2pd
2q
H
0.6/2.5
0.8/0.8
0.2/0.8
50/50
morpholine
thiomorpholine
cyclopropyl
tert-butyl
104
2r
78
3ad
3bd
CIPe
OFXf
H
H
>200
>200
60
>125
0.4/0.4
1.5/1.5
0.4/0.4
0.8/0.8
>200
bMinimum inhibitory concentration (µM). cMinimum bactericidal concentration (µM). dRif. 18. eCIP: ciprofloxacin; fOFX: ofloxacin
a Compound dose (µM) required to reduce the viability of MT-4 cells by 50%, as determined by the MTT method.
M. avium complex. Particularly, 1-tert-butyl-7-morpholin-
4-yl derivative 2n (MIC50 = 0.2 µM) resulted 7-fold more
active than derivative 2g (MIC50 = 1.5 µM), 5-fold more
active than ciprofloxacin (MIC50 = 1 µM) and 15-fold more
active than ofloxacin (MIC50 = 3 µM) against M. tuberculo-
sis, while 1-cyclopropyl-7-thiomorpholin-4-yl derivative 2r
was slightly less potent than ciprofloxacin and as potent as
ofloxacin against M. avium complex. The other test com-
pounds resulted essentially inactive.
noticed that, when the nitro group is replaced by fluorine or
amino group, the antimicrobial activity of quinolones dimin-
ishes or vanishes, thus suggesting the presence of strong
electronwithdrawing groups at quinolone C6 position as es-
sential for biological activity.
5. Experimental
Contrary to ciprofloxacin and ofloxacin, test compounds
were more active against gram-positive than gram-negative
bacteria [33-35] except for 7-morpholin-4-yl derivatives 2n
and 2q, which showed to be active both against gram-positive
and -negative bacteria. Derivative 2g resulted up to 30-fold
more potent than reference drugs against gram-positive bac-
teria, whereas 1-cyclopropyl derivatives 2h and 2p-r exhib-
ited a lower but still comparable activity respect to ciprof-
loxacin and ofloxacin.
5.1. Chemistry
Melting points were determined on a Buchi 530 melting
point apparatus and are uncorrected. Infrared (IR) spectra
(KBr) were recorded on a Perkin-Elmer 310 instrument. 1H
nuclear magnetic resonance (NMR) spectra were recorded at
200 MHz on a Bruker AC 200 spectrometer; chemical shifts
are reported in d (ppm) units relative to the internal reference
tetramethylsilane (Me4Si). All compounds were routinely
checked by thin-layer chromatography (TLC) and 1H NMR.
TLC was performed on aluminum-backed silica gel plates
(Merck DC-Alufolien Kieselgel 60 F254) with spots visual-
ized by UV light. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods.
In conclusion, the results of our studies confirm the impor-
tance of tert-butyl group as antimycobacterial pharmaco-
phore, as previously reported [33-35]. Interestingly, even if
conferring to test derivatives lower efficacy than the tert-
butyl moiety, the 1-cyclopropyl group increased the potency
of molecules against M. avium complex. It is also to be

G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
467
Concentration of solutions after reactions and extractions
involved the use of a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over
anhydrous sodium sulfate. Analytical results are
within 0.40% of the theoretical values. All chemicals were
purchased from Aldrich Chimica, Milan (Italy), or Lancaster
Synthesis GmbH, Milan (Italy), and were of the highest
purity.
5.1.3. General procedure for the preparation of 3-(4-(cyclo)
alkylamino-2-chloro-5-nitrophenyl)-3-oxopropionic acid ethyl
esters 6a-d
Example: Synthesis of 3-(4-tert-Butylamino-2-chloro-5-
nitrophenyl)-3-oxopropionic acid ethyl ester 6b.
A
mixture of 3-(2-chloro-4-fluoro-5-nitrophenyl)-3-
oxopropionic acid ethyl ester (1.73 mmol), dry acetonitrile
(30 ml), anhydrous potassium carbonate (1.73 mmol) and
tert-butylamine (1.89 mmol) was stirred at room temperature
overnight. The reaction mixture was diluted with ethyl ac-
etate (150 ml), washed with brine and dried (Na2SO4). After
filtration and evaporation of the solvent in vacuum, the resi-
due was purified by chromatography on silica gel column
(n-hexane/ethyl acetate/methanol 12/3/1) to give the TLC
pure desired ester. ( yield: 96%); 1H NMR (CDCl3) d 1.24-
1.31 (t, 3H, CH2CH3), 1.54 (s, 9H, t-butyl), 4.00 (s, 2H,
COCH2CO), 4.10-4.27 (q, 2H, CH2CH3), 7.13 (s, 1H, H3 –
Ar), 8.70 (br. s, 1H, NH, exchanged with D2O), 8.83 (s, 1H,
H6 – Ar).
5.1.1. Synthesis of 2-chloro-4-fluoro-5-nitrobenzoic acid 7
To a stirred solution of 2-chloro-4-fluorobenzoic acid
(28.64 mmol) in sulphuric acid 96 % (40 ml), nitric acid 65%
(42.96 mmol) was added and the resulting mixture was
stirred at room temperature for 1 h. The mixture was then
diluted with water and extracted with ethyl acetate (3 x
50 ml) and the combined organic extracts were washed with
brine (3 x 40 ml), dried (Na2SO4), filtered and evaporated
under reduced pressure to yield a TLC pure solid. (yield:
96 %); 1H NMR (CDCl3) d 7.44-7.49 (d, 1H, H3 – Ar), 8.66
(br. s, 1H, OH, exchanged with D2O), 8.76-8.80 (d, 1H, H6 –
Ar).
Compounds 6a-d were obtained as TLC pure solids.
Chemical and physical data are reported in Table 4.
5.1.4. General procedure for the preparation of unsymme-
trically disubstituted 1,4-dihydro-6-nitro-4-oxoquinoline-3-
carboxylic acid ethyl esters 5k-p
5.1.2. Synthesis of 3-(2-chloro-4-fluoro-5-nitrophenyl)-3-
oxopropionic acid ethyl ester 8
A solution of 2-chloro-4-fluoro-5-nitrobenzoic acid
(28.42 mmol) in thionyl chloride (10 ml) was refluxed for 2 h.
After cooling to room temperature, the mixture was concen-
trated under reduced pressure. Toluene was added (3 x 10 ml)
and the mixture concentrated again to yield the correspond-
ing acyl chloride as a TLC pure residual oil. Potassium ethyl
malonate (59.13 mmol) was placed in a flask under a nitrogen
blanket. Acetonitrile (60 ml) was added and the mixture
stirred and cooled to 10-15 °C. To this mixture was added
triethylamine (92.65 mmol) followed by magnesium chlo-
ride (65.36 mmol) and stirring continued at 20-25 °C for
2.5 h. The resulting slurry was re-cooled to 0 ° and a solution
of the acyl chloride in dry acetonitrile (10 ml) was added
dropwise over 25 min followed by the addition of more
triethylamine (9.27 mmol). The mixture allowed to stir over-
night at room temperature and the next cooled to 0° C before
adding 13 % aq HCl (15 ml) cautiously while keeping the
temperature below 25 °C. The two phases were separated and
the organic layer was evaporated under reduced pressure to
remove acetonitrile while the aqueous layer was back-
extracted with ethyl acetate (3 x 40 ml). The combined
organic extracts were washed with NaHCO3 saturated solu-
tion (2 x 40 ml) followed by brine (2 x 40 ml) and then
filtered, dried (Na2SO4) and concentrated under reduced
pressure to give the required product as a TLC pure solid.
(yield: 97 %); 1H NMR (CDCl3) d 1.28-1.35 (t, 3H,
CH2CH3), 4.01 (s, 2H, COCH2CO), 4.22-4.32 (q, 2H,
CH2CH3), 7.38-7.43 (d, 1H, H3 – Ar), 8.31-8.41 (m, 1H,
H6 – Ar).
Example: Synthesis of 7-tert-butylamino-1-cyclopropyl-
1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid ethyl
ester 5n
To a stirred solution of 3-(4-tert-butylamino-2-chloro-5-
nitrophenyl)-3-oxopropionic acid ethyl ester 6b (1.92 mmol)
in acetic anhydride (5 ml) triethyl orthoformate (2.98 mmol)
was added and the resulting mixture was refluxed for 1 h.
After cooling to room temperature, the mixture was concen-
trated under reduced pressure. Toluene was added (3 x 10 ml)
and the mixture concentrated again. The residual oil was
dissolved in DMSO (3 ml) and a solution of cyclopropy-
lamine (2.12 mmol) in DMSO was added while maintaining
the temperature between 15-20 °C. The resulting solution
was stirred at room temperature for 1 h. Then anhydrous
K2CO3 (2.12 mmol) was added and the mixture was heated to
90 °C overnight. After cooling to room temperature, the
mixture was diluted with water and filtered to collect a crude
precipitate which was used in the following hydrolysis step
without further purification (yield: 86 %); 1H NMR (DMSO-
d6) d 1.21-1.29 (m, 4H, CH2-cyclopropyl), 1.46-1.52 (t, 3H,
CH2CH3), 1.52 (t, 9H, t-butylamino, overlapped signal), 3.62
(m, 1H, CH-cyclopropyl), 4.14-4.24 (q, 2H, CH2CH3), 7.32
(s, 1H, H8-Ar), 8.12 (br. s, 1H, NH, exchanged with D2O),
8.36 (s, 1H, H5-Ar), 8.81 (s, 1H, H2 – Ar). IR (KBr): 3340
(NH), 1710 and 1630 (CO).
Compounds 5k-p were obtained as TLC pure solids and
were used without any further purification. Chemical and
physical data are reported in Table 4.

468
G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
Table 3
Chemical and physical data of compounds 1-3
Compd
1ab
1b
R
R1
R2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Yield (%)
49
Mp (°C)
>280
Crystallization solvent
acetonitrile
acetonitrile
DMF
Formulaa
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
16H15FN2O3
18H19FN2O3
20H23FN2O3
22H27FN2O3
18H23FN2O3
10H7N3O5
55
>280
1c
58
259-260
287-289
177-178
>280
1d
60
acetonitrile
benzene
1eb
2a
52
78
DMF
2bc
2c
ethyl
ethyl
77
>280
acetonitrile
acetonitrile
acetonitrile
acetonitrile
acetonitrile
DMF
14H15N3O5
14H13F2N3O5
14H12FN3O5
16H19N3O5
20H27N3O5
18H23N3O5
16H15N3O5
18H19N3O5
20H23N3O5
22H27N3O5
22H11F4N3O5
17H19N3O5
18H21N3O6
18H21N3O5S
17H19N3O5
17H17N3O6
17H17N3O5S
16H17N3O3
18H25N3O3
2-F-ethyl
ethenyl
2-F-ethyl
82
>280
2d
2e
2f
2gb
2hb
2i
2j
2k
2-F-ethyl
83
261-263
>280
iso-propyl
iso-amyl
iso-propyl
iso-amyl
79
76
>280
tert-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
tert-butyl
tert-butyl
tert-butyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
tert-butyl
tert-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
cyclopropyl
morpholine
thiomorpholine
tert-butyl
morpholine
thiomorpholine
cyclopropyl
tert-butyl
75
>280
88
>280
DMF
77
258-260
232-234
280-281
>280
acetonitrile
acetonitrile
ethanol 95°
ethanol 95°
acetonitrile
benzene/acetonitrile
acetonitrile
acetonitrile
benzene/acetonitrile
acetonitrile
acetonitrile
acetonitrile
79
80
2l
80
2mb
2n
82
>280
68
>280
2o
75
>280
2pb
2q
H
85
>280
72
198-200
238-239
>280
2r
64
3ab
3bb
H
H
90
89
>280
a Analytical results for C, H, N were within 0.4% of the calculated values.
b Rif. 18.
c Directly obtained from the 3-(2-chloro-4-fluoro-5-nitrophenyl)-3-oxopropionic acid ethyl ester .
5.1.5. General procedure for the preparation of symmetri-
cally disubstituted 1,4-dihydro-6-fluoro- and -6-nitro-4-
oxoquinoline-3-carboxylic acid ethyl esters 4a-e and 5a-j
Compounds 5a and 5b were obtained as different products
by reacting 2-fluoroethylamine according to this procedure.
Compounds 4a-e and 5a-j were obtained as TLC pure
solids and were used without any further purification.
Chemical and physical data are reported in Table 4.
Example: Synthesis of 1-tert-butyl-7-tert-butylamino-1,4-
dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid ethyl es-
ter 5e
5.1.6. General procedure for the preparation of 1,4-dihydro-
1,7-disubstituted-6-fluoro- and -6-nitro-4-oxoquinoline-3-car-
boxylic acids 1a-d, 2a,c,d, and 2h-l
To
a
stirred solution of 3-(2-chloro-4-fluoro-5-
nitrophenyl)-3-oxopropionic acid ethyl ester (3.41 mmol) in
acetic anhydride (8 ml) triethyl orthoformate (2.98 mmol)
was added and the resulting mixture was refluxed for 1 h.
After cooling to room temperature, the mixture was concen-
trated under reduced pressure. Toluene was added (3 x 10 ml)
and the mixture concentrated again. The residual oil was
dissolved in DMSO (5 ml) and a solution of tert-butylamine
(7.50 mmol) in DMSO was added while maintaining the
temperature between 15-20 °C. The resulting solution was
stirred at room temperature for 1 h. Then anhydrous K2CO3
(7.50 mmol) was added and the mixture was heated to 90 °C
overnight. After cooling to room temperature, the mixture
was diluted with water and filtered to collect a crude precipi-
tate which was used in the following hydrolysis step without
Example: Synthesis of 1-cyclopropyl-7-cyclopro-
pylamino-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic
acid 2h
To a solution of 1-cyclopropyl-7-cyclopropylamino-1,4-
dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid ethyl es-
ter 5f (10.27 mmol) in acetic acid (50 ml) and water (40 ml),
concentrated sulphuric acid (7 ml) was added and the result-
ing mixture was refluxed for 1.5 h.After cooling, the reaction
mixture was diluted with water and extracted with ethyl
acetate (3 x 50 ml) and the combined organic extracts were
washed with brine (3 x 40 ml) and dried (Na2SO4). After
filtration the solvent was evaporated under reduced pressure
to yield a TLC pure solid residue, which was recrystallized
from DMF (yield: 88%). 1H NMR (CF3COOD) d: 0.90-0.94
(m, 2H, CH2 C7-cyclopropylamino), 1.23-1.26 (m, 2H, CH2
N1-cyclopropyl), 1.46-1.49 (m, 2H, CH2 C7-cyclo-
propylamino), 1.70-1.73 (m, 2H, CH2 N1-cyclopropyl),
1
further purification ( yield: 93 %); H NMR (DMSO-d6) d
1.22-1.29 (t, 3H, CH2CH3), 1.51 (s, 9H,C7-t-butylamino),
1.80 (s, 9H, N1-t-butyl), 4.14-4.25 (q, 2H, CH2CH3), 7.09 (s,
1H, H8-Ar), 8.11 (br. s, 1H, NH, exchanged with D2O), 8.69
(s, 1H, H5-Ar), 8.89 (s, 1H, H2 – Ar).

G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
469
Table 4
Chemical and physical data of compounds 4-8
Compd
4ab
4b
4c
4d
4eb
5a
5b
5c
5d
5eb
5fb
5g
5h
5i
5j
5kb
5l
5m
5nb
5o
R
R1
R2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Yield (%)
85
Mp (°C)
218-219
248-249
207-208
244-245
208-210
245-246
218-220
205-207
191-192
>280
Crystallization solvent
acetonitrile
acetonitrile
acetonitrile
acetonitrile
cyclohexane-benzene
acetonitrile
ethyl acetate
benzene
Formulaa
18H19FN2O3
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
2-F-ethyl
ethenyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
tert-butyl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
86
20H23FN2O3
22H27FN2O3
24H31FN2O3
20H27FN2O3
16H17F2N3O5
16H16FN3O5
18H23N3O5
20H27N3O5
20H27N3O5
18H19N3O5
20H23N3O5
22H27N3O5
24H31N3O5
24H15F4N3O5
19H23N3O5
20H25N3O6
20H25N3O5S
19H23N3O5
19H21N3O6
19H21N3O5S
14H15ClN2O5
15H19ClN2O5
15H17ClN2O6
15H17ClN2O5S
87
90
89
2-F-ethyl
50c
45c
88
2-F-ethyl
iso-propyl
iso-amyl
iso-propyl
iso-amyl
89
acetonitrile
cyclohexane-benzene
DMF
tert-butyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
tert-butyl
tert-butyl
tert-butyl
cyclopropyl
cyclopropyl
cyclopropyl
tert-butyl
93
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-F2-phenyl
cyclopropyl
morpholine
thiomorpholine
tert-butyl
92
>280
90
266-267
247-248
191-192
244-245
>280
acetonitrile
cyclohexane-benzene
acetonitrile
acetonitrile
acetonitrile
benzene
92
93
90
83
82
>280
74
>280
acetonitrile
acetonitrile
acetonitrile
benzene
H
86
>280
morpholine
thiomorpholine
cyclopropyl
tert-butyl
78
248-250 dec
241-243 dec
72-73
5p
6a
6b
6c
6d
7
8
76
H
H
95
n-hexane
96
95-96
n-hexane
morpholine
thiomorpholine
88
172-174
160-162
166-168
63-64
benzene
86
benzene
96
benzene
C7H3ClFNO4
11H9ClFNO5
97
cyclohexane-diethyl ether
C
a Analytical results for C, H, N were within 0.4% of the calculated values.
b Rif. 18.
c Derivatives 5a and 5b were obtained as different products from the same reaction.
2.85-2.93 (m, 1H, CH C7-cyclopropylamino), 3.97-4.06 (m,
1H, CH N1-cyclopropyl), 8.23 (s, 1H, C8-H), 9.34 (s, 1H,
C5-H), 9.52 (s, 1H, C2–H). IR (KBr): 3350 (NH), 2900 (OH),
1705 (COOH), 1615 (CO), 1460-1370 (NO2) cm-1.
9H, N1-t-butyl), 2.22 (s, 9H, C7-t-butylamino), 8.10 (s, 1H,
H8-Ar), 8.11 (br. s, 1H, NH, exchanged with D2O), 9.67 (s,
1H, H5-Ar), 9.71 (s, 1H, H2 – Ar). IR (KBr): 3200 (NH),
2900 (OH), 1690 (COOH), 1635 (CO), 1450-1360 (NO2)
cm-1.
Compound 2a was obtained by this procedure starting
from 1-tert-butyl-7-tert-butylamino-1,4-dihydro-6-nitro-4-
oxoquinoline-3-carboxylic acid ethyl ester 5e. Compounds
1a-d, 2a,c,d, and 2h-l were obtained as TLC pure solids and
chemical and physical data are reported in Table 3.
Compounds 1e, 2e-g and 2m-r were obtained as TLC pure
solids and chemical and physical data are reported in Table 3.
5.1.8. Synthesis of 1,4-dihydro-1-ethyl-7-ethylamino-6-nitro-
4-oxoquinoline-3-carboxylic acid 2b
5.1.7. General procedure for the preparation of 1,4-dihydro-
1,7-disubstituted-6-fluoro- and -6-nitro-4-oxoquinoline-3-
carboxylic acids 1e, 2e-g, and 2m-r
To a stirred solution of 3-(2-chloro-4-fluoro-5-nitro-
phenyl)-3-oxopropionic acid ethyl ester (3.41 mmol) in ace-
tic anhydride (8 ml) triethyl orthoformate (2.98 mmol) was
added and the resulting mixture was refluxed for 1 h. After
cooling to room temperature, the mixture was concentrated
under reduced pressure. Toluene was added (3 x 10 ml) and
the mixture concentrated again. The residual oil was dis-
solved in DMSO (5 ml) and ethylamine hydrochloride
(7.50 mmol) and anhydrous K2CO3 (7.50 mmol) was added
while maintaining the temperature between 15-20 °C. The
resulting solution was stirred at room temperature for 1 h.
More anhydrous K2CO3 (7.50 mmol) was added and the
mixture was heated to 90 °C overnight.After cooling to room
temperature, the mixture was diluted with water, neutralized
Example: Synthesis of 1-tert-butyl-7-tert-butylamino-1,4-
dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid 2g
To a solution of NaOH (11.29 mmol) in water (30 ml)
1-tert-butyl-7-tert-butylamino-1,4-dihydro-6-nitro-4-oxoqui-
noline-3-carboxylic acid ethyl ester 5e (2.26 mmol) was
added and the resulting mixture was refluxed for 3 h. After
cooling, the reaction mixture was diluted with water and
acidified with HCl 2N. After stirring for about 10 min, the
resulting slurry was filtered to collect a precipitate which was
recrystallized from DMF to yield the title compound as TLC
1
pure solid. (yield: 75 %) H NMR (CF3COOD) d: 1.78 (s,

470
G. Sbardella et al. / IL FARMACO 59 (2004) 463–471
and filtered to collect a TLC pure solid, which was recrystal-
lized from acetonitrile (yield: 77%).
determined by subcultivating in Triptosio agar samples from
cultures with no apparent growth.
5.1.9. General procedure for the preparation of 6-amino-
1,4-dihydro-1,7-disubstituted-4-oxoquinoline-3-carboxylic
acids 3a,b
5.2.4. Antimybacterial Assays
M. tuberculosis 27294 and M. avium complex (MAC)
were ATCC strains. MICs were assessed in microtiter plates
by adding 20 ml aliquots of a culture suspension [whose
turbidity was equal to that of a no. 1 McFarland standard
containing 108 colony forming units (CFU)/ml] to 80 ml of
Middlebrook 7H9 medium containing 0.5% glycerol and
10% albumin-dextrose-catalase (ADC) and various concen-
trations of test compounds. Plates were then incubated for
9 days at 37 °C. At the end of incubation, the number of
viable mycobacteria was determined by the MTT method, as
already reported [36].
Example: Synthesis of 6-amino-1-tert-butyl-7-tert-butyl-
amino-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid
3b
To a stirred solution of 1-tert-butyl-7-tert-butylamino-
1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid 2g
(1.88 mmol) in ethanol 95° (4 ml) a solution of SnCl2·2H2O
(6.58 mmol) in 37% hydrochloric acid (2 ml) was cautiously
added and the resulting mixture was refluxed for 30 min.
After cooling, the reaction mixture was diluted with water,
made neutral (pH ≈ 7) with KOH 2N and extracted with ethyl
acetate (3 x 50 ml). The combined organic extracts were
washed with brine (3 x 40 ml) and dried over anhydrous
Na2SO4. After filtration and evaporation of the solvent under
reduced pressure a residue was obtained which was triturated
with ethanol/diethyl ether to give the desired compound as a
TLC pure solid (yield: 89 %). 1H NMR (DMSO-d6) d: 1.48
(s, 9H, N1-t-butyl), 1.92 (s, 9H, C7-t-butylamino), 5.68-5.78
(m, 2H, NH2, exchanged with D2O), 7.17 (s, 1H, NH, ex-
changed with D2O), 7.28 (s, 1H, H8-Ar), 7.76 (s, 1H, H5-Ar),
8.92 (s, 1H, H2 –Ar). IR (KBr): 3300 (NH), 2900 (OH), 1690
(COOH), 1600 (CO) cm-1.
Microbial and cell growth at each drug concentration were
expressed as percentage of untreated controls and concentra-
tions resulting in 50% (CC50, MIC50) or 90% (MIC90)
growth inhibition was determined by linear regression analy-
sis.
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