Synthesis of 3- or 4-phenyl-1,8-naphthyridine derivatives and evaluation of antimycobacterial and antimicrobial activity

Article abstract of DOI:10.1016/S0014-827X(03)00144-7

A series of 3- or 4-phenyl-1,8-naphthyridine derivatives variously substituted in the positions 2, 6 and 7 were synthesized and evaluated for in vitro evaluation for their antimycobacterial activity as part of a TAACF TB screening program under the direction of the US National Institute of Health, NIAID division. Several compounds showed an interesting activity when tested at a concentration of 6.25 μg/ml against Mycobacterium tuberculosis H ₃₇Rv and in particular compounds 2a, 4a,d, 8a,d and 8i, exhibit a % inhibition from 91 to 99. Among these, compounds 2a, 8a and 8d appeared to have a good activity with minimum inhibitory concentrations (MICs) of 6.25 μg/ml. On the basis of the biological results, the most effective substituent in position 2 or 7 seems to be the piperidinyl group. The introduction of a morpholinyl group either in position 2 or 7 of the heterocycle ring caused a decrease in activity. The 1,8-naphthyridine derivatives were also tested in vitro for their antimicrobial activity against Staphylococcus aureus as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria.

Full text of DOI:10.1016/S0014-827X(03)00144-7

Il Farmaco 58 (2003) 859Á866
/
www.elsevier.com/locate/farmac
Synthesis of 3- or 4-phenyl-1,8-naphthyridine derivatives and
evaluation of antimycobacterial and antimicrobial activity
Muwaffag Badawneh a, Laura Bellini b, Tiziana Cavallini b, Jalal Al jamal a,
Clementina Manera b,*, Giuseppe Saccomanni b, Pier Luigi Ferrarini b
a Philadelphia University, P.O. Box 1101, Sweileh, Jordan
b Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, I-56126 Pisa, Italy
Received 23 December 2002; accepted 22 March 2003
Abstract
A series of 3- or 4-phenyl-1,8-naphthyridine derivatives variously substituted in the positions 2, 6 and 7 were synthesized and
evaluated for in vitro evaluation for their antimycobacterial activity as part of a TAACF TB screening program under the direction
of the US National Institute of Health, NIAID division. Several compounds showed an interesting activity when tested at a
concentration of 6.25 mg/ml against Mycobacterium tuberculosis H37Rv and in particular compounds 2a, 4a,d, 8a,d and 8i, exhibit a
% inhibition from 91 to 99. Among these, compounds 2a, 8a and 8d appeared to have a good activity with minimum inhibitory
concentrations (MICs) of 6.25 mg/ml. On the basis of the biological results, the most effective substituent in position 2 or 7 seems to
be the piperidinyl group. The introduction of a morpholinyl group either in position 2 or 7 of the heterocycle ring caused a decrease
in activity. The 1,8-naphthyridine derivatives were also tested in vitro for their antimicrobial activity against Staphylococcus aureus
as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Tuberculostatic agents; Antimycobacterial agents; Antimicrobial agents; 1,8-Naphthyridines; Piperidinyl; Morpholinyl; Ethylpiperazinyl;
Phenylpiperazinyl
1. Introduction
novel chemotherapeutic agents to hinder the emergence
of resistance and, ideally, to shorten the duration of
therapy of this disease [8,9].
Contrary to general expectations, the incidence of
mycobacterial disease has significantly increased world-
wide since 1990 and in spite of the therapeutic protocols
used so far, tuberculosis continues to represent one of
In a previous paper [10], we reported the preparation
and the antimycobacterial activity of some 4-phenyl-1,8-
naphthyridine derivatives variously substituted in posi-
tions 2 and 7 tested in vitro at a concentration of 12.5
mg/ml against M. Tuberculosis H37Rv. Some of these
the major threats to public health in the world [1Á4].
/
Furthermore, other mycobacteria, especially the Myco-
bacterium avium complex, have emerged as important
pathogens due mainly to the AIDS epidemic [5]. The
prevalence of HIV infection, and the emergence of drug-
resistant and multi-drug-resistant strains of Mycobac-
terium tuberculosis are contributing to the worsening
impact of the disease [6]. The recent emergence of drug-
resistant M. tuberculosis has also become a serious
concern [7]. There is therefore a pressing need to develop
compounds showed an activity with an inhibition ꢀ
/
50%. More recently [11], we described the synthesis
and antimycobacterial activity of some 1,8-naphthyr-
idine derivatives variously substituted in positions 2, 3, 4
and 7. Several compounds, when tested in vitro at a
concentration of 6.25 mg/ml against M. Tuberculosis
H37Rv showed an interesting activity with % inhibition
in the range 38Á96%. These studies showed that the
/
most effective substituents in position 2 or 7 of the 1,8-
naphthyridine nucleus seem to be the piperidinyl or N-
carbethoxypiperazinyl groups.
* Corresponding author.
E-mail address: manera@farm.unipi.it (C. Manera).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00144-7

860
M. Badawneh et al. / Il Farmaco 58 (2003) 859Á866
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Now we report the antituberculosis activity of a new
series of 3- or 4-phenyl-1,8-naphthyridine derivatives
variously substituted in positions 2, 6 and 7.
Furthermore, it was recently reported that some 1,8-
naphthyridine derivatives show an interesting bacterici-
dal activity [12]. For this reason the 1,8-naphthyridine
derivatives reported in this paper were also tested in
vitro for their antimicrobial activity against Staphylo-
coccus aureus as Gram-positive bacteria and Escherichia
coli as Gram-negative bacteria.
2. Chemistry
Scheme 2.
When the 2,7-dichloro-4-phenyl-1,8-naphthyridine (1)
[13] was treated with piperidine, morpholine, ethylpi-
perazine and phenylpiperazine in a sealed tube at
140 8C, the 2,7-dicycloalkylamino derivatives 2 were
obtained (Scheme 1).
Under the same conditions the reaction of the 7-
amino-2-chloro-4-phenyl-1,8-naphthyridine (3) [14] with
the suitable cycloalkylamine afforded the 2-cycloalkyla-
mino derivatives 4 (Scheme 2). Diazotization of 7-
amino-2-cycloalkylamino-4-phenyl-1,8-naphthyridines
FTIR spectrometer. 1H NMR spectra were recorded
with a Bruker AC-200 spectrometer in d units from
TMS as an internal standard. Analytical TLC was
carried out on Merck 0.2 mm precoated silica-gel glass
plates (60 F-254) and location of spots was detected by
illumination with an UV lamp. Elemental analyses of all
compounds synthesized for C, H and N were within 9
/
0.4% of the theoretical values and were performed on a
Carlo Erba elemental analyzer model 1106 apparatus.
4, carried out at ꢀ12 8C in sulfuric acid, gave in a good
/
yield the corresponding 7-hydroxy derivatives 5, which
were converted, by reaction with phosphoryl chloride
under reflux, into the corresponding 7-chloro-2-cy-
cloalkylamino-4-phenyl-1,8-naphthyridines 6. These
last compounds 6 were then treated with sodium
methoxide to give the corresponding 7-methoxy deriva-
tives 7 (Scheme 2).
3.1.1. General procedure for the preparation of 2,7-
dicycloalkylamino-4-phenyl-1,8-naphthyridine derivatives
2aÁd
/
A mixture of 2,7-dichloro-4-phenyl-1,8-naphthyridine
(1) (3.64 mmol) and the suitable cycloalkylamine (16.4
mmol) was heated at 140 8C in a sealed tube for 24 h.
After cooling the reaction mixture was treated with H2O
and the solid obtained was collected by filtration,
washed with diethyl ether and purified by crystallization
All the compounds synthesized were characterized by
1
elemental analysis, IR and H NMR.
from petroleum ether 100Á
/
140 8C to give the title
compounds 2aÁd.
/
3. Experimental protocols
3.1.1.1. 2,7-Di(piperidin-1-yl)-4-phenyl-1,8-
3.1. Chemistry
naphthyridine (2a). 1.22 g, yield 90%; m.p. 125Á127 8C;
/
1H NMR (DMSO): d 7.57 (d, 1H, H5), 7.46 (m, 5H, Ar),
6.81 (d, 1H, H6), 6.70 (s, 1H, H3), 3.67 (m, 8H,
piperidinyl), 1.56 (m, 12H, piperazinyl). Anal.
C24H28N4 (C, H, N).
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. IR spectra in Nujol
mulls were recorded on an ATI Mattson Genesis Series
3.1.1.2. 2,7-Di(morpholin-1-yl)-4-phenyl-1,8-
naphthyridine (2b). 1.18 g, yield 86%; m.p. 155Á157 8C;
/
1H NMR (DMSO): d 7.62 (d, 1H, H5), 7.49 (m, 5H, Ar),
6.89 (d, 1H, H6), 6.79 (s, 1H, H3), 3.68 (m, 8H,
morpholinyl). Anal. C22H24N4O2 (C, H, N).
3.1.1.3. 2,7-Di(4-ethylpiperazin-1-yl)-4-phenyl-1,8-
naphthyridine (2c). 1.3 g, yield 82%; m.p. 100Á102 8C;
/
1H NMR (DMSO): d 7.63 (d, 1H, H5), 7.47 (m, 5H, Ar)
6.82 (d, 1H, H6), 6.76 (s, 1H, H3), 3.66 (m, 8H,
Scheme 1.

M. Badawneh et al. / Il Farmaco 58 (2003) 859Á
/866
861
piperazinyl), 2.44 (m, 12H, piperazinyl and CH2), 1.03
(t, 6H, CH3). Anal. C26H34N6 (C, H, N).
3.1.3. General procedure for the preparation of 2-
cycloalkylamino-7-hydroxy-4-phenyl-1,8-naphthyridine
derivatives 5aÁ
Sodium nitrite (1.8 mmol) was added portion wise to
an ice-cooled solution (ꢀ12 8C) of the appropriate 7-
amino-2-cycloalkylamino-4-phenyl-1,8-naphthyridine
derivative 4aÁd (1.50 mmol) in concentrated sulfuric
acid (5 ml). The reaction mixture was stirred at room
temperature for 2 h and then treated with crushed ice
and then with concentrated NH4OH until the pH was 8.
The solid was collected by filtration, washed with H2O
and purified by flash chromatography eluting with ethyl
acetate for 5a and 5b, ethyl acetate/diethylamine 12:1 for
5c and ethyl acetate/diethylamine 10:1 for 5d.
/
d
3.1.1.4. 2,7-Di(4-phenylpiperazin-1-yl)-4-phenyl-1,8-
/
naphthyridine (2d). 1.5 g, yield 77%; m.p. 175Á178 8C;
/
1H NMR (CDCl3): d 7.58 (d, 1H, H5), 7.35 (m, 5H, Ar),
7.27 (m, 5H, Ar), 6.94 (m, 5H, Ar), 6.71 (d, 1H, H6),6.68
(s, 1H, H3), 3.99 (m, 4H, piperazinyl), 3.35 (m, 4H,
piperazinyl). Anal. C25H24N4O (C, H, N).
/
3.1.2. General procedure for the preparation of 7-amino-
2-cycloalkylamino-4-phenyl-1,8-naphthyridine derivatives
4aÁd
/
A
mixture
of
7-amino-2-chloro-4-phenyl-1,8-
naphthyridine (3) (3.9 mmol) and the suitable cycloalk-
ylamine (11.7 mmol) was heated at 140 8C in a sealed
tube for 24 h. After cooling, the reaction mixture was
treated with H2O, and the precipitate formed was
collected by filtration and purified by crystallization to
3.1.3.1. 7-Hydroxy-4-phenyl-2-(piperidin-1-yl)-1,8-
naphthyridine (5a). 0.38 g yield 37%; m.p. 242Á244 8C
/
1
(toluene). H NMR (DMSO): d 11.55 (brs, 1H, OH),
7.46 (m, 6H, Ar and H5), 6.59 (s, 1H, H3), 6.09 (d, 1H,
H6), 3.69 (s, 4H, piperidinyl), 1.56 (s, 6H, piperidinyl).
Anal. C19H19 N3O (C, H, N).
give compounds 4aÁc. To obtain 4d the reaction
/
mixture was treated with H2O and the solution was
extracted with CHCl3; the combined extracts were dried
(MgSO4) and evaporated to dryness under a vacuum to
obtain a solid, which was purified by crystallization.
3.1.3.2. 7-Hydroxy-2-(morpholin-1-yl)-4-phenyl-1,8-
naphthyridine (5b). 0.42 g, yield 32%; m.p. 254Á256 8C
/
1
(toluene). H NMR (DMSO): d 11.58 (brs, 1H, OH),
7.47 (m, 6H, Ar and H5), 6.62 (s, 1H, H3), 6.14 (d, 1H,
H6), 3.67 (s, 8H, morpholinyl). Anal. C18H17N3O2 (C,
H, N).
3.1.2.1. 7-Amino-4-phenyl-2-(piperidin-1-yl)-1,8-
naphthyridine (4a). 0.78 g, yield 65%; m.p. 232Á236 8C
/
1
(toluene). H NMR (CDCl3): d 7.66 (d, 1H, H5), 7.46
(m, 5H, Ar), 6.65 (s, 1H, H3), 6.36 (d, 1H, H6), 4.83 (brs,
2H, NH2), 3.80 (m. 4H, piperidinyl), 1.68 (m, 6H,
piperidinyl). Anal. C19H20N4 (C, H, N).
3.1.3.3. 7-Hydroxy-4-phenyl-2-(4-ethylpiperazin-1-yl)-
1,8-naphthyridine (5c). 0.54 g, yield 53%; m.p. 157Á
/
1
160 8C (toluene). H NMR (CDCl3): d 9.08 (brs, 1H,
OH), 7.58 (d, 1H, H5), 7.40 (m, 5H, Ar), 6.46 (s, 1H,
H3), 6.31 (d, 1H, H6), 3.75 (m, 4H, piperazinyl), 2.53 (m,
6H, piperazinyl and CH2), 1.14 (t, 3H, CH3). Anal.
C20H22N4O (C, H, N).
3.1.2.2. 7-Amino-2-(morpholin-1-yl)-4-phenyl-1,8-
naphthyridine (4b). 0.50 g, yield 49%; m.p. 153Á156 8C
/
1
(toluene). H NMR (CDCl3): d 7.53 (d, 1H, H5), 7.46
(m, 5H, Ar), 6.63 (s, 1H, H3), 6.43 (d, 1H, H6), 4.94 (brs,
2H, NH2), 3.81 (m, 8H, morpholinyl). Anal. C18H18N4O
(C, H, N).
3.1.3.4. 7-Hydroxy-4-phenyl-2-(4-phenylpiperazin-1-yl)-
1,8-naphthyridine (5d). 0.30 g, yield 33%; m.p. 253Á
/
1
256 8C (toluene). H NMR (CDCl3): d 9.08 (brs, 1H,
OH), 7.60 (d, 1H, H5), 7.43 (m, 6H, Ar), 6.96 (m, 4H,
Ar), 6.51 (s, 1H, H3), 6.33 (d, 1H, H6), 3.88 (m, 4H,
piperazinyl), 3.32 (m, 6H, piperazinyl). Anal.
C24H22N4O (C, H, N).
3.1.2.3. 7-Amino-4-phenyl-2-(4-ethylpiperazin-1-yl)-1,8-
naphthyridine (4c). 0.55 g, yield 42%; m.p. 198Á200 8C
/
1
(toluene). H NMR (CDCl3): d 7.69 (d, 1H, H5), 7.43
(m, 5H, Ar), 6.65 (s, 1H, H3), 6.39 (d, 1H, H6), 4.82 (brs,
2H, NH2), 3.85 (m, 4H, piperazinyl), 2.49 (m, 6H, CH2
and piperazinyl), 1.14 (t, 3H, CH3). Anal. C20H23N5 (C,
H, N).
3.1.4. General procedure for the preparation of 7-chloro-
2-cycloalkylamino-4-phenyl-1,8-naphthyridine derivatives
6aÁd
/
A mixture of 0.98 mmol of the appropriate 2-
cycloalkylamino-7-hydroxy-4-phenyl-1,8-naphthyridine
3.1.2.4. 7-Amino-4-phenyl-2-(4-phenylpiperazin-1-yl)-
1,8-naphthyridine (4d). 0.50 g, yield 34%; m.p. 262Á
/
derivative 5aÁd and 3 ml of phosphoryl chloride was
/
265 8C (toluene). 1H NMR (CDCl3): d 7.71 (d, 1H,
H5), 7.45 (m, 4H, Ar), 7.31 (m, 3H, Ar), 7.01 (m, 3H,
Ar), 6.70 (s, 1H, H3), 6.42 (d, 1H, H6), 4.85 (brs, 2H,
NH2), 4.00 (m, 4H, piperazinyl), 3.32 (m, 4H, piper-
azinyl). Anal. C24H23N5 (C, H, N).
heated at 130 8C for 3 h. The cooled mixture was poured
into crushed ice and treated with concentrated NH4OH
until the pH was 8. The resulting precipitate was
collected by filtration, washed with water and purified
by crystallization to give 6aÁ6d.
/

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M. Badawneh et al. / Il Farmaco 58 (2003) 859Á866
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3.1.4.1. 7-Chloro-4-phenyl-2-(piperidin-1-yl)-1,8-
naphthyridine (6a). 0.28 g, yield 87%; m.p. 143Á
(petroleum ether 100Á
140 8C). 1H NMR (CDCl3): d
7.82 (d, 1H, H5), 7.45 (m, 5H, Ar), 7.01 (d, 1H, H6), 6.92
(s, 1H, H3), 3.87 (m, 4H, piperidinyl), 1.71 (m, 6H,
piperidinyl). Anal. C19H18N3Cl (C, H, N).
141 8C. 1H NMR (CDCl3): d 7.80 (d, 1H, H5), 7.47
(m, 5H, Ar), 6.76 (s, 1H, H3), 6.60 (d, 1H, H6), 4.14 (m,
3H, OCH3), 3.88 (m, 4H, piperazinyl), 2.59 (m, 6H,
piperazinyl and CH2), 1.15 (t, 3H, CH3). Anal.
C21H24N4O (C, H, N).
/
146 8C
/
3.1.5.4. 7-Methoxy-4-phenyl-2-(4-phenylpiperazin-1-yl)-
3.1.4.2. 7-Chloro-2-(morpholin-1-yl)-4-phenyl-1,8-
1,8-naphthyridine (7d). 0.45 g yield 76%; m.p. 85Á88 8C.
/
naphthyridine (6b). 0.27 g yield 84%; m.p. 91Á
/
94 8C
1H NMR (CDCl3): d 7.83 (d, 1H, H5), 7.31 (m, 6H, Ar),
6.87 (m, 5H, Ar and H3), 6.63 (d, 1H, H6), 4.16 (m, 3H,
OCH3), 4.02 (m, 4H, piperazinyl), 3.35 (m, 4H, piper-
azinyl). Anal. C25H24N4O (C, H, N).
1
(petroleum ether 40Á
/
60 8C). H NMR (CDCl3): d 7.89
(d, 1H, H5), 7.45 (m, 5H, Ar), 7.10 (d, 1H, H6), 6.91 (s,
1H, H3), 3.86 (m, 8H, morpholinyl). Anal.
C18H16ClN3O (C, H, N).
3.2. Biological assay
3.1.4.3. 7-Chloro-4-phenyl-2-(4-ethylpiperazin-1-yl)-1,8-
naphthyridine (6c). 0.31 g yield 89%; m.p. 167Á
/
170 8C
3.2.1. Antimycobacterial activity
(petroleum ether 100Á
/
140 8C). 1H NMR (CDCl3): d
All of the compounds were evaluated for in vitro
antituberculosis activity against M. tuberculosis as part
of a TAACF TB screening program under the direction
of the US National Institute of Health, NIAID division.
Primary screening was conducted at a single concen-
tration, 6.25 mg/ml against M. tuberculosis H37Rv
(ATCC 27294) in BACTEC 12B medium using a broth
microdilution assay, the microplate Alamar Blue Assay
(MABA) [15]. The standard compound used in this
7.87 (d, 1H, H5), 7.48 (m, 5H, Ar), 7.08 (d, 1H, H6), 6.92
(s, 1H, H3), 3.94 (m, 4H, piperazinyl), 2.53 (m, 6H,
piperazinyl and CH2). Anal. C20H21ClN4 (C, H, N).
3.1.4.4. 7-Chloro-4-phenyl-2-(4-phenylpiperazin-1-yl)-
1,8-naphthyridine (6d). 0.36 g yield 93%; m.p. 90Á
/
93 8C
(petroleum ether 100Á
/
140 8C). 1H NMR (CDCl3): d
7.89 (d, 1H, H5), 7.42 (m, 7H, Ar and H6), 7.00 (m, 5H,
Ar and H3), 4.08 (m, 4H, piperazinyl), 3.47 (m, 4H,
piperazinyl). Anal. C24H21ClN4 (C, H, N).
primary assay was rifampicin (MICꢁ
Compounds effecting B90% inhibition in the pri-
mary screening (MICꢀ6.25 mg/ml) were not generally
/
0.25 mg/ml).
/
/
3.1.5. General procedure for the preparation of 2-
cycloalkylamino-7-methoxy-4-phenyl-1,8-naphthyridine
evaluated further. The active compounds were re-tested
by serial dilution beginning at 6.25 mg/ml against M.
tuberculosis H37Rv to determine the actual minimum
inhibitory concentration (MIC) in BABTEC 460. The
MIC is defined as the lowest concentration effecting a
reduction in fluorescence of 90% compared with con-
trols.
derivatives 7aÁd
/
A solution of 15 mmol of freshly prepared sodium
methoxide and 1.5 mmol of the appropriate 7-chloro-2-
cycloalkylamino-4-phenyl-1,8-naphthyridine derivative
6aÁd in 30 ml of absolute methanol was refluxed for
/
5.5 h. After cooling, water was added and the pH of the
mixture was adjusted to 8 with 10% HCl. The solid
obtained was collected by filtration and crystallized
3.2.2. Cytotoxicity assay
The compounds were screened by serial dilution to
assess toxicity on a VERO cell line, generally beginning
from petroleum ether 100Á
/
140 8C to give 7aÁd.
/
at 10ꢂ the MIC, if permitted by the sample solubility in
/
3.1.5.1. 7-Methoxy-4-phenyl-2-(piperidin-1-yl)-1,8-
naphthyridine (7a). 0.33 g yield 69%; m.p. 80Á
82 8C. 1H
NMR (CDCl3): d 7.78 (d, 1H, H5), 7.47 (m, 5H, Ar),
6.76 (s, 1H, H3), 6.57 (d, 1H, H6), 4.11 (m, 3H, OCH3),
3.81 (m, 4H, piperidinyl), 1.70 (m, 6H, piperidinyl).
Anal. C20H21N3O (C, H, N).
culture media. The selectivity index (SI) is defined as the
ratio of the IC50 measured in VERO cells to the MIC
described above.
/
3.2.3. Antimicrobial activity
The 1,8-naphthyridine derivatives were evaluated in
vitro for their antimicrobial activity against S. aureus as
Gram-positive bacteria and E. coli as Gram-negative
bacteria, using the cup diffusion technique [16].
The compounds were dissolved in DMSO at a
concentration of 1 mg/ml. Sterile nutrient agar (oxoid)
was incubated with the organisms tested. Each 100 ml of
the medium received 1 ml of 24 h broth culture and 3
drops of the test compounds were placed separately in
cups (8 mm diameter) cut in the agar. The plates were
incubated at 37 8C for 24 h. DMSO as a blank showed
3.1.5.2. 7-Methoxy-2-(morpholin-1-yl)-4-phenyl-1,8-
naphthyridine (7b). 0.35 g yield 72%; m.p. 85Á
87 8C. 1H
/
NMR (CDCl3): d 7.83 (d, 1H, H5), 7.48 (m, 5H, Ar),
6.74 (s, 1H, H3), 6.38 (d, 1H, H6), 4.14 (m, 3H, OCH3),
3.83 (m, 8H, morpholinyl). Anal. C19H19N3O2 (C, H,
N).
3.1.5.3. 7-Methoxy-4-phenyl-2-(4-ethylpiperazin-1-yl)-
1,8-naphthyridine (7c). 0.39 g yield 74%; m.p. 139Á
/

M. Badawneh et al. / Il Farmaco 58 (2003) 859Á
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863
no inhibition zone. A solution of 0.1% of penicillin G or
streptomycin sulfate in DMSO was used as the standard
for Gram-positive and Gram-negative bacteria, respec-
tively. The resulting inhibition zone diameters (I.Z.)
were measured in mm. For compounds, which exhibited
of 24 h test organism culture and incubated at 37 8C for
48 h.
4. Results and discussion
reasonable inhibition zones (]20 mm), the MIC was
/
determined. The organisms tested were grown in suita-
ble broth media for 24 h at 37 8C. The compounds
selected were dissolved in DMSO (400 mg/ml) and
twofold serial dilutions were prepared using the appro-
priate broth. The tubes were then inoculated with 100 ml
4.1. Antimycobacterial activity
The new compounds 2aÁ
d and compounds previously reported 8aÁ
[18], and 8q [19] were tested in vitro at a concentration
/
d, 4aÁ
/
d, 5aÁ
/
d, 6aÁ
/
d and 7aÁ
/
/n [17], 8o,p
Table 1
Antimycobacterial in vitro activity of the tested compounds expressed as % inhibition of M. tuberculosis H37 Rv at a concentration of 6.25 mg/ml
Comp.
R
R1
R2
R3
R4
% Inhibition
2a
2b
2c
2d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
8e
8f
Pip
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
Pip
99
0
Morph
Ethylpipz
Phenylpipz
Pip
H
Morph
Ethylpipz
Phenylpipz
NH2
NH2
NH2
NH2
OH
H
81
47
91
47
69
94
0
H
H
Morph
Ethylpipz
Phenylpipz
Pip
H
H
H
H
Morph
Ethylpipz
Phenylpipz
Pip
H
OH
OH
0
H
29
32
70
36
68
55
85
35
72
86
99
79
71
91
89
83
59
48
92
64
67
43
73
88
67
98 a
H
OH
Cl
H
Morph
Ethylpipz
Phenylpipz
Pip
H
Cl
Cl
H
H
Cl
H
OCH3
OCH3
OCH3
OCH3
Pip
Morph
Ethylpipz
Phenylpipz
Pip
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Pip
Pip
H
Pip
Pip
H
Pip
Pip
H
Cep
H
Pipz
Morph
NH2
Cep
Pip
Pip
H
8g
8h
8i
H
Cep
Cep
H
H
Pip
Morph
Cl
8l
Cep
Cep
H
8m
8n
8o
8p
8q
RMP
H
Cmp
Pipz
H
OCH3
Pipz
Pip
H
Pipz
Pipz
H
H
OCH3
Pip, piperidinyl; Morph, morpholinyl; Ethylpipz, ethylpiperazinyl; Phenylpipz, phenylpiperazinyl; Cep, ethylcarbethoxypiperazinyl; Cmp,
methylcarbethoxypiperazinyl. Pipz, piperazinyl.
a
At a concentration of 0.25 mg/ml.

864
M. Badawneh et al. / Il Farmaco 58 (2003) 859Á866
/
of 6.25 mg/ml against M. tuberculosis H37Rv by the
method described by Collins and Franzblau [15]. The
results of the biological evaluation, expressed as %
inhibition of the growth of mycobacterium, are sum-
marized in Table 1 and for the sake of comparison the %
inhibition of Rifampicin (RMP) is also included. All the
compounds tested proved to be less active, against M.
tuberculosis H37Rv, than Rifampicin. In particular,
compounds 2a, 4a,d, 8a,d and 8i showed an appreciable
activity with inhibition from 91 to 99%. Compounds 2c,
substituents in positions 2, 3, 4 and 7 of the 1,8-
naphthyridine nucleus.
On the basis of the biological results, the most
effective substituent in positions 2 and/or 7 seems to
be the piperidinyl group. This result is in agreement with
previous findings [11].
The ethylcarbethoxypiperazinyl group and the new
ethylpiperazinyl and phenylpiperazinyl groups seem to
be effective substituents in positions 2 and/or 7 of the
1,8-naphthyridine nucleus for the antimycobacterial
activity. Moreover, also the amino, chloro or methoxy
groups in position 7 of the heterocyclic ring seem to be
effective for the antimycobacterial activity. In fact, the
7-amino derivatives 4a,c,d, the 7-chloro derivatives
6a,c,d and the 7-methoxy derivatives 7a,c,d showed a
moderate activity with inhibition from 55 to 91%. The
analogous derivatives 4b, 6b and 7b showed a very poor
activity: most probably, the lack of activity is due to the
presence of the morpholinyl group in position 2 of the
1,8-naphthyridine ring.
A comparison of the activity of morpholinyl-substi-
tuted derivatives versus the activity of the other 1,8-
naphthyridine derivatives clearly indicates that the
introduction of a morpholinyl group either in position
2 or 7 of the heterocycle ring causes a decrease in activity
as previously reported [11].
Furthermore, the biological results showed that the
introduction of nitro or amino group in position 6 of the
2,7-dipiperidinyl-3-phenyl-1,8-naphthyridine (8a), com-
pounds 8b and 8c, respectively, resulted in a decrease of
the antimycobacterial activity.
Moreover, the biological results of this series of 1,8-
naphthyridine derivatives confirmed that, when a hy-
droxy group was introduced into position 7 of the 1,8-
naphthyridine nucleus, inactive compounds were ob-
tained, as also reported in a previous paper [10,11].
4c, 6a,c,d, 7a,c,d, 8b,c, 8eÁ
moderate activity, with inhibition from 50 to 90%,
whereas compounds 2b,d, 4b, 5aÁd, 6b, 7b, 8h and 8n,
with inhibition B50%, were found to possess little or no
/g,l,m,o,p and 8q, showed a
/
/
activity at the concentration assayed (Table 1).
Compounds 2a, 4a,d, 8a,d and 8i, which demon-
strated an inhibition ꢀ90% in the primary screening,
/
were retested at a lower concentration against M.
tuberculosis H37Rv to determine the actual MIC with
the same method used in the primary screening. The
results of the MIC evaluation (Table 2) indicate that
only compounds 2a, 8a and 8d showed an MIC of 6.25
mg/ml, whereas compounds 4a,d and 8i showed an MIC
ꢀ6.25 mg/ml.
/
Concurrent with the determination of MICs, com-
pounds 2a, 8a and 8d were tested for cytotoxicity (IC50)
on a VERO cell line. The cytotoxicity data indicate that
compounds 2a and 8a exhibit a high degree of toxicity
and a low selectivity index level (SI) (Table 2). In the
case of compound 8d, the cytotoxicity was not deter-
mined because of its insolubility in tissue culture media
(Table 2).
The biological results of this series of 1,8-naphthyr-
idine derivatives, which present a wide number of
structural variables, linked to several molecular regions,
allow us to confirm [10,11] or clarify the influence of the
Table 2
Antimycobacterial in vitro activity and cytotoxicity effect of compounds 2a, 4a,d, 8a,d,i
Comp.
R
R1
R2
R3
R4
MIC (mg/ml)
IC50 (mg/ml)
SI
2a
4a
Pip
Pip
H
Ph
Ph
Ph
H
H
H
H
H
H
H
Pip
6.25
2.49
a
0.40
nd a
nd a
H
NH2
NH2
Pip
ꢀ
ꢀ/
/
6.25
6.25
nd
4d
8a
Phenylpipz
Pip
H
nd a
2.74
Ph
Ph
Ph
6.25
6.25
0.44
b
b
8d
8i
RMP
Pip
Cep
H
Cep
Pip
H
ꢀ/
6.25
nd a
nd a
0.25
Pip, piperidinyl; Phenylpipz, phenylpiperazinyl; Cep, ethylcarbethoxypiperazinyl.
a
nd: not determined.
Insoluble in tissue culture media.
b

M. Badawneh et al. / Il Farmaco 58 (2003) 859Á
/866
865
Finally, the phenyl group in position 3 or 4 of the
heterocyclic ring seems to play an important role for the
antimycobacterial activity of 1,8-naphthyridine deriva-
tives, confirming that in these positions a large lipophilic
group is indispensable, as shown in a previous paper for
the benzylic group in position 3 [11].
bacteria and E. coli as Gram-negative bacteria (Table
3). Furthermore, the MIC values in mg/ml using twofold
serial dilution method were calculated for compounds
which exhibited reasonable inhibition zones (]20 mm).
/
The results of the biological evaluation indicate that
all the compounds tested were less active than the
reference standards. Compound 8b showed an appreci-
able activity against S. aureus with an MIC value of 25,
compounds 5a, 6a and 7d possessed a moderate activity
with an MIC value of 100 and compounds 4b, 5b, 8f and
8i, with MIC values from 140 to 200, showed a poor
4.2. Antimicrobial activity
The new compounds were evaluated in vitro for their
antibacterial activity against S. aureus as Gram-positive
Table 3
Inhibition zone diameters (I.Z.), values in mm and MIC values in mg/ml
Comp.
R
R1
R2
R3
R4
S. aureus
E. coli
I.Z.
I.Z.
MIC
MIC
25
2a
2b
2c
2d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
8e
8f
Pip
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
Pip
14
12
12
12
14
20
16
16
24
20
12
14
26
18
28
Morph
Ethylpipz
Phenylpipz
Pip
H
Morph
Ethylpipz
Phenylpipz
NH2
NH2
NH2
NH2
OH
Á/
H
12
18
20
33
36
32
H
H
100
25
Morph
Ethylpipz
Phenylpipz
Pip
H
200
H
12.5
50
H
H
100
200
Á/
26
Morph
Ethylpipz
Phenylpipz
Pip
H
OH
OH
200
H
Á/
14
H
OH
H
Cl
Cl
100
Á/
Morph
Ethylpipz
Phenylpipz
Pip
H
12
16
14
20
24
H
Cl
Cl
Á/
22
H
H
OCH3
OCH3
OCH3
OCH3
Pip
Á/
Á/
Á/
28
16
30
200
100
Morph
Ethylpipz
Phenylpipz
Pip
H
H
Á/
Á/
H
100
25
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
20
100
50
Pip
Pip
H
Pip
Pip
Á/
H
Á/
26
18
14
26
Pip
Pip
H
Cep
15
18
22
18
17
23
13
H
Pipz
Morph
NH2
Cep
Pip
Pip
H
160
140
130
8g
8h
8i
H
Á/
Cep
Cep
H
18
25
17
24
12
12
18
16
H
Pip
Morph
Cl
120
45
8l
Cep
H
8m
8n
8o
8p
Cep
Cmp
H
Á/
16
14
H
OCH3
Pipz
Pip
Pipz
H
Pipz
Pipz
H
Á/
12
42
H
OCH3
Penicillin G
Streptomycin
4
38
6
Pip, piperidinyl; Cep, ethylcarbethoxypiperazinyl; Ethylpipz, ethylpiperazinyl; Phenylpipz, phenylpiperazinyl; Morph, morpholinyl; Pipz,
piperazinyl; Cmp, methylcarbethoxypiperazinyl.

866
M. Badawneh et al. / Il Farmaco 58 (2003) 859Á866
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found to be inactive.
As regards activity against E. coli, compounds 2a,
/
[7] J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon,
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/
MIC value from 12.5 to 50. The compounds 4a, 5b, 7a,b,
8a,f and 8i showed a moderate activity with MIC values
from 100 to 200. The other 1,8-naphthyridine deriva-
tives were found to be inactive.
On the basis of these results, the 1,8-naphthyridine
derivatives seem to be more active against E. coli than
against S. aureus. At this time the biological results do
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Acknowledgements
The in vitro evaluation of the antituberculosis activity
was carried out in the Tuberculosis Antimicrobial
Acquisition & Coordinating Facility (TAACF) at the
National Institute of Allergy and Infectious Disease,
Southern Research Institute, GWL Hansen’s Disease
Center and Colorado State University, USA; we thank
J.A. Maddry, Ph.D. for his collaboration.
877Á
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