Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.06.098

A simple and efficient synthesis of 6-fluoro-4-oxopyrido[2,3-a]carbazole-3- carboxylic acids (13a-e) and a structurally related 6-fluoro-4- oxothieno[2′,3′:4,5]pyrrolo[3,2-h]quinoline (13f) was achieved via Stille arylation of 7-chloro-6-fluoro-8-nitro-4-oxoquinoline-3-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. The ability of 13a-f to inhibit the activity of DNA gyrase and topoisomerase IV was also investigated. The thieno isostere (13f) emerged as the most active antibacterial, while the 9-fluoro derivative (13e) was the most potent against multidrug-resistant staphylococci. Compounds 13a, 13c-f displayed growth inhibition against MCF-7 breast tumor and A549 non-small cell lung cancer cells coupled with an absence of cytotoxicity toward normal human-derm fibroblasts (HuDe). Compound 13e was the most active anticancer against MCF-7 cells, with greater potency than ellipticine (IC₅₀ 0.8 and 1.6 μM, respectively). The most active compounds in this series show promise as dual acting anticancer and antibacterial chemotherapeutics.

Full text of DOI:10.1016/j.bmc.2010.06.098

Bioorganic & Medicinal Chemistry 18 (2010) 5873–5884
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of tetracyclic fluoroquinolones
as antibacterial and anticancer agents
b
Salah A. Al-Trawneh a, Jalal A. Zahra a, Marwan R. Kamal a, Mustafa M. El-Abadelah a, , Franca Zani ,
*
Matteo Incerti b, Andrea Cavazzoni c, Roberta R. Alfieri c, Pier G. Petronini c, Paola Vicini b,
*
a Chemistry Department, Faculty of Science, The University of Jordan, Amman 11942, Jordan
b Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27/A, Parma 43100, Italy
c Dipartimento di Medicina Sperimentale, Sezione di Oncologia Sperimentale, Università degli Studi di Parma, Via Volturno 39, Parma 43100, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and efficient synthesis of 6-fluoro-4-oxopyrido[2,3-a]carbazole-3-carboxylic acids (13a–e) and
a structurally related 6-fluoro-4-oxothieno[20,30:4,5]pyrrolo[3,2-h]quinoline (13f) was achieved via Stille
Received 28 February 2010
Revised 25 June 2010
Accepted 29 June 2010
Available online 3 July 2010
arylation of 7-chloro-6-fluoro-8-nitro-4-oxoquinoline-3-carboxylate and
a subsequent microwave-
assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro anti-
microbial and antiproliferative activity. The ability of 13a–f to inhibit the activity of DNA gyrase and
topoisomerase IV was also investigated. The thieno isostere (13f) emerged as the most active antibacte-
rial, while the 9-fluoro derivative (13e) was the most potent against multidrug-resistant staphylococci.
Compounds 13a, 13c–f displayed growth inhibition against MCF-7 breast tumor and A549 non-small cell
lung cancer cells coupled with an absence of cytotoxicity toward normal human-derm fibroblasts (HuDe).
Compound 13e was the most active anticancer against MCF-7 cells, with greater potency than ellipticine
Keywords:
6-Fluoroquinolones
4-Oxo-1,4-dihydropyrido[2,3-a]carbazoles
4-Oxothieno[20,30:4,5]pyrrolo[3,2-
h]quinoline
(IC50 0.8 and 1.6
anticancer and antibacterial chemotherapeutics.
lM, respectively). The most active compounds in this series show promise as dual acting
Antibacterial activity
Anticancer activity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
their substituted derivatives have been the most successfully em-
ployed side chains as evidenced by the compounds currently on
the market. The substituent at C-8, and similarly the substituent
at C-5, affects the overall steric configuration and the number of
intracellular targets on bacterial type II topoisomerases.2d A fused
ring with a bridge between C-8 and N-1 is found in levofloxacin
and rufloxacin. Recently, last generation fluoroquinolones demon-
strated the favorable influence of an OCH3 substituent at position
8 on Gram positive and on anaerobic bacteria. Moreover, the opti-
mal substituent placed on C-8, combined with a bulky addition at
C-7 has been shown to markedly reduce the development of fluoro-
quinolone resistance in Staphylococcus aureus.3
From these observations, changes in substitution on the quino-
lone C-7 and C-8 positions appear to play a significant role in the tar-
get preference and may offer new insight into the structure–activity
relationship of the fluoroquinolone antibacterials. To the best of our
knowledge, fused rings with a bridge between the critical 7 and 8
positions has not yet been extensively investigated in this system.
As part of an ongoing program aimed at developing facile
synthesis of novel heterocycles [h]fused onto 1-cylopropyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, we have
recently explored the synthesis of some 9-cyclopropyl-4-fluoro-
6-oxoimidazo[4,5-h]quinoline-7-carboxylic acids4 and of 9-cyclo-
propyl-4-fluoro-6-oxo[1,2,5]thiadiazolo[3,4-h]quinoline-5-carbox-
ylic acid, which is endowed with strong antibacterial activity.5
After the discovery of the first fluoroquinolone, norfloxacin,1 in
1980 as an antibacterial agent with potent and broad spectrum
activity, several new members of this family (Fig. 1) have emerged
with enhanced activity against Gram positive and anaerobic bacte-
ria and improved pharmacokinetic profile. Much has been learned
about how molecular modifications of the core quinolone structure
affect the antibacterial profile. The structure–activity relationship of
quinolones has been the subject of extensive review.2 For most of
the current agents, a hydrogen at position 2, a carboxyl group at
position 3 and a keto group at position 4 in the bicyclic ring cannot
be changed without a significant loss of activity. Furthermore it ap-
pears that a cyclopropyl group is optimal at C-1. The fluorine atom at
position 6 imparts increased intracellular penetration and enhanced
DNA gyrase activity and some efficacy against Gram positive bacte-
ria. It can be considered a breakthrough for the enhanced Gram neg-
ative activity, which led to the group of the modern 6-fluoro
compounds (or second generation quinolones). The substituent at
position 7 greatly influences potency, spectrum and safety. A nitro-
gen heterocyclic moiety is optimal and piperazine, pyrrolidine and
* Corresponding author. Tel.: +39 0521 905051; fax: +39 0521 905006.
E-mail addresses: paola.vicini@unipr.it, pvicini@ipruniv.cce.unipr.it (P. Vicini).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.098

5874
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
O
N
NH2
O
O
F
COOH
F
COOH
F
COOH
H3C
N
N
N
N
N
HN
H3C
F
HN
HN
O
CH3
Sparfloxacin (3)
Ciprofloxacin (1)
Levofloxacin (2)
O
O
CH3
O
F
COOH
F
COOH
F
COOH
H3CO
N
N
N
N
N
N
N
N
HN
OCH3
HN
CH3
CH3
H2N
Gatifloxacin (5)
Gemifloxacin (6)
Grepafloxacin (4)
O
N
O
N
F
COOH
F
CO2H
N
N
N
OCH3
N
S
Me
Moxifloxacin (7)
Rufloxacin (8)
Figure 1. Structures of some fluoroquinolones in clinical use.
We report here the synthesis and in vitro evaluation of the
antimicrobial and antitumor properties of novel tetracyclic fluoro-
quinolones (Fig. 2) such as the variously substituted 1-cyclopro-
pyl-6-fluoro-4-oxo-1,4-dihydropyrido[2,3-a]carbazole-3-carboxylic
acids (13a–e) and the structurally related 1-cyclopropyl-6-fluoro-
4-oxo-4,10-dihydro-1H-thieno[20,30:4,5]pyrrolo[3,2-h]quinoline-
3-carboxylic acid (13f), a thiophene isostere of 13a.
The investigation of the antitumor activity of the tetracyclic
fluoroquinolones 13a–f was suggested by the use of the 4-quino-
lone-3-carboxylic acid motif as a multivalent scaffold in medicinal
chemistry,6 and the fact that the structure of 13a–e closely
resembles the pyrido[4,3-b]carbazole system that constitutes the
skeleton of the naturally occurring anticancer ellipticine and its
derivatives (Fig. 2: bold structures).7 Ellipticine 14a, an alkaloid
O
O
COOH
F
COOH
F
N
N
S
R2
NH
NH
R1
R2
13f
13a-e
13a R1 = H; R2 = H;
13b R1 = Me; R2 = H
13c R1 = OMe; R2 = H
13d R1 = OMe; R2 = Me
13e R1 = F; R2 = H
R1
OH
R2
R3
Me
Me
+
N
N
OAc
NH
NH
Me
Me
16
14a R1 = R3 = H; R2 = Me;
14b R1 = OMe; R2 = Me; R3 = H
14c R1 = OH; R2 = Me; R3 = H
15 R1 = R2 = H; R3 = Me
Figure 2. New synthesized fluoroquinolones (13a–f), ellipticines (14a–c, 16), and olivacine (15).

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
5875
isolated from Apocynaceae plants,8,9 such as Ochrosia elliptica labil,
and several of its derivatives (e.g., the natural analogs 9-methoxy-
ellipticine 14b, 9-hydroxyellipticine 14c, and the isomeric oliva-
cine 15) show promise in the treatment of osteolytic breast
cancer metastases, kidney sarcoma, brain tumors and myeloblastic
leukemia.10–17 A number of successful ellipticine analogues have
been designed and synthesized with improved cytotoxicity and
anticancer activities against a panel of cancer cell lines.18–20 The
derivative 9-hydroxy-2-methylellipticinium acetate 16 (elliptini-
um acetate), with a good solubility, has found application in the
treatment of breast, kidney, and thyroid cancer.21–23 The interest
in ellipticine and its derivatives for clinical purposes is mainly
due to their high efficiency against several types of cancer, limited
toxic side effects, and complete lack of hematological toxicity.24
The antiproliferative activity of all compounds investigated in
the present study was evaluated against MCF-7 (breast), A549
(non-small cell lung cancer) tumor cells, and normal human-derm
fibroblasts (HuDe). Compound 13e, with the best antiproliferative
activity, was further assessed, in comparison to ellipticine, by flow
cytometric analysis on cell cycle distributions. Moreover apoptosis
induction and p53 expression were studied to obtain further in-
sight, at the molecular level, into the mechanism of their action.
4-dihydropyrido[2,3-a]carbazole-3-carboxylic acids 13a–e and
1-cyclopropyl-6-fluoro-4-oxo-4,10-dihydro-1H-thieno[20,30:4,5]-
pyrrolo[3,2-h]quinoline-3-carboxylic acid 13f.
The new compounds 11–13 were characterized by elemental
analyses, IR, MS and NMR spectral data. These data, detailed in Sec-
tion 4, are consistent with the suggested structures. Thus, the mass
spectra display the correct molecular ion peaks for which the mea-
sured high resolution (HRMS) data are in good agreement with the
calculated values. DEPT and 2D (COSY, HMQC, HMBC) experiments
showed correlations that helped in the 1H- and 13C-signal assign-
ments to the different carbons and their attached, and/or neighbor-
ing hydrogens. For compounds 11a–f, long-range correlations are
observed between H-5 and each of C-8a, C-4 and C-7; likewise,
H-2 is correlated with C-8a, C-4, C-10, and CO2Et. For compounds
12a–e and 13a–e, similar long-range correlations are also observed
between H-5 and each of C-11b, C-4, and C-6a, as well as between
H-2 and each of C-11b, C-4, C-10, and CO2Et/CO2H. For compounds
12f and 13f similar correlations are likewise observed between
each of H-2, H-5, H-8, H-9, and the neighboring carbons. For com-
pounds 11–13, the carbons of the benzo-fused ring B are readily
identified by their doublet signals (with varying J-values) originat-
ing from coupling with the nearby fluorine atom at C-6.
2. Results and discussion
2.1. Synthesis
2.2. Biology
2.2.1. Antimicrobial activity
The antimicrobial activity of the novel heterocyclic compounds
13a–f was assayed against 16 standard microorganisms representa-
tive of Gram positive and Gram negative bacteria, yeasts, and molds.
Bearing in mind that the spread of resistant strains reduces the num-
ber of available chemotherapeutic agents, multidrug-resistant
bacterial species, such as clinical isolates of quinolone- and penicil-
lin-resistant S. aureus, Staphylococcus epidermidis, Acinetobacter
baumannii, Escherichia coli, and Pseudomonas aeruginosa, were in-
cluded in the investigation.
The inhibitory activity of 13a–f against bacteria is summarized
in Table 1, together with the results obtained for commercial quin-
olones ciprofloxacin, levofloxacin, moxifloxacin, and gemifloxacin,
which were used as reference drugs.
A generally applicable method for the synthesis of substituted
carbazoles is based on the phosphite-mediated Cadogan reductive
cyclization of the corresponding 2-nitrobiaryls.25–27 However, this
process demands drastic conditions and long reaction times, mak-
ing it a less-favored synthetic route. Recently, microwave irradia-
tion28–30 has emerged as a valuable tool in organic synthesis
whereby rate enhancements and improved yields are often attain-
able for a wide variety of reaction types performed under focused
conditions. In this context, microwave-enhanced Cadogan cycliza-
tion of nitro compounds, in combination with phosphite reagent,
has provided easy access to substituted carbazoles and other fused
heterocyclic systems.31 The requisite 2-nitrobiaryls and related
congeners are accessible via palladium-catalyzed cross-coupling
reactions of ortho-halonitroarenes with the appropriate organo-
metal partners, exemplified by aryl(trialkyl) stannanes32–34 of the
Stille reaction.35–43
Based on the above methodologies, the synthesis of the desired
6-fluoro-4-oxo-1,4-dihydropyrido[2,3-a]carbazole-3-carboxylic
acids (13a–e) and their ethyl esters (12a–e) was achieved by utiliz-
ing ethyl 7-chloro-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-
3-carboxylate (9)5,44,45 as the starting material, and constructing
the indole nucleus via a two-step procedure as illustrated in
Scheme 1. The first key-step involves palladium-catalyzed carbon–
carbon cross coupling between 9 and the appropriate aryltrimethyl
stannane (10a–e) (Stille reaction) with ultimate production of the
respective ethyl 7-aryl-8-nitro-4-oxo-1,4-dihydroquinoline-3-
carboxylate (11a–e). In a subsequent step, the latter compounds
undergo phosphite-mediated reductive cyclization (Cadogan
reaction) assisted by microwave irradiation to furnish the respec-
tive ethyl 4-oxo-4,11-dihydropyrido[2,3-a]carbazole-3-carboxyl-
ates 12a–e. Compound 12f is obtained through the same
procedure, using 2-thienyltrimethyl stannane (10f) (Scheme 2).34
This reaction entails the generation of a nitrene intermediate that
undergoes insertion into a 7-aryl C–H bond to form a fused ‘pyr-
role’ ring (C) embedded in the tetracyclic product. It is of note that
in absence of microwave irradiation, the phosphite-mediated nitro
group-deoxygenation step, leading to 12, does not proceed to any
detectable extent at 160 °C for 96 h. Acid-catalyzed hydrolysis of
the esters 12a–f produced the corresponding target 4-oxo-1,
All new compounds exhibited strong activity against Bacillus
subtilis, the most sensitive microorganism to the tested substances.
The minimum inhibitory concentrations (MIC) of 13a–d ranged
from 0.015 to 0.07
olones, while a higher effect was exerted by 13e and 13f (MIC
0.003 g/mL). In addition, 13a and, above all, 13e–f showed the
lg/mL and were close to those of standard quin-
l
best activity against wild staphylococci at the same concentrations
observed for the reference drugs. These bacteria were also signifi-
cantly inhibited by compounds 13c and 13d.
Of particular relevance is that compounds 13e and 13f had effec-
tive antibacterial properties against both multidrug-resistant
strains of S. aureus (SAR 72) and S. epidermidis with MIC values
1.5–12
lg/mL and 3–50
lg/mL, respectively. Compound 13a was
also a moderate inhibitor of SAR 72 at 12
lg/mL concentration. It is
worth noting that, against the above mentioned staphylococci, 13a
and 13f exhibited greater inhibitory activity than that of ciprofloxa-
cin and levofloxacin and comparable to that of moxifloxacin and
gemifloxacin, whereas 13e (MIC 1.5–3
lg/mL) was found to be more
potentthanall thereferencequinolones(MICfrom6to>100
lg/mL).
MIC values in Table 1 show that all the tested compounds exhib-
ited relatively better inhibition of Gram positive bacteria than Gram
negative ones. Among the latter, Haemophilus influenzae was found
to be the most sensitive (MIC 0.015–6 lg/mL) and it was inhibited
by 13a and 13e–f at the same concentrations of standard quino-
lones. A lower potency was exhibited by the above mentioned
compounds, relative to the standard quinolones, for the inhibition
of A. baumannii and E. coli and by 13f towards P. aeruginosa, while

5876
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
O
4
O
SnMe3
5
CO2Et
F
CO2Et
F
4a
8a
6
7
3
2
(i)
B
A
N
2"
(ii)
R2
R1
1"
+
3"
8
Cl
N
1
R2
R2
4"
1'
NO2
3'
6"
NO2
R1
5"
R2
2'
9
10a-e
11a-e
O
4
5
CO2R
4a
F
6
Compounds 10-13
3
2
B
A
6a
entry
a
b
c
d
e
7
6b
11b
N
1
11a
NH
C
8
R1
R2
H
Me
H
OMe OMe
Me
F
R2
1'
D
10a 11
H
H
H
3'
9
2'
10
R1
12a-e (R = Et
13a-e (R = H
R2
)
(iii)
)
Scheme 1. Reagents and conditions: (i) Pd(OAc)2, CsF, DMF, 70–100 °C; (ii) P(OEt)3, 200 °C (MW); (iii) 10% aq HCl, EtOH, reflux.
O
4
O
4
5
5
CO2R
4a
F
6
CO2R
4a
F
6
3
2
3
(ii)
(i)
6a
+
9
1
7
2
1
10b
6b
Me3Sn
N
8a
2''
10a
S
N
S
8
S
7
10
1'
NH
1'
NO2
8
9a
3''
10f
5''
2'
3'
9
3'
2'
4''
12f (R = Et)
13f (R = H
11f
(iii)
)
Scheme 2. Reagents and conditions: (i) Pd(OAc)2, CsF, DMF, 90 °C; (ii) P(OEt)3, 200 °C (MW); (iii) 10% aq HCl, EtOH, reflux.
no activity was noted against multidrug-resistant Gram negative
strains. Thus, 13f was the most active quinolone against all tested
microorganisms, with the exception of multidrug-resistant staphy-
lococci that were more significantly inhibited by 13e.
In order to reveal potential antifungal properties, the new com-
pounds were tested on Aspergillus niger, Candida tropicalis, and Sac-
charomyces cerevisiae, but no activity was observed at, or below,
conversely, the detrimental action of the electron-donating methyl
and methoxy groups (13b–d).
The structure–activity relationships analysis of the data reported
in Table 1 further shows that the thiophene isosteric replacement of
the D benzene moiety played a positive, crucial role in the antibac-
terial effectiveness of the tested compounds. Hence, 13f possessed
the highest inhibitory properties as compared to the corresponding
benzofused derivatives 13a–e. However, it is noteworthy that, in
the case of multidrug-resistant staphylococci, the presence of a flu-
oro substituted benzene ring (13e) contributed to an activity
enhancement, with respect to the thiophene analogue 13f.
the concentration of 100 lg/mL (data not shown).
Analysis of the structure–activity relationships shows that the
different substituents at ring (D), fused onto the pyrroloquinolone
scaffold, affect the antibacterial properties of the studied sub-
stances. Concerning the benzo-fused derivatives, bulky substitu-
ents on the benzene ring, such as a lipophilic methyl group or a
hydrophilic methoxy group, did not improve the antibacterial activ-
ity of the parent analogue 13a. In fact, MIC values of compound 13a
were always lower than those of compounds 13b–d. Among these,
the methyl derivative 13b was active only against B. subtilis. Better
activity was noted for the methoxy derivative 13c, which showed
excellent inhibition of all wild type Gram positive bacteria tested
and Gram negative H. influenzae and also a moderate effect against
A. baumannii. As expected, the introduction of two methyl groups in
13c, that produces 13d, led to decreased antibacterial activity. It is
worth noting that the most effective compounds contained a fluo-
rine atom. For instance, compound 13e, bearing fluorine in the para
position of the benzene ring, was the most active carbazole deriva-
tive. The electronic influence on the antibacterial activity of the se-
lected substituents in the series 13a–e, can be seen from the
favorable effect of the electron-withdrawing fluorine (13e) and,
2.2.2. DNA gyrase and topoisomerase IV inhibition
To elucidate the mechanism by which the novel tetracyclic flu-
oroquinolones induce antibacterial activity, the inhibitory activi-
ties of all the compounds 13a–f were examined against DNA
gyrase and topoisomerase IV isolated from E. coli (Table 2). Anti-
bacterial quinolones exert their activity by targeting Gram nega-
tive bacteria DNA gyrase and Gram positive bacteria
topoisomerase IV, and inhibiting DNA replication process.2 We
hypothesized that fluoroquinolones 13a–f could afford favorable
binding to the DNA–enzyme complex, but surprisingly we found
in most cases moderate inhibition of DNA gyrase (compounds
13a–c and 13e–f IC50 1.1–4.8
lg/mL) and a weak inhibition of
topoisomerase IV (compounds 13a and 13c–e IC50 28–48
lg/mL).
All of the tested compounds displayed higher IC50 values than
the reference ciprofloxacin and moxifloxacin, nevertheless most
of them, as reported below (Table 1), exhibit against Gram positive

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
5877
Table 1
Inhibitory activity against Gram positive and Gram negative bacteria, expressed as MIC (
l
g/mL)
Bacteriaa
Compoundb
13a
13b
13c
13d
13e
13f
CIP
LEV
MOX
GEM
Gram positive
BS
SA
SAR 72
SAR 4790
SE
0.015
0.3
12
>100
0.15
>100
0.07
0.07
3
>100
>100
3
0.07
6
>100
>100
25
0.003
0.07
1.5
>100
0.07
3
0.003
0.03
12
>100
0.03
50
0.03
0.3
100
>100
0.07
100
0.03
0.07
25
100
0.15
>100
0.015
0.03
12
50
0.07
50
0.007
0.015
6
50
0.015
50
>100
c
—
—
>100
—
SER
>100
>100
Gram negative
AB
ABR
EC
ECR
HI
PA
12
>100
50
>100
0.03
>100
—
>100
—
>100
—
>100
>100
—
50
>100
—
>100
—
6
>100
—
12
>100
25
>100
0.15
>100
—
3
>100
1.5
>100
0.015
50
0.7
0.15
>100
0.03
25
0.15
0.3
0.15
50
0.03
50
0.03
0.7
>100
0.3
100
>100
>100
—
1.5
>100
—
>100
0.015
100
0.15
0.07
25
0.015
50
0.015
0.07
>100
PAR
>100
50
a
BS, Bacillus subtilis ATCC 6633; SA, Staphylococcus aureus ATCC 6538; SAR 72 and SAR 4790, Staphylococcus aureus quinolone- and penicillin-resistant clinical isolates; SE,
Staphylococcus epidermidis ATCC 12228; SER, Staphylococcus epidermidis quinolone- and penicillin-resistant clinical isolate; AB, Acinetobacter baumannii ATCC 19606; ABR,
Acinetobacter baumannii quinolone- and penicillin-resistant clinical isolate; EC, Escherichia coli ATCC 8739; ECR, Escherichia coli quinolone- and penicillin-resistant clinical
isolate; HI, Haemophilus influenzae ATCC 19418; PA, Pseudomonas aeruginosa ATCC 9027; PAR, Pseudomonas aeruginosa quinolone- and penicillin-resistant clinical isolate.
b
CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin; GEM, gemifloxacin.
Not tested because inactive against the corresponding quinolone-sensitive microorganism.
c
Table 2
Table 3
Inhibitory activity against DNA gyrase and topoisomerase IV of E. coli, expressed as
Effects of compounds 13a–f and ellipticine on MCF-7, A549, and HuDE cell viability
50% inhibitory concentration (
lg/mL)
Compound
IC50 MCF-7 (
lM)
IC50 A549 (
lM)
IC50 HuDe (lM)
Compound
IC50
13a
13b
13c
13d
13e
13f
Ellipticine
3.6 1.09
>10
6.4 1.02
>10
4.9 1.01
>10
>10
>10
>10
>10
>10
>10
Gyrasea
Topoisomerase IVb
2.4 1.05
1.7 1.04
0.8 1.04
4.4 1.09
1.6 1.16
13°
13b
13c
13d
1.7
3.0
4.8
>30
1.8
35
>300
48
31
28
3
1.03
3.5 1.05
6.4 1.07
3.4 1.04
13e
13f
Ciprofloxacin
Moxifloxacin
1.1
0.34
0.85
100
4.6
5.0
Cells were treated with indicated compounds at concentrations ranging from 0.1 to
20 M for 72 h and then viability was determined by MTT assay. Concentration that
inhibits 50% (IC50) (e.g., the point at which viability is 50%) was extrapolated from
the dose–response curves. Representative results of at least three independent
experiments are reported.
l
Representative results of at least three independent experiments are reported.
a
E. coli DNA gyrase supercoiling assay.
E. coli topoisomerase IV decatenation assay.
b
decreased the antiproliferative activity on both cell lines (IC50
bacteria lower or comparable MICs in respect to that of the refer-
ence quinolones. Lack of correlation between the MICs and the
IC50s indicates that inhibition of bacterial cell growth, when
detected for compounds 13a–f, was not mainly caused by inhibi-
tion of the topoisomerase IV and suggests that other different
mechanisms could be involved in the antibacterial effect.
>10
with a hydrophilic and electron-donor methoxy group (to give
13c), resulted in increased potency (IC50 2.4 and 4.9 M against
MCF-7 and A549 cells, respectively), compared to 13a (IC50 3.6
and 6.4 M against MCF-7 and A549 cells, respectively). Compound
13e, bearing a fluoro substituent with electron-withdrawing prop-
erties, had a fourfold increased potency in breast cells (IC50 0.8 M)
compared to the unsubstituted analogue 13a (IC50 3.6 M) and was
even more active than the reference ellipticine (IC50 1.6 M). A sig-
nificantly increased activity was also observed for 13e against A549
cells (IC50 3.5 vs 6.4 M). Interestingly, the 4-methoxy-3,5-di-
methyl derivative 13d was more potent than the 4-methoxy substi-
tuted carbazole 13c, showing that the presence of two small
lipophilic and electron-donor methyl groups in the 3 and 5 posi-
tions was beneficial. This led us to speculate that the biological tar-
get responsible for the antiproliferative activity of the
fluoroquinolones/carbazoles under study has two additional drug-
binding sites, with two small hydrophobic pockets at the 3 to 5 ben-
zene positions distance.
lM). By contrast, substitution of the parent compound 13a
l
l
l
2.2.3. Antitumor activity
l
The antitumor activity of compounds 13a–f was assayed with
respect to ellipticine by evaluating cell proliferation in MCF-7
breast cancer and in A549 non-small cell lung cancer (NSCLC) cell
lines and cell cycle distribution, apoptosis induction and p53
expression in MCF-7 cell line.
l
l
After 72 h, all the tested compounds showed significant inhibi-
tion of cell proliferation in a dose-dependent manner (Table 3).
Ellipticine showed IC50 of 1.6
(13c–e) had comparable IC50 to the reference substance, ranging
between 0.8 and 2.4 M against MCF-7 cells and between 3 and
4.9 M against A549 cells. In contrast, compound 13b showed a re-
duced effect on cell proliferation against both cell lines (IC50
>10 M).
lM. The most interesting compounds
l
l
Replacement of the D benzene ring of compound 13a with its
heterocyclic bioisoster thiophene (13f) did not significantly modify
the antiproliferative activity, further evidence for the bioisosteric
equivalence between benzene and thiophene rings.
To examine the mechanism responsible for cell growth inhibi-
tion, cell cycle distribution was evaluated using flow cytometric
l
The structure–activity relationships analysis of the data
reported in Table 3 shows that among carbazole analogues 13a–e,
a lipophilic and electron-donor substituent, such as a methyl group
(13b) at the para position of the D benzene ring, significantly

5878
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
cells treated with 5 lM for 24 h were analyzed by flow cytometry.
As shown in Figure 3, both ellipticine and 13e caused a significant
increase in the proportion of cells in G2/M phase with a decrease in
G0/G1 phase.
Using fluorescence microscopy analysis with Hoechst 33342/PI
staining (Fig. 4), we demonstrated that treatment of MCF-7 cells
with ellipticine and with compound 13e at 5 lM for 48 h was asso-
ciated with the induction of cell death. Western blotting analysis
revealed that programmed cell death by apoptosis was involved
in the loss of viability. As shown in Figure 5, the activation and
cleavage of caspase 7 (MCF-7 cells are caspase 3 null) was detected
in MCF-7 cells treated with ellipticine and with 13e.
It has been reported that in MCF-7 ellipticine treatment
resulted in an increase of p53 and KIP1/p27.12 As shown by
Western blot analysis in Figure 6, ellipticine treatment induced
p53 accumulation in MCF-7 cells expressing wild type p53 protein.
In contrast, compound 13e induced only a modest increase of p53
protein, suggesting that other mechanisms were involved in its
antiproliferative effect.
Figure 3. Determination of cell cycle distribution. MCF-7 cells were incubated in
the absence or in the presence of 5 lM of ellipticine and compound 13e, and after
24 h cells were stained with propidium iodide and analyzed by flow cytometry for
cell cycle–phase distribution. N = 3. Values are means SD *P <0.05, **P <0.01, ***P
<0.001.
The compounds were also evaluated against normal human-
derm fibroblasts (HuDe) and, as shown in the Table 3, no effect
on cell proliferation and viability was detected until 10 lM indicat-
ing a selectivity of action, toward tumor cells coupled with a lack of
cytotoxicity towards normal cells.
In summary, the selective antiproliferative effect observed for
the fluoroquinolone 13e as a representative of the novel fluoroquin-
olones with a carbazole core (13a–e) or with a bioisosterically thie-
no related structure (13f) seems to be due to multiple mechanisms.
3. Conclusion
Novel 1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydropyrido[2,3-a]-
carbazole-3-carboxylic acids (13a–e) and the structurally related
1-cyclopropyl-6-fluoro-4-oxo-4,10-dihydro-1H-thieno[20,30:4,5]
pyrrolo[3,2-h]quinoline-3-carboxylic acid (13f) were synthesized
by efficient synthetic routes and their antibacterial and antiprolif-
erative activity assessed. The six tetracyclic fluoroquinolones
exhibited high antibacterial activity against Gram positive strains,
including a few resistant ones. Furthermore, most of them had high
antiproliferative activity against breast MCF-7 and lung A549 tu-
mor cell lines, and were devoid of cytoxicity against normal cells.
13e emerged as the most active antibacterial compound against
multidrug-resistant staphylococci and the most potent antiprolif-
erative compound against MCF-7 cells.
Figure 4. Determination of cell death after treatment with ellipticine and
compound 13e. MCF-7 cells were treated with ellipticine and compound 13e at
5 lM. Cell death was quantitated after 48 h by fluorescence microscopy on Hoechst
33342/PI stained cells. Data are expressed as percent values. Columns show means
of three separate experiments, bars SD. **P <0.01.
While the investigation at the molecular level identified only in
part the targets and the binding modes of these new molecules
need to be further elucidated, the combination of potent activity
against Gram positive bacteria and cancer cell lines, the absence
of cytoxicity against normal cells, make these agents of interest
in the search for potential cancer chemotherapeutics. These com-
pounds, possessing both anticancer and antibacterial activity, have
a promising therapeutic potential due to their selective cytoxicity
coupled with the ability to reduce the danger of bacterial infections
in the frequently immunocompromised cancer patient. This is sup-
ported by recent clinical data,49 demonstrating a positive effect of
quinolone antimicrobials on cancer patients’ survival, and makes
the multiple targeted approach, as here, a promising avenue for
drug development.
Figure 5. Cleavage of pro-caspase-7 after treatment with ellipticine and compound
13e. MCF-7 cells were incubated with each compound at 5 lM for 24 h. Cells lysates
were analyzed by Western blotting to assess the expression of pro-caspase-7, the
p30 fragment and actin proteins. Representative blot of three independent
experiments is reported.
Figure 6. Modulation of p53 protein expression during treatment with ellipticine
and compound 13e. MCF-7 cells were incubated with each compound at 5 lM for
24 h. Cells lysates were analyzed by Western blotting to assess the expression of
p53 and actin proteins. Representative blot of three independent experiments is
reported.
4. Experimental
4.1. Chemistry
2,4-Dichloro-5-fluoro-3-nitrobenzoic acid, ethyl 3-(N,N-
dimethylamino)acrylate, cyclopropylamine, palladium(II) acetate,
cesium fluoride, trimethyltin chloride, and triethyl phosphite were
analysis in MCF-7 cells. To determine the effect of ellipticine and
the most active compound 13e on MCF-7 cell cycle distribution,

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
5879
4
purchased from Acros. Bromobenzene, 4-bromotoluene, 4-bromo-
anisole, 4-bromofluorobenzene and 4-bromo-2,6-dimethylanisole
were purchased from Aldrich. Silica gel for column chromatogra-
phy was purchased from Macherey-Nagel GmbH & Co (Germany).
Melting points (uncorrected) were determined on a Stuart sci-
entific melting point apparatus in open capillary tubes. 1H and
13C NMR spectra were recorded on a 300 MHz spectrometer (Bru-
ker DPX-300) in DMSO-d6 or CDCl3 with TMS as the internal stan-
dard. Chemical shifts are expressed in d units; 1H–1H, H–F, and C–F
coupling constants are given in Hertz. Electron-impact mass spec-
tra (EIMS) were obtained using a Finnegan MAT TSQ-70 spectrom-
eter at 70 eV; ion source temperature: 200 °C. High resolution
mass spectra (HRMS) were measured by electrospray ionization
(ESI) technique on a Bruker APEX-IV instrument. The samples were
dissolved in acetonitrile, diluted in spray solution (methanol/water
1:1 v/v mixed with 0.1% formic acid) and infused using a syringe
(CO2Et), 171.3 (d, JC–F = 2 Hz, C-4); HRMS (ESI) m/z: calcd for
C
21H18FN2O5 [M+H]+: 397.11997. Found 397.11945; EIMS m/z
(%): 396 (M+, 34), 379 (70), 351 (42), 324 (72), 307 (78), 279
(96), 277 (100%), 264 (62), 251 (50), 222 (57), 208 (38), 203 (36),
176 (26), 133 (18), 120 (13).
4.1.2.2. Ethyl 1-cyclopropyl-6-fluoro-7-(4-methylphenyl)-8-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (11b). Yellow
solid (reaction temperature: 90 °C; reaction time: 10 h; ratio of the
eluting mixture: 1:5, v/v). Yield 0.78 g (68%); mp 217–219 °C. Anal.
Calcd for C22H19FN2O5 (410.40): C, 64.39; H, 4.67; N, 6.83. Found:
C, 64.29; H, 4.29; N, 6.46. IR (KBr)
1635, 1613, 1542, 1459, 1384, 1366, 1340, 1308, 1277, 1193,
1169, 1135, 1105, 1037, 1022, 951, 803, and 768 cmꢀ1 1H NMR
mmax: 3114, 3079, 2986, 1728,
;
(300 MHz, CDCl3): d 0.99–1.11 (m, 4H, 2 H-20/2 H-30), 1.40 (t,
J = 7.1 Hz, 3H, CH3CH2O–), 2.40 (s, 3H, C(400)–CH3), 3.58–3.67 (m,
1H, H-10), 4.40 (q, J = 7.1 Hz, 2H, –OCH2Me), 7.18 (d, J = 8.1 Hz,
2H, H-200/H-600), 7.27 (d, J = 8.1 Hz, 2H, H-300/H-500), 8.38 (d,
3JH–F = 8.5 Hz, 1H, H-5), 8.65 (s, 1H, H-2); 13C NMR (75 MHz,
CDCl3): d 10.9 (C-20/C-30), 14.4 (CH3CH2O–), 21.4 (C(400)–CH3),
pump with a flow rate of 2 lL/min. External calibration was con-
ducted using Arginine cluster in a mass range m/z 175–871. IR
spectra were recorded as KBr discs on a Nicolet Impact-400 FT-IR
spectrophotometer. Microwave irradiation experiments, con-
ducted for the Cadogan-nitrene insertion reactions, were carried
out with a Biotage Initiator 2.0 Microwave Synthesizer instrument.
Elemental analyses were performed on a Euro Vector Elemental
Analyzer (EA 3000 A).
2
37.8 (C-10), 61.4 (–OCH2Me), 111.7 (C-3), 115.6 (d, JC–F = 24.8 Hz,
C-5), 125.9 (C-100), 128.5 (C-200/C-600), 129.5 (C-8a), 129.6 (C-300/C-
2
3
500), 130.2 (d, JC–F = 23.2 Hz, C-7), 131.2 (d, JC–F = 6.8 Hz, C-4a),
1
139.9 (C-400), 141.6 (C-8), 151.7 (C-2), 156.1 (d, JC–F = 248 Hz, C-
4
6), 164.6 (CO2Et), 171.4 (d, JC–F = 2.0 Hz, C-4); HRMS (ESI) m/z:
calcd for C22H20FN2O5 [M+H]+: 411.13562. Found 411.13501; EIMS
m/z (%): 410 (M+, 31), 393 (70), 365 (42), 338 (66), 321 (89), 308
(62), 291 (100%), 278 (75), 265 (65), 236 (49), 222 (38), 216 (23),
183 (21), 133 (15), 118 (12).
4.1.1. Synthesisofethyl7-chloro-1-cyclopropyl-6-fluoro-8-nitro-
4-oxo-1,4-dihydroquinoline-3-carboxylate (9)
This compound was prepared from 2,4-dichloro-5-fluoro-3-
nitrobenzoic acid and ethyl 3-(N,N-dimethylamino)acrylate,
according to the literature procedure.5,44,45
4.1.2.3. Ethyl 1-cyclopropyl-6-fluoro-7-(4-methoxyphenyl)-8-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (11c). Yellow
solid (reaction temperature: 75 °C; reaction time: 10 h; ratio of
the eluting mixture: 1:5, v/v). Yield 0.75 g (62%); mp 207–208 °C.
Anal. Calcd for C22H19FN2O6 (426.40): C, 61.97; H, 4.49; N, 6.57.
4.1.2. General procedure for the synthesis of ethyl 7-aryl-1-cyclo-
propyl-6-fluoro-8-nitro-4-oxo-1,4-dihydroquinoline-3-carbox-
ylates (11a–e)
The
appropriate
aryltrimethyl
stannane
(10a–e)32,33
(3.2 mmol), Pd(OAc)2 (0.065 g, 0.29 mmol), and CsF (0.44 g,
2.9 mmol) were successively added to a stirred solution of 9
(2.8 mmol) in DMF (4 mL). The reaction mixture was heated at
70–100 °C under nitrogen atmosphere for 8–16 h. The resulting
solution was then cooled to rt and quenched with water (10 mL),
producing a black gummy precipitate. After filtration, the latter
crude product was dissolved in CHCl3, filtered, dried over anhy-
drous Na2SO4, and the solvent was evaporated under reduced pres-
sure to give a brown residue which was purified by column
chromatography using silica gel and eluting with chloroform/ethyl
acetate. The final product was crystallized from dichloromethane/
n-hexane.
Found: C, 61.57; H, 4.36; N, 6.20. IR (KBr)
2935, 1733, 1611, 1539, 1523, 1458, 1344, 1298, 1248, 1176,
1111, 1027, 836, 806, and 695 cmꢀ1 1H NMR (300 MHz, CDCl3):
mmax: 3092, 3012, 2989,
;
d
1.00–1.12 (m, 4H, 2
H-20/2 H-30), 1.40 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 3.59–3.67 (m, 1H, H-10), 3.84 (s, 3H, C(400)–OCH3),
4.39 (q, J = 7.1 Hz, 2H, –OCH2Me), 6.98 (d, J = 8.6 Hz, 2H, H-300/H-
3
500), 7.23 (d, J = 8.6 Hz, 2H, H-200/H-600), 8.37 (d, JH–F = 8.5 Hz, 1H,
13
H-5), 8.65 (s, 1H, H-2); C NMR (75 MHz, CDCl3): d 11.0 (C-20/C-
30), 14.4 (CH3CH2O–), 37.8 (C-10), 55.4 (C(400)–OCH3), 61.4
2
(–OCH2Me), 111.6 (C-3), 114.3 (C-300/C-500), 115.5 (d, JC–F
=
2
25.3 Hz, C-5), 120.7 (C-100), 129.9 (d, JC–F = 22.8 Hz, C-7), 130.1
3
(C-200/C-600), 131.1 (d, JC–F = 6.6 Hz C-4a), 131.2 (C-8a), 141.7 (d,
3JC–F = 1.4 Hz, C-8), 151.7 (C-2), 156.3 (d, JC–F = 248 Hz, C-6),
1
4.1.2.1. Ethyl 1-cyclopropyl-6-fluoro-8-nitro-4-oxo-7-phenyl-
4
160.7 (C-400), 164.6 (CO2Et), 171.4 (d, JC–F = 1.8 Hz, C-4); HRMS
1,4-dihydroquinoline-3-carboxylate (11a).
Yellow solid
(ESI) m/z: calcd for
C
22H20FN2O6 [M+H]+: 427.13054. Found
(reaction temperature: 100 °C; reaction time: 16 h; ratio of the
eluting mixture: 1:2, v/v). Yield 0.50 g (45%); mp 205–206 °C. Anal.
Calcd for C21H17FN2O5 (396.37): C, 63.63; H, 4.32; N, 7.07. Found:
427.12997; EIMS m/z (%): 426 (M+, 42), 409 (76), 368 (29), 354
(54), 337 (98), 308 (100%), 294 (80), 281 (63), 253 (48), 239 (30),
195 (24), 169 (18), 132 (16), 111 (13).
C, 63.21; H, 4.04; N, 6.83. IR (KBr)
1614, 1541, 1460, 1441, 1367, 1337, 1311, 1271, 1228, 1195,
1173, 1138, 1105, 1030, 855, 801, 760, and 698 cmꢀ1 1H NMR
mmax: 3098, 2993, 1729, 1638,
4.1.2.4. Ethyl 1-cyclopropyl-6-fluoro-7-(4-methoxy-3,5-dimeth-
ylphenyl)-8-nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate
(11d). Yellow solid (reaction temperature: 70 °C; reaction time:
12 h; ratio of the eluting mixture: 1:3, v/v). Yield 0.9 g (70%); mp
243–245 °C. Anal. Calcd for C24H23FN2O6 (454.45): C, 63.43; H,
;
(300 MHz, CDCl3): 1.01–1.11 (m, 4H, 2 H-20 2 H-30), 1.39 (t, 3H,
J = 7.1, CH3CH2O–), 3.59–3.66 (m, 1H, H-10), 4.38 (q, J = 7.1 Hz,
2H, –OCH2Me), 7.28 (dd, J = 7.0, 1.4 Hz, 2H, H-200/H-600), 7.44 (dd,
J = 6.0, 1.4 Hz, 1H, H-400), 7.46 (dd, J = 6.0, 7.0 Hz, 2H, H-300/H-500),
3
8.34 (d, JH–F = 8.5 Hz, 1H, H-5), 8.64 (s, 1H, H-2); 13C NMR
5.10; N, 6.16. Found: C, 63.09; H, 4.96; N, 5.96. IR (KBr) mmax
:
(75 MHz, CDCl3): d 10.9 (C-20/C-30), 14.4 (CH3CH2O–), 37.9 (C-10),
3073, 3008, 2970, 1731, 1613, 1599, 1536, 1487, 1453, 1365,
2
1341, 1311, 1258, 1231, 1198, 1175, 1134, 1110, 1050, 1028,
61.4 (–OCH2Me), 111.7 (C-3), 115.6 (d, JC–F = 24.8 Hz, C-5), 128.6
1010, 807, and 769 cmꢀ1 1H NMR (300 MHz, CDCl3): d 1.01–1.10
;
3
(C-400), 128.7 (C-200/C-600), 129.0 (d, JC–F = 6.5 Hz, C-4a), 129.7 (C-
2
(m, 4H, 2 H-20/2 H-30), 1.39 (t, J = 7.1 Hz, 3H, CH3CH2O–), 2.28 (s,
300/C-500), 130.0 (C-100), 130.2 (C-8a), 131.4 (d, JC–F = 22.5 Hz, C-7),
1
6H, C (300)–CH3/C(500)–CH3), 3.58–3.66 (m, 1H, H-10), 3.74 (s, 3H,
141.6 (C-8), 151.7 (C-2), 156.0 (d, JC–F = 249 Hz, C-6), 164.4

5880
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
C(400)–OCH3), 4.38 (q, J = 7.1 Hz, 2H, –OCH2Me), 6.92 (s, 2H, H-200/H-
4.1.3. General procedure for synthesis of ethyl 1-cyclopropyl-6-
fluoro-4-oxo-4,11-dihydro-1H-pyrido[2,3-a]carbazole-3-car-
boxylates (12a–e) and ethyl 1-cyclopropyl-6-fluoro-4-oxo-4,10-
dihydro-1H-thieno[20,30:4,5]pyrrolo[3,2-h]quinoline-3-
carboxylate (12f)
The appropriate ethyl 7-aryl-1-cyclopropyl-6-fluoro-8-nitro-4-
oxo-1,4-dihydroquinoline-3-carboxylate (11a–f) (0.55–0.85 mmol)
and P(OEt)3 (3–4 mL) were placed in a 10 mL biotage reactor glass
vial. The vial was sealed tightly with an aluminum Teflon crimpÒ
top and the mixture irradiated with microwaves (initial power
level, 400 W) for 1.5–2 h at a preselected temperature of 200 °C.
The vial was then cooled to 20 °C in a gas jet and excess P(OEt)3
was evaporated in vacuo; the residual solid was treated with CHCl3
(3 ꢁ 4 mL). The respective title products 12a–f were collected by
suction filtration and crystallized from DMF as light yellow solids.
3
600), 8.32 (d, JH–F = 8.5 Hz, 1H, H-5), 8.63 (s, 1H, H-2); 13C NMR
(75 MHz, CDCl3): d 1.0 (C-20/C-30), 14.4 (CH3CH2O–), 16.2 (s, C
(300)–CH3/C(500)–CH3), 37.9 (C-10), 59.8 (C(400)–OCH3), 61.4
2
(–OCH2Me), 111.6 (C-3), 114.7 (C-300/C-500), 115.4 (d, JC–F
=
3
25.1 Hz, C-5), 124.0 (C-100), 129.1 (C-200/C-600), 129.9 (d, JC–F = 2.8
Hz, C-8a), 130.1 (d, JC–F = 23.3 Hz, C-7), 131.1 (d, JC–F = 6.8 Hz,
2
3
1
C-4a), 141.5 (C-8), 151.7 (C-2), 156.1 d, JC–F = 248 Hz, C-6), 158.2
(C-400), 164.5 (CO2Et), 171.4 (C-4); HRMS (ESI) m/z: calcd for
C
24H24FN2O6 [M+H]+: 455.16184. Found 455.16130; EIMS m/z (%):
454 (M+, 34), 437 (50), 396 (22), 382 (40), 365 (60), 334 (46), 322
(64), 289 (52), 262 (80), 245 (94), 233 (86), 216 (100%), 203 (92),
176 (71), 162 (53), 133 (55), 120 (38), 107 (34).
4.1.2.5.
Ethyl
1-cyclopropyl-6-fluoro-7-(4-fluorophenyl)-8-
nitro-4-oxo-1,4-dihydroquinoline-3-carboxylate (11e). Yellow
solid (reaction temperature: 80 °C; reaction time: 16 h; ratio of the
eluting mixture: 1:2, v/v). Yield 0.4 g (34%); mp 224–226 °C. Anal.
Calcd for C21H16F2N2O5 (414.36): C, 60.87; H, 3.89; N, 6.76. Found:
4.1.3.1. Ethyl 1-cyclopropyl-6-fluoro-4-oxo-4,11-dihydro-1H-
pyrido[2,3-a]carbazole-3-carboxylate (12a).
Prepared from
11a (0.30 g, 0.75 mmol); irradiation time: 1.75 h. Yield 0.10 g
(36%); mp 278–280 °C. Anal. Calcd for C21H17FN2O3 (364.37): C,
69.22; H, 4.70; N, 7.69. Found: C, 69.05; H, 4.62; N, 7.53. IR (KBr)
C, 60.35; H, 3.72; N, 6.53. IR (KBr)
mmax: 3096, 3050, 3015, 2977,
1735, 1638, 1616, 1534, 1512, 1451, 1387, 1341, 1308, 1284, 1270,
1230, 1174, 1158, 1136, 1103, 1035, 832, 804, and 790 cmꢀ1 1H
;
mmax: 3481, 3072, 2970, 1738, 1718, 1606, 1568, 1486, 1448,
NMR (300 MHz, CDCl3): d 1.01–1.10 (m, 4H, 2 H-20/2 H-30), 1.40
(t, J = 7.1 Hz, 3H, CH3CH2O–), 3.57–3.66 (m, 1H, H-10), 4.39 (q,
J = 7.1 Hz, 2H, –OCH2Me), 7.16 (dd, 3JH–F = 8.5 Hz, J = 8.7 Hz, 2H, H-
1422, 1366, 1347, 1257, 1227, 1171, 1111, 1037, 802, 743 and
657 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.19–1.22 (m, 2H)
;
and 1.40–1.47 (m, 2H) (2 H-20/2 H-30), 1.27 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 4.21 (q, J = 7.1 Hz, 2H, –CH2Me), 4.44–4.53 (m, 1H,
H-10), 7.32 (ddd, J = 7.7, 7.5, 1.1 Hz, 1H, H-8), 7.55 (ddd, J = 8.2,
4
300/H-500), 7.28 (dd, JH–F = 3 Hz, J = 8.7 Hz, 2H, H-200/H-600), 8.38 (d,
3JH–F = 8.5 Hz, 1H, H-5), 8.65 (s, 1H, H-2); 13C NMR (75 MHz, CDCl3):
d 11.0 (C-20/C-30), 14.4 (CH3CH2O–), 37.9 (C-10), 61.5 (–OCH2Me),
3
7.7, 1.0 Hz, 1H, H-9), 7.66 (d, JH–F = 10.8 Hz, 1H, H-5), 7.80 (dd,
2
2
J = 8.2, 1.1 Hz, 1H, H-10), 8.15 (dd, J = 7.5, 1.0 Hz, 1H, H-7), 8.55
(s, 1H, H-2), 11.65 (s, 1H, N(11)–H); 13C NMR (75 MHz, DMSO-
d6): d 10.2 (C-20/C-30), 14.8 (CH3CH2O–), 38.2 (C-10), 60.3 (–CH2Me),
111.8 (C-3), 115.7 (d, JC–F = 24.8 Hz, C-5), 116.1 (d, JC–F = 22 Hz,
4
2
C-300/C-500), 124.7 (d, JC–F = 2.6 Hz, C-100), 129.0 (d, JC–F = 23.2 Hz,
4
3
C-7), 129.9 (d, JC–F = 2.7 Hz, C-8a), 130.7 (d, JC–F = 8.5 Hz, C-200/C-
2
3
600), 131.5 (d, JC–F = 6.8 Hz, C-4a), 141.7 (C-8), 151.7 (C-2), 156.0
100.6 (d, JC–F = 20.9 Hz, C-5), 109.8 (C-3), 113.0 (C-10), 115.3 (d,
3
2JC–F = 23.1 Hz, C-6a), 118.8 (d, JC–F = 2.3 Hz, C-6b), 121.2 (C-8),
(d, 1JC–F = 249 Hz, C-6), 163.6 (d, 1JC–F = 249 Hz, C-400), 164.4 (CO2Et),
171.2 (d, 4JC–F 2.1 Hz, C-4); HRMS (ESI) m/z: calcd for C21H17F2N2O5
[M+H]+: 415.11055. Found 415.11001; EIMS m/z (%): 414 (M+, 28),
397 (62), 369 (42), 342 (78), 325 (89), 313 (86), 296 (100%), 282
(69), 269 (60), 240 (67), 226 (52), 201 (42), 188 (33), 175 (25), 133
(18), 120 (13).
4
122.4 (d, J = 3.9 Hz, C-7), 126.7 (d, JC–F = 1.7 Hz, C-11b), 127.5 (C-
3
3
9), 127.7 (d, JC–F = 6.9 Hz, C-4a), 130.8 (d, JC–F = 10.0 Hz, C-11a),
1
140.5 (C-10a), 148.7 (C-2), 155.7 (d, JC–F = 244 Hz, C-6), 164.9
4
(CO2Et), 172.3 (d, JC–F = 2.1 Hz, C-4); HRMS (ESI) m/z: calcd for
C
21H18FN2O3 [M+H]+): 365.13015. Found: 365.12949; EIMS m/z
(%): 364 (M+, 30), 319 (7), 292 (100%), 291 (30), 263 (21), 236
(14), 221 (13), 131 (6).
4.1.2.6. Ethyl 1-cyclopropyl-6-fluoro-8-nitro-4-oxo-7-(thien-2-
yl)-1,4-dihydroquinoline-3-carboxylate (11f). This compound
was prepared from 2-thienyltrimethyl stannane (10f)34 (0.8 g,
3.24 mmol) and 9 (1.0 g, 2.82 mmol) by following similar proce-
dure described above for compounds 11a–e. Yellow solid (reaction
temperature: 90 °C; reaction time: 8 h; ratio of the eluting mixture
1:1, v/v). Yield 0.45 g (40%); mp: 193–195 °C. Anal. Calcd for
4.1.3.2. Ethyl 1-cyclopropyl-6-fluoro-9-methyl-4-oxo-4,11-dihy-
dro-1H-pyrido[2,3-a] carbazole-3-carboxylate (12b). Prepared
from 11b (0.25 g, 0.61 mmol); irradiation time: 1.5 h. Yield 0.14 g
(61%); mp 293–295 °C. Anal. Calcd for C22H19FN2O3 (378.40): C,
69.83; H, 5.06; N, 7.40. Found: C, 69.58; H, 4.93; N, 7.24. IR (KBr)
mmax: 3210, 2970, 1738, 1721, 1633, 1597, 1563, 1486, 1453,
C
19H15FN2O5S (402.40): C, 56.71; H, 3.76; N, 6.96. Found: C,
56.55; H, 3.52; N, 6.57. IR (KBr) max: 3107, 2989, 1728, 1638,
1612, 1542, 1459, 1386, 1366, 1332, 1314, 1267, 1241, 1173,
1366, 1347, 1323, 1233, 1173, 1114, 1039, 861, 801, and
m
633 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.18–1.22 (m, 2H)
;
and 1.40–1.46 (m, 2H) (2 H-20/2 H-30), 1.27 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 2.51 (s, 3H, C(9)–CH3), 4.21 (q, J = 7.1 Hz, 2H, –
OCH2Me), 4.41–4.51 (m, 1H, H-10), 7.14 (d, J = 8 Hz, 1H, H-8), 7.58
1135, 1099, 1046, 1032, 820, 801, 715, 693 cmꢀ1
;
1H NMR
(300 MHz, CDCl3): d 1.04–1.13 (m, 4H, 2 H-20/2 H-30), 1.39 (t,
J = 7.1 Hz, 3H, CH3CH2O–), 3.58–3.67 (m, 1H, H-10), 4.38 (q,
J = 7.1 Hz, 2H, –OCH2Me), 7.13 (m, 2H, H-300 + H-400), 7.56 (m, 1H,
3
(s, 1H, H-10), 7.64 (d, JH–F = 10.8 Hz, 1H, H-5), 8.01 (d, J = 8 Hz,
3
1H, H-7), 8.53 (s, 1H, H-2), 11.46 (s, 1H, N(11)–H); 13C NMR
H-500), 8.36 (d, JH–F = 8.6 Hz, 1H, H-5), 8.64 (s, 1H, H-2); 13C NMR
(75 MHz, DMSO-d6): d 10.2 (C-20/C-30), 14.8 (CH3CH2O–), 22.3
(75 MHz, CDCl3): d 11.1 (C-20/C-30), 14.4 (CH3CH2O–), 37.8 (C-10),
2
2
(C(9)–CH3), 38.2 (C-10), 60.3 (–OCH2Me), 100.6 (d, JC–F = 20.9 Hz,
61.5 (–OCH2Me), 111.6 (C-3), 115.5 (d, JC–F = 24.8 Hz, C-5), 123.5
2
2
C-5), 109.8 (C-3), 112.6 (C-10), 115.5 (d, JC–F = 22.1 Hz, C-6a),
(d, JC–F = 29 Hz, C-7), 127.0 (C-200), 127.4, 130.1 (C-300/C-400),
3
4
3
116.7 (d, JC–F = 2.3 Hz, C-6b), 122.1 (d, J = 2.4 Hz, C-7), 122.9
129.4 (C-500), 130.0 (d, JC–F = 2.9 Hz, C-8a), 131.8 (d, JC–F = 6.9 Hz,
4
3
1
(C-8), 126.6 (d, JC–F = 1.6 Hz, C-11b), 127.4 (d, JC–F = 8 Hz, C-4a),
C-4a), 142.0 (C-8), 151.8 (C-2), 156.2 (d, JC–F = 250 Hz, C-6),
3
130.7 (d, JC–F = 10.2 Hz, C-11a), 137.3 (C-9), 141.0 (C-10a), 148.5
164.4 (CO2Et), 171.2 (C-4); HRMS (ESI) m/z: calcd for C19H16FN2O5S
[M+H]+: 403.0764. Found 403.07586; EIMS m/z (%): 402 (M+, 70),
385 (79), 357 (51), 344 (57), 330 (100%), 313 (91), 284 (81), 272
(54), 246 (51), 214 (47), 202 (24), 189 (17), 145 (14), 132 (13),
105 (8).
1
(C-2), 155.5 (d, JC–F = 243 Hz, C-6), 165.0 (CO2Et), 172.3 (d,
4JC–F = 2.1 Hz, C-4); HRMS (ESI) m/z: calcd for C22H20FN2O3
[M+H]+: 379.14581. Found: 379.14524; EIMS m/z (%): 378 (M+,
37), 333 (9), 307 (22), 306 (100%), 277 (20), 250 (12), 221 (8).

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
5881
4.1.3.3. Ethyl 1-cyclopropyl-6-fluoro-9-methoxy-4-oxo-4,11-
dihydro-1H-pyrido[2,3-a]carbazole-3-carboxylate (12c). Pre-
pared from 11c (0.30 g, 0.70 mmol); irradiation time: 1 h. Yield
20/C-30), 14.8 (CH3CH2O–), 38.2 (C-10), 60.3 (–OCH2Me), 99.2 (d,
2
2JC–F = 26.4 Hz, C-10), 101.1 (d, JC–F = 20.7 Hz, C-5), 109.7 (d,
2
2JC–F = 24.5 Hz, C-8), 109.8 (C-3), 115.1 (d, JC–F = 25.9 Hz, C-6a),
3
3
0.13 g (47%); mp 286–288 °C. Anal. Calcd for
(394.40): C, 67.00; H, 4.86; N, 7.10. Found: 66.76; H, 4.57; N, 6.82.
IR (KBr) max; 3471, 3076, 2989, 2934, 2836, 1709, 1636, 1607,
1571, 1488, 1451, 1422, 1342, 1322, 1274, 1233, 1196, 1165,
1140, 1109, 1066, 1037, 801 and 636 cmꢀ1 1H NMR (300 MHz,
C
22H19FN2O4
115.7 (d, JC–F = 2.9 Hz, C-6b), 124.1 (dd, JC–F = 10.2 Hz, J = 3.8 Hz,
4
3
C-7), 126.6 (d, JC–F = 1.6 Hz, C-11b), 127.5 (d, JC–F = 7.0 Hz, C-4a),
3
3
m
131.6 (d, JC–F = 8.9 Hz, C-11a), 141.3 (d, JC–F = 11.4 Hz, C-10a),
1
1
148.7 (C-2), 155.3 (d, JC–F = 244 Hz, C-6), 162.1 (d, JC–F = 240 Hz,
4
;
C-9), 164.9 (CO2Et), 172.3 (d, JC–F = 2 Hz, C-4); HRMS (ESI) m/z:
DMSO-d6): d 1.18–1.25 (m, 2H) and 1.41–1.48 (m, 2H) (2 H-20/2
H-30), 1.26 (t, J = 7.1 Hz, 3H, CH3CH2O–), 3.86 (s, 3H, C(9)–OCH3),
4.18 (q, J = 7.1 Hz, 2H, –OCH2Me), 4.36–4.46 (m, 1H, H-10), 6.91
(dd, J = 8.6, 2 Hz, 1H, H-8), 7.25 (d, J = 2.1 Hz, 1H, H-10), 7.58 (d,
3JH–F = 10.8 Hz, 1H, H-5), 7.95 (d, J = 8.6 Hz, 1H, H-7), 8.48 (s, 1H,
H-2), 11.47 (s, 1H, N(11)–H); 13C NMR (75 MHz, DMSO-d6): d 10.2
(C-20/C-30), 14.8 (CH3CH2O–), 38.2 (C-10), 55.9 (C(9)–OCH3), 60.3
(–OCH2Me), 95.5 (C-10), 100.8 (d, 2JC–F = 20.9 Hz, C-5), 109.6 (C-3),
calcd for C21H17F2N2O3 [M+H]+: 383.12072. Found: 383.12023;
EIMS m/z (%): 382 (M+, 26), 316 (11), 310 (100%), 281 (25), 205
(17), 149 (38), 85 (32), 71 (62).
4.1.3.6. Ethyl 1-cyclopropyl-6-fluoro-4-oxo-4,10-dihydro-1H-thi-
eno[20,30:4,5]pyrrolo[3,2-h]quinoline-3-carboxylate (12f). Pre-
pared from 11f (0.25 g, 0.62 mmol); irradiation time: 1.5 h. Yield
0.11 g (48%); mp: 301–303 °C. Anal. Calcd for
(370.40): C, 61.61; H, 4.08; N, 7.56. Found: C, 61.52; H, 4.23; N,
7.36. IR (KBr) max; 3337, 3086, 2971, 2941, 1714, 1629, 1597,
1570, 1521, 1486, 1448, 1366, 1333, 1234, 1195, 1175, 1102, 1039,
C19H15FN2O3S
3
2
111.4 (C-8), 112.7 (d, JC–F = 2.0 Hz, C-6b), 115.7 (d, JC–F = 23.4 Hz,
4
C-6a), 123.3 (d, J = 3.9 Hz, C-7), 126.4 (d, JC–F = 1.6 Hz, C-11b),
126.7 (d, JC–F = 6.2 Hz, C-4a), 130.4 (d, JC–F = 10.4 Hz, C-11a),
142.1 (C-10a), 148.6 (C-2), 155.2 (d, JC–F = 242 Hz, C-6), 159.9
m
3
3
1
801, 741 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.18–1.22 (m, 2H)
;
4
and 1.38–1.45 (m, 2H) (2 H-20/2 H-30), 1.26 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 4.20 (q, J = 7.1 Hz, 2H, –OCH2Me), 4.32–4.42 (m, 1H, H-
(C-9), 165.0 (CO2Et), 172.4 (d, JC–F = 2.1 Hz, C-4); HRMS (ESI) m/z:
calcd for C22H20FN2O4 [M+H]+: 395.14071. Found: 395.13998; EIMS
m/z (%): 394 (M+, 26), 349 (9), 323 (22), 322 (100%), 293 (16), 279
(12), 209 (6).
3
10), 7.32 (d, J = 5.2 Hz, 1H, H-9), 7.62 (d, JH–F = 10.5 Hz, 1H, H-5),
7.82 (d, J = 5.2 Hz, 1H, H-8), 8.50 (s, 1H, H-2), 11.89 (s, 1H, N(10)–
H); 13C NMR (75 MHz, DMSO-d6):
d
10.2 (C-20/C-30), 14.8
2
(CH3CH2O–), 38.1 (C-10), 60.3 (–OCH2Me), 100.3 (d, JC–F = 20.3 Hz,
4.1.3.4. Ethyl 1-cyclopropyl-6-fluoro-9-methoxy-8,10-dimethyl-
4-oxo-4,11-dihydro-1H-pyrido[2,3-a]carbazole-3-carboxylate
(12d). Prepared from 11d (0.25 g, 0.55 mmol); irradiation time:
2 h. Yield 0.12 g (52%); mp 233–235 °C. Anal. Calcd for
C-5), 109.9 (C-3), 113.2 (C-9), 115.1 (d, 2JC–F = 26.1 Hz, C-6a), 125.2
3
(d, JC–F = 6.0 Hz, C-4a), 127.7 (C-10b), 129.1 (C-10a), 131.2 (d,
3JC–F = 10.4 Hz, C-6b), 131.9 (C-8), 146.4 (C-9a), 148.2 (C-2), 152.7
(d, 1JC–F = 242 Hz, C-6), 165.0 (CO2Et), 172.3 (C-4); HRMS (ESI) m/z:
calcd for C19H16FN2O3S [M+H]+: 371.08657. Found: 371.08598; EIMS
m/z (%): 370 (M+, 27), 356 (8), 325 (16), 298 (100%), 269 (19), 242
(12), 228 (14).
C
24H23FN2O4 (422.45): C, 68.24; H, 5.49; N, 6.63. Found: C, 68.22;
H, 5.37; N, 6.56. IR (KBr) max; 3555, 3478, 3092, 2970, 2932,
1723, 1632, 1614, 1571, 1484, 1447, 1423, 1407, 1366, 1338,
1227, 1170, 1109, 1054, 1031, 1004 and 802 cmꢀ1 1H NMR
m
;
(300 MHz, DMSO-d6): d 1.21–1.27 (m, 2H) and 1.31–1.41 (m, 2H)
(2 H-20/2 H-30), 1.26 (t, J = 7.1 Hz, 3H, CH3CH2O–), 2.37 (s, 3H,
C(8)–CH3), 2.56 (s, 3H, C(10)–CH3), 3.74 (s, 3H, C(9)–OCH3), 4.19
(q, J = 7.1 Hz, 2H, –OCH2Me), 4.70–4.80 (m, 1H, H-10), 7.60 (d,
3JH–F = 10.7 Hz, 1H, H-5), 7.76 (s, 1H, H-7), 8.53 (s, 1H, H-2), 10.45
4.1.4. General procedure for synthesis of 1-cyclopropyl-6-fluoro-
4-oxo-4,11-dihydro-1H-pyrido[2,3-a]carbazole-3-carboxylic acids
(13a–e) and 1-cyclopropyl-6-fluoro-4-oxo-4,10-dihydro-1H-thi-
eno[20,30:4,5]pyrrolo[3,2-h]quinoline-3-carboxylic acid (13f)
A suspension of the appropriate ester 12a–f (1 mmol) in 10% aq
HCl (8 mL) and ethanol (10 mL) was refluxed for 20–24 h. The
solvents were evaporated in vacuo from the reaction mixture and
the residual solid product was washed with methanol (3–5 mL),
collected by in vacuo filtration, dried and crystallized from
DMSO.
13
(s, 1H, N(11)–H); C NMR (75 MHz, DMSO-d6): d 10.4 (C-20/C-30),
11.2 (C(10)–CH3), 14.8 (CH3CH2O–), 17.0 (C(8)–CH3), 37.8 (C-10),
2
60.3 (–OCH2Me), 60.6 (C(9)–OCH3), 101.1 (d, JC–F = 20.6 Hz, C-5),
3
109.7 (C-3), 114.4 (C-10), 115.2 (d, JC–F = 1.8 Hz, C-6b), 116.1 (d,
2JC–F = 23.4 Hz, C-6a), 121.0 (d, J = 3.8 Hz, C-7), 125.2 (C-8), 126.6
4
3
(d, JC–F = 1.5 Hz, C-11b), 127.2 (d, JC–F = 6.2 Hz, C-4a), 131.2 (d,
3JC–F = 10.4 Hz, C-11a), 139.6 (C-10a), 148.8 (C-2), 155.4 (d,
1JC–F = 244 Hz, C-6), 156.7 (C-9), 165.0 (CO2Et), 172.3 (d, JC–F
=
4
2 Hz, C-4); HRMS (ESI) m/z: calcd for C24H24FN2O4 [M + H]+:
423.17201. Found: 423.17146; HRMS (ESI): found [M+H]+, re-
quires; EIMS m/z (%): 422 (M+, 33), 396 (10), 382 (20), 365 (30),
350 (100%), 335 (41), 321 (40), 307 (26), 294 (21), 279 (18), 237
(12), 210 (8), 175 (10), 146 (8).
4.1.4.1.
1-Cyclopropyl-6-fluoro-4-oxo-4,11-dihydro-1H-pyr-
ido[2,3-a]carbazole-3-carboxylic acid (13a).
(95%); mp: 341–342 °C (dec). Anal. Calcd for
Yield 0.32 g
19H13FN2O3
C
(336.32): C, 67.86; H, 3.90; N, 8.33. Found: C, 67.62; H, 3.75; N,
8.21. IR (KBr) max; 3335, 1703, 1637, 1597, 1572, 1514, 1481,
m
1455, 1339, 812, 750, 721 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
;
1.28–1.32 (m, 2H) and 1.46–1.52 (m, 2H) (2 H-20/2 H-30), 4.59–
4.1.3.5. Ethyl 1-cyclopropyl-6,9-difluoro-4-oxo-4,11-dihydro-
4.69 (m, 1H, H-10), 7.36 (dd, J = 7.6, 7.3 Hz, 1H, H-8), 7.60 (dd,
1H-pyrido[2,3-a]carbazole-3-carboxylate
(12e).
Prepared
3
J = 7.8, 7.3 Hz, 1H, H-9), 7.75 (d, JH–F = 10.3 Hz, 1H, H-5), 7.86 (d,
from 11e (0.35 g, 0.85 mmol); irradiation time: 2 h. Yield 0.13 g
(40%); mp 291–293 °C. Anal. Calcd for C21H16F2N2O3 (382.36): C,
65.97; H, 4.22; N, 7.33. Found: C, 65.69; H, 4.03; N, 7.12. IR (KBr)
J = 7.8 Hz, 1H, H-10), 8.18 (d, J = 7.6 Hz, 1H, H-7), 8.79 (s, 1H, H-2),
11.97 (s, 1H, N(11)–H), 15.37 (s, 1H, CO2H); 13C NMR (75 MHz,
DMSO-d6): d 10.2 (C-20/C-30), 39.5 (C-10), 99.7 (d, 2JC–F = 21.4 Hz, C-
m
max; 3549, 3473, 3401, 3073, 2985, 2922, 1706, 1634, 1606,
2
5), 107.3 (C-3), 113.3 (C-10), 116.3 (d, JC–F = 22.5 Hz, C-6a), 118.6
1569, 1485, 1449, 1423, 1341, 1227, 1172, 1107, 1034, 869, 802
3
and 636 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.18–1.25 (m,
;
(d, JC–F = 4.6 Hz, C-6b), 121.6 (C-8), 122.6 (d, J = 3.9 Hz, C-7),
124.6 (d, 3JC–F = 8.4 Hz, C-4a), 127.9 (d, 4JC–F = 1.4 Hz, C-11b), 128.2
2H) and 1.39–1.48 (m, 2H) (2 H-20/2 H-30), 1.27 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 4.21 (q, J = 7.1 Hz, 2H, –OCH2Me), 4.40–4.48 (m, 1H,
3
(C-9), 130.6 (d, JC–F = 10.7 Hz, C-11a), 140.5 (C-10a), 148.4 (C-2),
1
4
3
H-10), 7.16 (ddd, JH–F = 10 Hz, J = 8.6, 2.1 Hz, 1H, H-8), 7.51 (dd,
156.3 (d, JC–F = 247 Hz, C-6), 166.4 (CO2H), 177.3 (d, JC–F = 3 Hz,
C-4); HRMS (ESI) m/z: calcd for C19H12FN2O3 [MꢀH]+: 335.08374.
Found: 335.08417; EIMS m/z (%): 336 (M+, 18), 292 (100%), 263
(31), 236 (24), 223 (28), 222 (12), 196 (9), 130 (6).
3
3JH–F = 10 Hz, J = 2.1 Hz, 1H, H-10), 7.66 (d, JH–F = 10.7 Hz, 1H, H-
4
5), 8.13 (dd, J = 8.6 Hz, JH–F = 5.4 Hz, 1H, H-7), 8.53 (s, 1H, H-2),
11.72 (s, 1H, N(11)–H); 13C NMR (75 MHz, DMSO-d6): d 10.2 (C-

5882
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
4.1.4.2. 1-Cyclopropyl-6-fluoro-9-methyl-4-oxo-4,11-dihydro-
1H-pyrido[2,3-a]carbazole-3-carboxylic acid (13b). Yield
0.33 g (94%); mp: 340–342 °C. Anal. Calcd for 20H15FN2O3
(350.35): C, 68.57; H, 4.32; N, 7.99. Found: C, 68.41; H, 4.24; N,
7.86. IR (KBr) max; 3339, 3016, 2970, 1738, 1634, 1594, 1566,
1519, 1441, 1367, 1338, 1229, 1217, 1124, 1034, 855, 799, 767,
4.1.4.5. 1-Cyclopropyl-6,9-difluoro-4-oxo-4,11-dihydro-1H-pyr-
ido[2,3-a]carbazole-3-carboxylic acid (13e).
(93%); mp: 320–322 °C (dec). Anal. Calcd for
Yield 0.33 g
C19H12F2N2O3
C
(354.31): C, 64.41; H, 3.41; N, 7.90. Found: C, 64.22; H, 3.35; N,
7.78. IR (KBr) max: 3317, 1695, 1641, 1597, 1575, 1510, 1477,
1441, 1423, 1332, 1310, 1219, 1107, 1034, 801 cmꢀ1 1H NMR
m
m
;
740, 643 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): d 1.25–1.29 (m,
(300 MHz, DMSO-d6): d 1.27–1.32 (m, 2H) and 1.47–1.54 (m, 2H)
3
2H) and 1.47–1.53 (m, 2H) (2 H-20/2 H-30), 2.52 (s, 3H, C(9)–CH3),
(2 H-20/2 H-30), 4.73–4.84 (m, 1H, H-10), 7.22 (dd, JH–F = 9 Hz,
3
4.63–4.73 (m, 1H, H-10), 7.19 (d, J = 7.9 Hz, 1H, H-8), 7.67 (s, 1H,
J = 8.3 Hz, 1H, H-8), 7.55 (d, JH–F = 10 Hz, 1H, H-10), 7.73 (d,
3
4
H-10), 7.73 (d, JH–F = 10.3 Hz, 1H, H-5), 8.05 (d, J = 7.9 Hz, 1H, H-
3JH–F = 10.2 Hz, 1H, H-5), 8.16 (dd, J = 8.3 Hz, JH–F = 5.5 Hz, 1H, H-
7), 8.77 (s, 1H, H-2), 11.85 (s, 1H, N(11)–H), 15.34 (s, 1H, CO2H);
7), 8.78 (s, 1H, H-2), 11.98 (br s, 1H, N(11)–H), 15.28 (s, 1H,
13
13C NMR (75 MHz, DMSO-d6): d 10.2 (C-20/C-30), 22.4 (C(9)–CH3),
CO2H); C NMR (75 MHz, DMSO-d6): d 10.2 (C-20/C-30), 39.4 (C-
2
2
2
39.3 (C-10), 99.7 (d, JC–F = 21.2 Hz, C-5), 107.4 (C-3), 112.9 (C-10),
10), 99.3 (d, JC–F = 26.2 Hz, C-10), 100.3 (d, JC–F = 21.2 Hz, C-5),
2
2
116.4 (d, JC–F = 18.5 Hz, C-6a), 116.6 (C-6b), 122.2 (d, J = 3.5 Hz,
107.4 (C-3), 110.4 (d, JC–F = 24.8 Hz, C-8), 115.5 (C-6b), 116.1 (d,
3
3
C-7), 123.4 (C-8), 124.3 (d, JC–F = 8.4 Hz, C-4a), 127.8 (C-11b),
2JC–F = 23.6 Hz, C-6a), 124.4 (d, JC–F = 8.4 Hz, C-4a), 124.5 (dd,
3
3
130.5 (d, JC–F = 10.6 Hz, C-11a), 138.1 (C-9), 141.2 (C-10a), 148.3
3JC–F = 8.5 Hz, J = 3 Hz, C-7), 127.8 (C-11b), 131.2 (d, JC–F
=
1
3
(C-2), 156.1 (d, JC–F = 247 Hz, C-6), 166.4 (CO2H), 177.3 (d,
11.2 Hz, C-11a), 141.2 (d, JC–F = 13.2 Hz, C-10a), 148.6 (C-2),
155.9 (d, JC–F = 247 Hz, C-6), 162.4 (d, JC–F = 241 Hz, C-9), 166.3
4JC–F = 3 Hz, C-4); HRMS (ESI) m/z: calcd for C20H16FN2O3 [M+H]+:
351.11450. Found: 351.11399; EIMS m/z (%): 350 (M+, 22), 306
(100%), 291 (38), 277 (36), 237 (30), 210 (10), 145 (9).
1
1
4
(CO2H), 177.3 (d, JC–F = 2.3 Hz, C-4); HRMS (ESI) m/z: calcd for
C
19H11F2N2O3 [MꢀH]+: 353.07432. Found: 353.07485; EIMS m/z
(%): 354 (M+, 10), 310 (100%), 295 (25), 281 (39), 254 (32), 241
(39), 214 (13), 200 (9), 141 (6).
4.1.4.3. 1-Cyclopropyl-6-fluoro-9-methoxy-4-oxo-4,11-dihydro-
1H-pyrido[2,3-a]carbazole-3-carboxylic acid (13c).
Yield
0.35 g (96%); mp: 325–326 °C (dec). Anal. Calcd for C20H15FN2O4
(366.35): C, 65.57; H, 4.13; N, 7.64. Found: C, 65.41; H, 4.24; N,
4.1.4.6. 1-Cyclopropyl-6-fluoro-4-oxo-4,10-dihydro-1H-thieno-
[20,30:4,5]pyrrolo[3,2-h]quinoline-3-carboxylic acid (13f). Yield
7.86. IR (KBr)
m
max: 3324, 3106, 3088, 3000, 2953, 2829, 1703,
0.31 g (91%); mp: 356–357 °C. Anal. Calcd for
(342.35): C, 59.64; H, 3.24; N, 8.18. Found: C, 59.45; H, 3.11; N,
7.89. IR (KBr) max: 3346, 3106, 3091, 3011, 1724, 1630, 1550,
1517, 1470, 1445, 1354, 1328, 1245, 1216, 1186, 1092, 1041, 863,
C17H11FN2O3S
1637, 1597, 1568, 1525, 1452, 1426, 1343, 1314, 1270, 1230,
1205, 1172, 1110, 1034, 834, 801, 743 cmꢀ1
;
1H NMR (300 MHz,
m
DMSO-d6): d 1.29–1.34 (m, 2H) and 1.47–1.54 (m, 2H) (2 H-20/2
H-30), 3.88 (s, 3H, –OCH3), 4.61–4.71 (m, 1H, H-10), 6.99 (d,
710, 667 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.25–1.30 (m, 2H)
;
3
J = 8.6 Hz, 1H, H-8), 7.31 (s, 1H, H-10), 7.75 (d, JH–F = 10.4 Hz, 1H,
and 1.47–1.53 (m, 2H) (2 H-20/2 H-30), 4.51–4.61 (m, 1H, H-10),
3
H-5), 8.05 (d, J = 8.6 Hz, 1H, H-7), 8.79 (s, 1H, H-2), 11.77 (s, 1H,
N(11)–H), 15.38 (s, 1H, CO2H); 13C NMR (75 MHz, DMSO-d6): d
10.2 (C-20/C-30), 39.4 (C-10), 55.9 (C(9)–OCH3), 95.5 (C-10), 100.0
7.38 (d, J = 5.2 Hz, 1H, H-9), 7.71 (d, JH–F = 10.1 Hz, 1H, H-5), 7.89
(d, J = 5.2 Hz, 1H, H-8), 8.74 (s, 1H, H-2), 12.25 (s, 1H, N(10)–H),
13
15.50 (s, 1H, CO2H); C NMR (75 MHz, DMSO-d6): d 10.2 (C-20/C-
2
3
2
(d, JC–F = 21.5 Hz, C-5), 107.2 (C-3), 112.1 (C-8), 112.5 (d, JC–F
=
30), 39.3 (C-10), 99.4 (d, JC–F = 20.5 Hz, C-5), 107.4 (C-3), 113.4 (C-
2
2
3
1.5 Hz, C-6b), 116.9 (d, JC–F = 23.4 Hz, C-6a), 123.5 (d, J = 3.9 Hz,
9), 116.0 (d, JC–F = 25.4 Hz, C-6a), 122.2 (d, JC–F = 8.0 Hz, C-4a),
3
3
C-7), 123.6 (d, JC–F = 8.3 Hz, C-4a), 127.7 (C-11b), 130.3 (d,
129.0 (C-10b), 113.7 (C-10a), 130.9 (d, JC–F = 11.0 Hz, C-6b), 133.0
(C-8), 147.1 (C-9a), 147.9 (C-2), 153.4 (d, JC–F = 245 Hz, C-6), 166.4
3JC–F = 10.7 Hz, C-11a), 142.3 (C-10a), 148.3 (C-2), 155.8 (d,
1
1JC–F = 246 Hz, C-6), 160.3 (C-9), 166.4 (CO2H), 177.3 (d, JC–F
=
(CO2H), 177.2 (C–4); HRMS (ESI) m/z: calcd for C17H10FN2O3S
[MꢀH]+: 341.04016. Found: 341.04059; EIMS m/z (%): 342 (M+,
23), 298 (100%), 283 (13), 269 (27), 242 (20), 229 (21), 228 (12),
202 (9), 158 (5), 148 (5).
4
2.9 Hz, C-4); HRMS (ESI) m/z: calcd for C20H14FN2O4 [MꢀH]+:
365.09431. Found: 365.09482; EIMS m/z (%): 366 (M+, 2), 323
(29), 322 (100%), 293 (28), 279 (23), 253 (20), 223 (10), 210 (24),
147 (6), 139 (5).
4.2. Biology
4.1.4.4.
oxo-4,11-dihydro-1H-pyrido[2,3-a]carbazole-3-carboxylic acid
(13d). Yield 0.37 g (94%); mp: 310–312 °C (dec). Anal. Calcd
for C22H19FN2O4 (394.40): C, 67.00; H, 4.86; N, 7.10. Found: C,
67.12; H, 4.63; N, 7.02. IR (KBr) max: 3422, 3098, 2920, 1710,
1630, 1601, 1572, 1510, 1445, 1328, 1219, 1194, 1121, 965, 863,
1-Cyclopropyl-6-fluoro-9-methoxy-8,10-dimethyl-4-
4.2.1. Antimicrobial activity
The newly synthesized compounds were tested for their in vitro
antimicrobial properties against the following microorganisms:
Gram positive bacteria (B. subtilis ATCC 6633, S. aureus ATCC
6538, S. epidermidis ATCC 12228, quinolone- and penicillin-
resistant clinical isolates of both S. aureus and S. epidermidis), Gram
negative bacteria (A. baumannii ATCC 19606, E. coli ATCC 8739, H.
influenzae ATCC 19418, P. aeruginosa ATCC 9027, quinolone- and
penicillin-resistant clinical isolates of all A. baumannii, E. coli and
P. aeruginosa strains), yeasts (C. tropicalis ATCC 1369, S. cerevisiae
ATCC 9763), and mold (A. niger ATCC 6275). The screening was per-
formed according to the broth dilution method.46 Three types of
specific culture media were used in this study: Haemophilus Test
Medium for H. influenzae, Mueller Hinton Broth for the other bac-
teria, and Sabouraud Dextrose Broth for fungi. Test compounds
were dissolved in dimethyl sulfoxide and diluted in the growth
m
808 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d 1.30–1.35 (m, 2H)
;
and 1.39–1.46 (m, 2H) (2 H-20/2 H-30), 2.40 (s, 3H, C(8)–CH3),
2.59 (s, 3H, C(10)–CH3), 3.76 (s, 3H, C(9)–OCH3), 4.92–5.02 (m,
3
1H, H-10), 7.75 (d, JH–F = 10.3 Hz, 1H, H-5), 7.85 (s, 1H, H-7), 8.81
(s, 1H, H-2), 10.71 (s, 1H, N(11)–H), 15.34 (s, 1H, CO2H); 13C
NMR (75 MHz, DMSO-d6): d 10.5 (C-20/C-30), 11.3 (C(10)–CH3),
2
17.0 (C(8)–CH3), 39.1 (C-10), 60.7 (C(9)–OCH3), 100.2 (d, JC–F
=
3
21.2 Hz, C-5), 107.4 (C-3), 114.7 (C-10), 115.1 (d, JC–F = 2.5 Hz,
C-6b), 117.0 (d, JC–F = 22.8 Hz, C-6a), 121.1 (d, J = 3.8 Hz, C-7),
124.2 (d, JC–F = 8.1 Hz, C-4a), 125.8 (C-8), 127.8 (C-11b), 131.0
2
3
3
(d, JC–F = 10.4 Hz, C-11a), 139.8 (C-10a), 148.7 (C-2), 156.0 (d,
4
1JC–F = 247 Hz, C-6), 157.2 (C-9), 166.4 (CO2H), 177.3 (d, JC–F
=
media to give the final concentration which ranged from 100 lg/
2.5 Hz, C-4); HRMS (ESI) m/z: calcd for C22H18FN2O4 [MꢀH]+:
393.12561. Found: 393.12606; EIMS m/z (%): 394 (M+, 27), 350
(100%), 335 (46), 320 (25), 307 (24), 279 (14), 266 (10), 237 (12),
223 (7), 175 (10), 125 (5).
mL to 0.00015
l
g/mL. The final size of inocula was 5 ꢁ 105 CFU/
mL and 1 ꢁ 103 CFU/mL for bacteria and for fungi, respectively.
The minimum inhibitory concentrations (MIC, lg/mL) were deter-
mined after 24 h incubation at 37 °C for antibacterial activity and

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 18 (2010) 5873–5884
5883
after 48 h at 30 °C for antifungal activity. MICs were considered to
be the lowest concentrations of the tested compounds which
inhibited the reproduction of the tested microorganisms.
Drug-free tubes and organism-free tubes were kept as growth
controls and purity controls, respectively. Controls containing only
solvent showed that dimethyl sulfoxide, at the concentrations
studied, had no effect on the microorganisms. Ciprofloxacin, levo-
floxacin, moxifloxacin, and gemifloxacin were used as standard
antibacterial drugs.
Cell viability was assessed, after 3 days of treatment, with tetra-
zolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), obtained from Sigma (Dorset, UK), and assayed
as previously described.47 Representative results of at least three
independent experiments were used for evaluation of dose–
response curves, calculated from experimental points using single
or double Hill functions (Graph Pad Prism Software 5, San Diego
California USA, www.graphpad.com). IC50 concentrations were
obtained from the dose–response curves by extrapolation.
In all assays, the drugs were dissolved in DMSO immediately
before the addition to cell cultures. The final concentration of
DMSO never exceeded 0.1% (v/v), and equal amounts of the solvent
were added to control cells.
All compounds were tested in triplicate and the experiments
were repeated at least three times.
4.2.2. DNA gyrase and topoisomerase IV inhibition
The inhibitory activity of new fluoroquinolones against target
enzymes was detected by using E. coli DNA gyrase and topoisomer-
ase IV (Inspiralis, Norwich, UK).
DNA gyrase activity was measured in a supercoiling assay with
relaxed pBR322 DNA as a substrate. The reaction mixtures con-
tained 35 mM Tris–HCl pH 7.5, 24 mM KCl, 4 mM MgCl2, 2 mM
dithiothreitol, 1.8 mM spermidine, 1 mM ATP, 6.5% w/v glycerol,
4.2.5. Cell cycle analysis
Distribution of the cells in the cell cycle was determined by pro-
pidium iodide staining and flow cytometry analysis (EPICS XL-MCL
cytometer; Beckman Coulter). Cells (5 ꢁ 105) were harvested,
washed in PBS, fixed in cold 70% ethanol, and then stained with
40 lg/mL propidium iodide while treating with RNase. Analysis
0.1 mg/mL albumin, 16 ng/
l
L relaxed pBR322 DNA, 0.026 U/
l
L of
was done with a Beckman Coulter EPICS XL-MCL cytometer (Beck-
man Coulter). Cell cycle distributions were analyzed by Multi-
Cycle DNA Content and Cell Cycle Analysis Software (Phoenix Flow
Systems, Inc.) as previously described.48
gyrase protein, and different amounts of each fluoroquinolone.
All mixtures were incubated for 30 min at 37 °C and then the reac-
tions were stopped by adding chloroform/isoamyl alcohol 24:1
solution and bromophenol blue loading dye. After a brief vortex,
the blue aqua phase was analyzed by electrophoresis in 1.0% w/v
agarose gel.
Topoisomerase IV activity was measured by decatenation assay
using kinetoplast DNA as a substrate. The reaction mixtures con-
tained 40 mM HEPES–KOH pH 7.6, 100 mM potassium glutamate,
4.2.6. Western blot analysis
Procedures for protein extraction, solubilization, and protein
analysis by 1D PAGE are described elsewhere.48 Briefly, 30–50
lg
proteins from lysates were resolved by 5–15% SDS–PAGE and
transferred to PDVF membranes. The membranes were then incu-
bated with primary antibody, washed and then incubated with
HRP-anti-mouse or HRP-anti-rabbit antibodies. Immunoreactive
bands were visualized using an enhanced chemiluminescence
system.
10 mM Mg(OAc)2, 10 mM dithiothreitol, 1 mM ATP, 50
lg/mL
albumin, 6.6 ng/ L kDNA, 0.016 U/ L topoisomerase IV protein,
l
l
and different amounts of each fluoroquinolone. All mixtures were
incubated for 30 min at 37 °C and then the reactions were stopped
by adding chloroform/isoamyl alcohol 24:1 solution and bromo-
phenol blue loading dye. After a brief vortex, the blue aqua phase
was analyzed by electrophoresis in 1.0% w/v agarose gel.
The products of the reactions were quantitated using a Bio-Rad
gel documentation system and the 50% inhibitory concentration
(IC50, lg/mL) was determined as the drug concentration that re-
duced by 50% the supercoiling and the decatenation activity of
the cited enzymes observed with drug-free controls. Ciprofloxacin
and moxifloxacin were used as reference fluoroquinolones.
Results were confirmed in at least three independent
experiments.
4.2.7. Detection of apoptosis
Apoptosis was assessed by: (a) morphology on stained (Hoechst
33342, PI) or unstained cells using light-, phase contrast- and fluo-
rescence microscopy; (b) activation of caspase-7 (detection of
cleavage products) by Western Blotting procedure as previously
described.48
Acknowledgment
We wish to thank the Higher Council for Science and Technol-
ogy (Amman, Jordan) for financial support.
4.2.3. Antibodies and reagents
Caspase-7 antibody was obtained from Cell Signaling Technol-
ogy (Beverly, MA, USA); monoclonal anti-p53 antibody (DO-1)
was obtained from Santa Cruz Biotechnology (CA, USA); monoclo-
nal anti-actin antibody was obtained from Sigma (Dorset, UK).
Horseradish peroxidase-conjugated (HRP) secondary antibodies
and the enhanced chemiluminescence system (ECL) were from
Millipore (Millipore, MA, USA). Reagents for electrophoresis and
blotting analysis were obtained from Bio-Rad Laboratories. Ellipti-
cine was from Sigma (Dorset, UK).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.098. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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