Synthesis and biological evaluation of tetracyclic thienopyridones as antibacterial and antitumor agents

Article abstract of DOI:10.1016/j.bmc.2011.03.018

A facile synthesis of model 4-oxopyrido[3′,2′:4,5]thieno[3,2-b] indole-3-carboxylic acids 9a-e was achieved via Stille arylation of 2-chloro-3-nitro-4-oxothieno[2,3-b]pyridine-5-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan re

Full text of DOI:10.1016/j.bmc.2011.03.018

Bioorganic & Medicinal Chemistry 19 (2011) 2541–2548
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of tetracyclic thienopyridones as
antibacterial and antitumor agents
Salah A. Al-Trawneh a, Mustafa M. El-Abadelah a, , Jalal A. Zahra a, Samir A. Al-Taweel b, Franca Zani c,
⇑
Matteo Incerti c, Andrea Cavazzoni d, Paola Vicini c,
⇑
a Chemistry Department, Faculty of Science, The University of Jordan, Amman 11942, Jordan
b Chemistry Department, Faculty of Science, Mu’tah University, Karak 61710, Jordan
c Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27/A, Parma 43124, Italy
d Dipartimento di Medicina Sperimentale, Sezione di Oncologia Sperimentale, Università degli Studi di Parma, Via Volturno 39, Parma 43125, Italy
a r t i c l e i n f o
a b s t r a c t
A
facile synthesis of model 4-oxopyrido[30,20:4,5]thieno[3,2-b]indole-3-carboxylic acids 9a–e was
Article history:
Received 19 January 2011
Revised 5 March 2011
Accepted 8 March 2011
Available online 12 March 2011
achieved via Stille arylation of 2-chloro-3-nitro-4-oxothieno[2,3-b]pyridine-5-carboxylate and a subse-
quent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for
their in vitro antimicrobial and antiproliferative activity. Compounds 9a–c and 9e exhibited very high
potency against Gram positive Bacillus subtilis and Bacillus megaterium at concentrations 0.000015–
0.007 lg/mL. They also displayed excellent activity towards other Gram positive bacilli and staphylococci
Keywords:
and Gram negative Haemophilus influenzae, being in most cases superior or equal to commercial fluoro-
quinolones. Both 9a and 9c were inhibitors of the DNA gyrase activity. As concerns antitumor properties,
compounds 9b–e showed growth inhibition of MCF-7 breast tumor and A549 non-small cell lung cancer
4-Oxothieno[2,3-b]pyridines
4-Oxopyrido[30,20:4,5]thieno[3,2-b]indole-
3-carboxylic acids
Antibacterial activity
Anticancer activity
cells with IC50 1.6–2.8
normal cells. These compounds are promising as dual acting chemotherapeutics.
Ó 2011 Elsevier Ltd. All rights reserved.
lM and 2.6–6.9 lM, respectively, coupled with absence of cytotoxicity towards
1. Introduction
O
O
F
CO2H
The fluoroquinolones constitute a major class of antibacterial
chemotherapeutic agents which have a broad spectrum against
Gram positive and Gram negative bacteria.1–3 Examples include
norfloxacin 1a2 and ciprofloxacin 1b3 (Fig. 1) which were the first
two quinolones marketed consecutively in 1986 and 1987 for hu-
man use. Since 1986, more than twenty fluoroquinolones have
been approved by FDA and most of them remain on the market.1f
On the other hand, several 4-oxothieno[2,3-b]pyridine-5-
carboxylic acids (e.g. 2a–e, Fig. 1), potential bioisosters of quino-
lone antibacterials, were prepared and bioassayed.4–11 Substitution
at the N(7)-position of thienopyridones has been reported for alkyl
groups, for example, compounds 2a4 and 2b5,6 which exhibited
good level of activity against Gram negative bacteria. N(7)-Aryl
and -heteroaryl substitution has also been achieved, exemplified
by compounds 2c12 and 2d13 that exhibited good level of activity
against Gram negative and Gram positive bacterial strains. In
addition, selected N(7)-azacyclohexyl derivatives and related
CO2H
Cl
N
N
R
S
N
R
HN
2c (R = 2,4-C6H3F2)
2d(R = 2-thienyl)
2e (R = NMe2)
2a(R= Et)
2b(R = c-C3H5)
1a(R = Et)
1b(R = c-C3H5)
Figure 1. Structures of some fluoroquinolones (1a–b) and thieno[2,3-b]pyridones
(2a–e).
congeners, such as 7-(N,N-dimethylamino) derivative 2e, have
been prepared;14 the latter exhibited good level of activity, espe-
cially against Klebsiella pneumoniae and Salmonella paratyphi A
(MIC 0.5 and 1.0 l
g/mL, respectively).14
Quite recently, we have also explored the synthesis of some
tetracyclic fluoroquinolones (Fig. 2) such as 4-oxopyrido[2,3-a]
carbazole-3-carboxylic acids (3),15 and 4-oxothieno[20,30:4,5]-pyr-
rolo[3,2-h]quinoline-3-carboxylic acid (4).15,16
As part of an ongoing program aimed at developing facile
synthesis of novel antibacterial agents, we directed our attention
⇑
Corresponding authors. Address: Pharmaceutical Department, University of
Parma, Viale G. P. Usberti 27/A, Parma 43100, Italy. Tel.: +39 0521 905051; fax: +39
0521 905006 (P.V.); tel.: +962 6 5355000x22143; fax: +962 6 534 8932 (M.M.E.-A.).
E-mail addresses: mustelab@ju.edu.jo (M.M. El-Abadelah), paola.vicini@unipr.it,
pvicini@ipruniv.cce.unipr.it (P. Vicini).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.018

2542
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
2-thienyltrimethyl stannane (6a–e)19–21 (Stille reaction)22–30 with
O
O
ultimate production of the respective ethyl 2-aryl-7-cyclopropyl-
3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate
7a–e. In a subsequent step, the latter compounds undergo phos-
phite-mediated reductive cyclization (Cadogan reaction) assisted
by microwave irradiation to furnish the respective ethyl 4-oxopyr-
ido[30,20:4,5]thieno[3,2-b]indole-3-carboxylates 8a–e. This reac-
CO2H
CO2H
F
F
A
A
N
B
B
N
C
S
C
D
NH
D
NH
tion entails the generation of
a nitrene intermediate that
R
3
4
undergoes insertion into an 7-thienyl C–H bond to form a fused
pyrrole 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 8, does not proceed to
any detectable extent at 160 °C for 96 h. Acid-catalyzed hydrolysis
of the esters 8a–e produced the corresponding target 4-oxothie-
no[20,30:4,5]pyrrolo[3,2-h]quinoline-3-carboxylic acids 9a–e.
The new compounds 7–9 were characterized by elemental anal-
yses, IR, MS and NMR spectral data. These data, detailed in the
experimental part, are consistent with the suggested structures.
Thus, the mass spectra display the correct molecular ion peaks
for which the measured 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 assignments to the different carbons and their at-
tached, and/or neighboring hydrogens. In compounds 7e–9e, the
carbons of the benzo-fused ring D are readily identified by their
doublet signals (with varying JC–F-values) originating from cou-
pling with the nearby fluorine atom at C-7.
(R: H; Me; OMe; F)
Figure 2. Structures of some tetracyclic fluoroquinolones.
towards 4-oxothieno[2,3-b]pyridine-5-carboxylic acid derivatives
that are bioisosters of fluoroquinolone antibacterials. To the best
of our knowledge, fused rings with a bridge between the critical
C-2 and C-3 positions have not yet been investigated in this 4-oxo-
thieno[2,3-b]pyridine system. At the same time, the designed com-
pounds are isosteric with pyridocarbazoles, a class to which the
natural ellipticine antitumor drug belongs.15 Accordingly, we re-
port herein the synthesis and in vitro evaluation of the antimicro-
bial and antitumor properties of novel tetracyclic thienopyridones
such as the variously substituted 4-oxopyrido[30,20:4,5]thieno[3,2-b]
indole-3-carboxylic acids 9a–e, shown in Scheme 1.
2. Result and discussion
2.1. Synthesis
2.2. Biology
Microwave-enhanced Cadogan cyclization of ortho-nitrobiaryls,
in combination with phosphite reagent, has provided easy access
to substituted carbazoles and other fused heterocyclic systems.17,18
Based on the above methodology, the synthesis of the target 4-oxo-
pyrido[30,20:4,5]thieno[3,2-b]indole-3-carboxylic acids 9a–e and
their ethyl esters 8a–e was achieved by utilizing ethyl 2-chloro-
7-cyclopropyl-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-
5-carboxylate (5)5,6 as the starting material, and constructing the
fused thieno[b]indole nuclei via a two-step procedure as illustrated
in Scheme 1. The first key-step involves palladium-catalyzed
carbon–carbon cross coupling between 5 and the appropriate
2.2.1. Antimicrobial activity
The new thienopyridones 9a–e were screened for their in vitro
antimicrobial activity against model Gram positive and Gram neg-
ative bacteria, including multidrug-resistant species, yeasts and
mould. The minimum inhibitory concentrations are listed in Table 1
and are compared with the results obtained for standard antibac-
terial quinolones ciprofloxacin, levofloxacin, moxifloxacin and
gemifloxacin.
All the investigated compounds presented remarkable antibac-
terial properties against Gram positive bacteria, both bacilli and
O
N
SnMe3
O2N
O
R2
O2N
2
4
CO2Et
3
2
"
CO2Et
3a
7a
3"
(i)
R2
5
6
(ii)
Cl
R3
1"
4"
+
1
7
N
S
S
6"
R2
5"
R2
R3
1
'
7a-e
5
6a-e
2'
3'
R2
compounds 7 - 9
6
H5
N
R3
R2
5a
9a
O
4
7
8
entry
R3
a
b
c
d
e
4b
CO2R1
D
C
4a
3
2
H
H
Me
H
OMe OMe
Me
F
H
B
9b
A
9
H
R2
10a
S
N1
10
1'
2'
3'
8a-e(R1 = Et)
9a-e(R1 = H)
(iii)
Scheme 1. Reagents and conditions: (i) Pd(OAc)2, CsF, DMF, 50–65 °C; (ii) P(OEt)3, 175–200 °C (MW); (iii) 10% aq HCl, EtOH, reflux.

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
2543
Table 1
Inhibitory activity of compounds 9a–e against bacteria and fungi, expressed as MIC (lg/mL)
Microorganisma
Compoundb
9a
9b
9c
9d
9e
CIP
LEV
MOX
GEM
Gram positive bacteria
BC
BM
BS
BTK
SA
SAR 72
SAR 4790
SE
0.007
0.000015
0.000015
0.007
0.003
0.7
>100
0.03
3
0.3
0.03
3
3
3
1.5
6
3
>100
3
0.007
0.000015
0.000015
0.003
0.003
0.7
>100
0.03
1.5
0.3
0.15
0.03
0.03
0.07
0.07
25
100
0.15
>100
0.3
0.07
0.0003
0.007
0.015
0.015
6
50
0.015
50
0.007
0.0003
0.15
0.07
12
>100
0.3
25
0.00003
0.0003
0.007
0.015
1.5
>100
0.15
6
0.007
0.03
0.03
0.3
100
>100
0.07
100
0.003
0.015
0.03
0.03
12
50
0.07
50
SER
3
Gram negative bacteria
AB
ABR
EC
ECR
HI
PA
0.07
>100
25
>100
0.03
>100
0.7
3
>100
—
>100
—
6
0.15
>100
50
>100
0.15
>100
0.7
0.15
>100
0.03
25
0.15
0.3
0.15
50
0.03
50
0.03
0.7
0.3
c
>100
>100
—
1.5
>100
>100
>100
—
0.15
>100
>100
0.015
100
0.15
0.07
100
0.015
50
0.015
0.07
>100
Fungi
SC
CT
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
AN
a
BC, Bacillus cereus ATCC 11778; BM, Bacillus megaterium ATCC 19213; BS, Bacillus subtilis ATCC 6633; BTK, Bacillus thuringiensis var. kurstaki BGSC 4D1; 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 pen-
icillin-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; SC, Saccharomyces cerevisiae ATCC 9763; CT, Candida tropicalis ATCC 1369; AN, Aspergillus niger ATCC 6275.
b
CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin; GEM, gemifloxacin.
Not tested because inactive against the corresponding quinolone-sensitive microorganism.
c
staphylococci, in most cases at concentrations lower than refer-
ence drugs. Interestingly, compounds 9a and 9e exhibited unprec-
higher than those of standard substances. However, none of the
tested thienopyridones inhibited P. aeruginosa and multidrug-
resistant Gram negative strains even at the higher concentration
edented powerful activity at 0.000015
megaterium and Bacillus subtilis. These microorganisms were also
inhibited by 9b and 9c at 0.00003–0.007 g/mL, so appearing to
lg/mL against Bacillus
of 100 lg/mL.
l
It is worthwhile to note that 9e was the only compound exhib-
iting antifungal properties, when compared to the other thieno-
pyridones and quinolones tested. This chemical displayed against
yeast Saccharomyces cerevisiae an effect equal to that of standard
be the most sensitive strains. Furthermore, compounds 9a–c and
9e displayed excellent activity against other tested bacilli and wild
staphylococci. The inhibition of Bacillus cereus, Bacillus thuringiensis
and Staphylococcus aureus was achieved by compounds 9a, 9c and
antifungal miconazole (MIC 12
active than the reference drug against Candida tropicalis and Asper-
gillus niger (MIC of miconazole 6 and 3 g/mL, respectively).
lg/mL), but it was found to be less
9e at concentrations of 0.003–0.03
those of standard quinolones (MICs 0.015–0.3
against Staphylococcus epidermidis compounds 9a and 9e at
0.03 g/mL were superior to reference ciprofloxacin, levofloxacin
and moxifloxacin (MICs 0.07–0.15 g/mL). As concerns compound
9d, it showed significant activity against all tested bacteria at con-
centrations 1.5–6 g/mL.
l
g/mL, generally lower than
lg/mL), and also
l
The structure–activity relationships analysis of the data re-
ported in Table 1 shows that the thiophene isosteric replacement
of the fluorobenzene (ring B) in the tetracyclic structure of fluoro-
quinolones 315 played a positive role in the antibacterial effective-
ness, with a general increase of activity, especially against bacilli,
and including resistant S. aureus SAR 72 and S. epidermidis strains.
Overall, 9a, unsubstituted, and 9e, carrying an electron-withdrawing
fluorine group with low bulkiness and a comparable size to the
hydrogen atom, showed higher effectiveness against both Gram
positive and Gram negative bacteria, as compared to methyl and
methoxy substituted thienopyridones 9b–d. MIC values exhibited
by 9e were equal to those of 9a with a slight enhancement in the
activity against Gram positive B. thuringiensis and resistant S. epide-
rmidis strain and a weak reduction against Gram negative A. bau-
mannii, E. coli and H. influenzae. It is worth noting that fluoro
substituted 9e is also endowed with a certain antifungal activity.
The introduction in the para position of the benzene ring D of bulky
and electron-donating substituents, such as a hydrophilic methoxy
group and, in a more extent, a lipophilic methyl group, leads to
comparatively less active compounds 9c and 9b. The antibacterial
properties of the latters are further considerably reduced in three-
substituted 9d, except when multidrug-resistant S. aureus SAR 72
and S. epidermidis strains were tested. In the studied thienopyri-
dones the presence of a fluorine atom on the benzene ring D of
l
l
l
Of particular relevance is that thienopyridones exerted strong
antibacterial effectiveness against both multidrug-resistant strains
of S. aureus (SAR 72) and S. epidermidis, their minimum inhibitory
concentrations ranging from 0.7 to 25 lg/mL and displaying for
compounds 9a and 9c–e higher potency as compared to the stan-
dard quinolones used in this study.
Almost all of the studied thienopyridones exhibited also a
marked degree of activity against Gram negative bacteria. Among
these, Acinetobacter baumannii and, especially, Haemophilus influ-
enzae were found to be more sensitive in comparison to Escherichia
coli and Pseudomonas aeruginosa. Unsubstituted 9a displayed the
highest activity, inhibiting the growth of H. influenzae and A. bau-
mannii at concentrations of 0.03 and 0.07
comparable or lower than those of the reference quinolones.
Strong effect was shown by fluoro derivative 9e at 0.15 g/mL,
while compounds 9b and 9c were found to exhibit high to good
antibacterial properties (MIC 0.15–3 g/mL). A significant inhibi-
tion was also observed for 9d towards H. influenzae. Only 9a and
9e showed activity against E. coli with MIC values 25–50 g/mL,
lg/mL, respectively,
l
l
l

2544
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
Table 2
and between 2.6 and 2.7 lM against A549 cells. Compound 9a
showed the lowest effect on cell proliferation against both cell
Inhibitory activity of compounds 9a and 9c against DNA gyrase and topoisomerase IV
of E. coli, expressed as 50% inhibitory concentration (
l
g/mL)
lines (IC50 10 and 5.2 M against A549 and MCF-7 cells, respec-
l
Compound
IC50
tively). Replacement of the B benzene ring of compounds 3 with
its heterocyclic bioisoster thiophene (9a–e) did not significantly
modify the antiproliferative activity as a further evidence for the
bioisosteric equivalence between benzene and thiophene rings.
Gyrasea
Topoisomerase IVb
9a
9c
1.0
1.1
0.34
0.85
>24
>24
4.60
5.00
Ciprofloxacin
Moxifloxacin
3. Conclusion
a
E. coli DNA gyrase supercoiling assay.
E. coli topoisomerase IV decatenation assay.
b
Novel 1-cyclopropyl-4-oxopyrido[30,20:4,5]thieno[3,2-b]indole-
3-carboxylic acids 9a–e were synthesized by efficient synthetic
routes and their antimicrobial and antitumor properties assessed.
Four of these five tetracyclic thieno[2,3-b]pyridones showed
unprecedented powerful activity against B. megaterium and B. sub-
tilis, excellent activity against S. aureus and also against Gram neg-
ative H. influenzae, in addition to activity against few resistant
Gram positive bacteria. Furthermore, these compounds had high
anticancer activity against breast MCF-7 and lung A549 tumor cell
lines, and were devoid of cytotoxicity against normal cells.
The combination of potent activity against bacteria and cancer
cell lines, together with the absence of cytotoxicity against normal
cells, makes these agents of interest in the search for novel poten-
tial antimicrobials able to reduce the danger of bacterial infections
in the frequently immunocompromised cancer patients.31
the tetracyclic ring system has not the significant impact on the
antibacterial properties as exhibited in the fluoroquinolones 3,
where the para-fluoroderivative is more active than the unsubsti-
tuted analogue, and the most active derivative among the com-
pounds 3. The introduction of bulky and multiple substituents
seems detrimental for the antimicrobial activity tested, as already
observed for the tetracyclic isosters 3.
2.2.2. DNA gyrase and topoisomerase IV inhibition
Bacterial DNA gyrase of Gram negative strains and topoisomer-
ase IV of Gram positive ones are well-characterized clinically vali-
dated targets of the quinolone antibacterials.1 Therefore, the
enzymatic inhibition of compounds 9a and 9c, possessing potent
antibacterial activity against a wide spectrum of microorganisms
and structurally related to quinolones, was tested against DNA gyr-
ase and topoisomerase IV isolated from E. coli. Data reported in Ta-
ble 2 show that both the tested compounds displayed inhibitory
4. Experimental
4.1. Chemistry
properties towards DNA gyrase, with IC50 values 1.0–1.1
(ciprofloxacin and moxifloxacin IC50 0.34 and 0.85 g/mL, respec-
tively), whereas they did not inhibit topoisomerase IV up to the
concentration of 24 g/mL. The different target affinity suggests
lg/mL
Synthetic starting material, reagents and solvents were pur-
chased from Aldrich Chemical Co. Silica gel for column chromatog-
l
raphy was purchased from Macherey-Nagel GmbH
& Co
l
(Germany). Melting points (uncorrected) were determined on a
Stuart scientific melting point apparatus in open capillary tubes.
1H and 13C NMR spectra were recorded on a 300 MHz spectrometer
(Bruker DPX-300) with TMS as the internal standard. Chemical
shifts are expressed in d units; 1H–1H, H–F and C–F coupling con-
stants are given in hertz (Hz). Electron-impact mass spectra (EIMS)
were obtained using a Finnegan MAT TSQ-70 spectrometer at
70 eV; ion source temperature 200 °C. High resolution mass spec-
tra (HRMS) were measured by electrospray ionization (ESI) tech-
that the great antibacterial potency exhibited by thienopyridones
against Gram positive organisms was not due to inhibition of topo-
isomerase IV and hints that other mechanisms are involved in the
antibacterial effect.
2.2.3. Antitumor activity
The antitumor activity of compounds 9a–e was assayed with re-
spect to ellipticine by evaluating cell proliferation in MCF-7 breast
cancer, in A549 non-small cell lung cancer (NSCLC) cell lines and in
normal human-derm fibroblasts (HuDe). After 72 h cell viability
was determined: against HuDe cells no effect was detected until
nique on
a Bruker APEX-2 instrument. The samples were
dissolved in acetonitrile, diluted in spray solution (methanol/water
1:1 v/v + 0.1% formic acid) and infused using a syringe pump with a
flow rate of 2 lL/min. External calibration was conducted using
Arginine cluster in a mass range m/z 175–871. IR spectra were re-
corded as KBr discs on a Nicolet Impact-400 FT-IR spectrophotom-
eter. Microwave irradiation experiments, conducted for the
Cadogan–nitrene insertion reactions, were carried out with a Bio-
tage Initiator 2.0 Microwave Synthesizer Instrument. Elemental
analyses were performed on a Euro Vector Elemental Analyzer
(EA 3000 A).
10
pounds showed significant inhibition of cell proliferation in a dose-
dependent manner (Table 3). Ellipticine showed IC50 of 1.6 M and
3.4 M against MCF-7 and A549 cells, respectively. The most po-
tent compounds 9c and 9e had comparable IC50 to the reference
lM, while against both the tumor cell lines all the tested com-
l
l
substance, ranging between 1.6 and 1.9 lM against MCF-7 cells,
Table 3
Effects of compounds 9a–e and ellipticine on MCF-7, A549 and HuDE cell lines
Compound IC50 MCF-7,
cancer)
lM (breast
IC50 A549,
(NSCLC)
lM
IC50 HuDe,
l
M
4.1.1. Ethyl 2-chloro-7-cyclopropyl-3-nitro-4-oxo-4,7-
dihydrothieno[2,3-b]pyridine-5-carboxylate (5)
This compound was prepared from 3-acetyl-2,5-dichlorothio-
phene, dimethyl carbonate, triethyl orthoformate and cyclopropyl-
amine by following the literature procedures.5,6
9a
5.2 1.09
2.8 1.05
1.6 1.04
2.5 1.05
1.9 1.04
1.6 1.06
10 1.04
6.9 1.06
2.6 1.02
4.7 1.05
2.7 1.02
3.4 1.04
>10
>10
>10
>10
>10
>10
9b
9c
9d
9e
Ellipticine
4.1.2. General procedure for the synthesis of ethyl 2-aryl-7-
cyclopropyl-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-
carboxylates (7a–e)
Cells were treated with the indicated compounds at concentrations ranging from
0.1 to 20 M for 72 h and then viability was determined by MTT assay. Concen-
tration 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 3
independent experiments are reported.
l
The appropriate trimethyl(aryl)stannane (6a–e)19–21 (3.9–
6.9 mmol), Pd(OAc)2 (0.065 g, 0.29 mmol), and CsF (0.44 g,

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
2545
2.9 mmol) were successively added to a stirred solution of ethyl
2-chloro-7-cyclopropyl-3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyri-
dine-5-carboxylate (5) (1.0 g, 2.9 mmol) in DMF (4 mL). The reac-
tion mixture was heated at 50–65 °C under nitrogen atmosphere
for 12–15 h. The resulting solution was then cooled to rt and
quenched with water (10 ml), whereby a black gummy precipitate
was obtained. The latter product was treated with CHCl3 and fil-
tered to remove the insoluble matter. The filtrate was dried over
anhydrous Na2SO4 and evaporated under reduced pressure to give
a brown residue. This crude product was purified by column chro-
matography using silica gel and eluting with chloroform/ethyl ace-
tate to give 7a–e as yellow solid products.
3.76 (m, 1H, H-10), 3.79 (s, 3H, OCH3), 4.20 (q, J = 7.1 Hz,
2H, –OCH2Me), 7.07 (d, J = 8.7 Hz, 2H, H-300/H-500), 7.42 (d,
J = 8.7 Hz, 2H, H-200/H-600), 8.33 (s, 1H, H-6). 13C NMR (75 MHz,
DMSO-d6): d 7.6 (C-20/C-30), 14.7 (CH3CH2O–), 37.2 (C-10), 56.0
(OCH3), 60.8 (–OCH2Me), 115.7 (C-300/C-500), 116.1 (C-5), 120.3 (C-
3a), 120.6 (C-100), 130.0 (C-200/C-600), 130.5 (C-2), 140.3 (C-3),
146.2 (C-6), 149.3 (C-7a), 161.4 (C-400), 164.4 (CO2Et), 168.1 (C-4).
HRMS (ESI): found 415.09591 ([M+H]+), C20H19N2O6S requires
415.09583; found 829.18334 ([2M+H]+), C40H37N4O12S2 requires
829.18439; m/z (EI): 414 (M+, 23), 369 (6), 342 (100), 338 (6),
326 (6), 297 (12), 270 (17), 252 (6), 151 (7), 124 (9), 123 (12),
109 (9).
4.1.2.1. Ethyl 7-cyclopropyl-3-nitro-4-oxo-2-phenyl-4,7-dihy-
drothieno[2,3-b]pyridine-5-carboxylate (7a). Reaction tempera-
ture: 60 °C; reaction time: 14 h; ratio of the eluting mixture:
1:2, v/v. Yield 65%; mp 220–223 °C (dec). Anal. Calcd for
4.1.2.4. Ethyl 7-cyclopropyl-2-(4-methoxy-3,5-dimethylphenyl)-
3-nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate
(7d). Reaction temperature: 55 °C; reaction time: 15 h; ratio of
the eluting solvent mixture: 1:1, v/v. Yield 61%; mp 290–292 °C
(dec). Anal. Calcd for C22H22N2O6S (442.49): C, 59.72; H, 5.01;
N, 6.33; S, 7.25. Found: C, 60.02; H, 5.13; N, 6.52; S, 7.16. IR:
mmax (KBr)/cmꢀ1 3022, 2968, 2949, 1743, 1626, 1535, 1448,
1372, 1237, 1216, 1008 and 799; 1H NMR (300 MHz, DMSO-d6):
d 1.15 (m, 2H) and 1.27 (m, 2H) (H2-20/H2-30), 1.24 (t, J = 7.1 Hz,
3H, CH3CH2O–), 2.23 (s, 6H, C(300)–CH3/C(500)–CH3), 3.68 (s, 3H,-
OCH3), 3.73 (m, 1H, H-10), 4.20 (q, J = 7.1 Hz, 2H, –OCH2Me), 7.15
(s, 2H, H-200/H-600), 8.33 (s, 1H, H-6). 13C NMR (75 MHz, DMSO-
d6): d 7.6 (C-20/C-30), 14.7 (CH3CH2O–), 16.3 (C(300)–CH3/C(500)–
CH3), 37.2 (C-10), 60.0 (–OCH3), 60.8 (–OCH2Me), 116.1 (C-5),
123.0 (C-3a), 123.6 (C-100), 128.8 (C-200/C-600), 129.1 (C-2), 132.6
(C-300/C-500), 138.2 (C-3), 146.3 (C-6), 149.5 (C-7a), 159.0 (C-400),
164.4 (CO2Et), 168.2 (C-4). HRMS (ESI): found 465.10901
([M+Na]+), C22H22N2O6SNa requires 465.10908; m/z (EI): 442 (M+,
23), 412 (6), 397 (12), 370 (100), 325 (8), 310 (6), 280 (5), 163
(3), 115 (2).
C
19H16N2O5S (384.41): C, 59.37; H, 4.19; N, 7.29; S, 8.34. Found:
C, 59.35; H, 4.27; N, 6.98; S, 8.21. IR: mmax (KBr)/cmꢀ1 2971,
1728, 1699, 1623, 1535, 1499, 1452, 1372, 1321, 1241, 1150,
1074, 1034, 801, 799, 760 and 694; 1H NMR (300 MHz, DMSO-
d6): d 1.16 (m, 2H) and 1.28 (m, 2H) (H2-20/H2-30), 1.24 (t,
J = 7.1 Hz, 3H, CH3CH2O–), 3.75 (m, 1H, H-10), 4.20 (q, J = 7.1 Hz,
2H, –OCH2Me), 7.48 (m, 5H, H-200/H-600, H-300/H-500, H-400), 8.34 (s,
13
1H, H-6). C NMR (75 MHz, DMSO-d6): d 7.6 (C-20/C-30), 14.7
(CH3CH2O–), 37.2 (C-10), 60.8 (–OCH2Me), 116.1 (C-5), 128.3 (C-
2), 128.5 (C-200/C-600), 130.2 (C-300/C-500), 141.6 (C-3), 120.7 (C-100),
131.1 (C-3a), 130.9 (C-400), 146.4 (C-6), 149.9 (C-7a), 164.3 (CO2Et),
168.2 (C-4). HRMS (ESI): found 385.08583 ([M+H]+), C19H17N2O5S
requires 385.08527; found 407.06777 ([M+Na]+), C19H16N2O5SNa
requires 407.06721; found 791.14567 ([2M+Na]+), C38H32N4O10S2-
Na requires 791.14521; m/z (EI): 384 (M+, 7), 339 (8), 312 (100),
296 (6), 267 (23), 250 (22), 236 (18), 225 (11), 185 (7), 165 (6),
105 (8).
4.1.2.5. Ethyl 7-cyclopropyl-2-(4-fluorophenyl)-3-nitro-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate (7e). Reaction
temperature: 50 °C; reaction time: 15 h; ratio of the eluting
solvent mixture: 1:1, v/v. Yield 65%; mp 275–277 °C (dec). Anal.
Calcd for: C19H15N2FO5S (402.40) C, 56.71; H, 3.76; N, 6.96;
S, 7.97. Found: C, 56.61; H, 3.66; N, 7.14; S, 7.78. IR: mmax
(KBr)/cmꢀ1 3011, 2968, 1724, 1615, 1543, 1492, 1452, 1372,
1321, 1252, 1230, 1168, 1077, 864, 837, 801 and 784; 1H NMR
(300 MHz, DMSO-d6): d 1.15 (m, 2H) and 1.28 (m, 2H) (H2-20/H2-
30), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O–), 3.74 (m, 1H, H-10), 4.20 (q,
4.1.2.2. Ethyl 7-cyclopropyl-2-(4-methylphenyl)-3-nitro-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate (7b). Reaction
temperature: 55 °C; reaction time: 12 h; ratio of the eluting
solvent mixture: 1:2, v/v. Yield 70%; mp 282–284 °C (dec) Anal.
Calcd for C20H18N2O5S (398.44): C, 60.29; H, 4.55; N, 7.03;
S, 8.05. Found: C, 59.98; H, 4.41; N, 6.91; S, 7.76. IR: mmax
(KBr)/cmꢀ1 2924, 1728, 1615, 1535, 1499, 1452, 1325, 1248,
1150, 1070 and 805; 1H NMR (300 MHz, DMSO-d6): d 1.14
(m, 2H) and 1.27 (m, 2H) (H2-20/H2–30), 1.23 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 2.33 (s, 3H, C(400)–CH3), 3.74 (m, 1H, H-10), 4.19
(q, J = 7.1 Hz, 2H, –OCH2Me), 7.32 (d, J = 8.2 Hz, 2H, H-300/H-500),
7.36 (d, J = 8.2 Hz, 2H, H-200/H-600), 8.34 (s, 1H, H-6). 13C NMR
(75 MHz, DMSO-d6): d 7.6 (C-20/C-30), 14.7 (CH3CH2O–), 21.3
(C(400)–CH3), 37.2 (C-10), 60.8 (–OCH2Me), 116.1 (C-5), 123.1
(C-3a), 125.4 (C-100), 128.2 (C-200/C-600), 128.4 (C-2), 130.8 (C-300/
C-500), 141.0 (C-3), 141.0 (C-400), 146.4 (C-6), 149.6 (C-7a), 164.4
(CO2Et), 168.2 (C-4). HRMS (ESI): found 421.08389 ([M+Na]+),
3
J = 7.1 Hz, 2H, –OCH2Me), 7.37 (dd, JH–F = 8.8 Hz, J = 8.7 Hz, 2H,
4
H-300/H-500), 7.54 (dd, JC–F = 5 Hz, J = 8.7 Hz, 2H, H-200/H-600), 8.35
13
(s, 1H, H-6). C NMR (75 MHz, DMSO-d6): d 7.6 (C-20/C-30), 14.6
(CH3CH2O–), 37.2 (C-10), 60.8 (-OCH2Me), 116.1 (C-5), 117.3 (d,
4
2JC–F = 22 Hz, C-300/C-500), 122.9 (C-3a), 124.7 (d, JC–F = 2 Hz, C-100),
3
129.3 (C-2), 131.1 (d, JC–F = 8.7 Hz, C-200/C-600), 141.5 (C-3), 146.5
1
(C-6), 149.9 (C-7a), 163.6 (d, JC–F = 246 Hz, C-400), 164.3 (CO2Et),
168.2 (C-4). HRMS (ESI): found 403.07631([M+H]+), C19H16N2FO5S
requires 403.07585; found 425.05799 ([M+Na]+), C19H15N2FO5SNa
requires 425.05779; found 827.12762 ([2M+Na]+), C38H30N4
F2O10S2Na requires 827.12636; m/z (EI): 402 (M+, 8), 357 (7), 330
(100), 314 (6), 285 (11), 254 (15), 214 (6), 163 (6), 139 (10), 123
(11), 95 (6).
C
C
20H18N2O5SNa requires 421.08286; found 819.17760 ([2M+Na]+),
40H36N4O10S2Na requires 819.17651; m/z (EI): 398 (M+, 10), 353
(3), 350 (7), 326 (100), 279 (6), 250 (6), 195 (6), 182 (14), 165
(11), 152 (6), 135 (9), 107 (7), 83 (9).
4.1.2.3. Ethyl 7-cyclopropyl-2-(4-methoxyphenyl)-3-nitro-4-oxo-
4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate (7c). Reaction
temperature: 65 °C; reaction time: 15 h; ratio of the eluting
solvent mixture: 1:2, v/v. Yield 42%; mp 285–288 °C (dec).
Anal. Calcd for C20H18N2O6S (414.44): C, 57.96; H, 4.38; N, 6.76;
S, 7.74. Found: C, 57.64; H, 4.08; N, 6.84; S, 7.97%. IR: mmax (KBr)/
cmꢀ1 2997, 1699, 1630, 1605, 1539, 1499, 1445, 1248, 1183,
1027, 841 and 798; 1H NMR (300 MHz, DMSO-d6): d 1.15 (m, 2H)
and 1.27 (m, 2H) (H2-20/H2-30), 1.24 (t, J = 7.1 Hz, 3H, CH3CH2O–),
4.1.3. General procedure for the synthesis of ethyl 1-
cyclopropyl-4-oxo-1,5-dihydro-4H-pyrido[30,20:4,5]thieno[3,2-
b]indole-3-carboxylates (8a–e)
A mixture of the appropriate ethyl 2-aryl-7-cyclopropyl-3-
nitro-4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxylate (7a–e)
(0.3 mmol) and P(OEt)3 (3–4 mL) was placed in a 10 mL glass vial.
The vial was sealed tightly with an aluminum Teflon crimpÒ top
and the mixture therein irradiated with microwaves (power level,

2546
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
400 W) for 1.75 h at preselected temperature of 175–200 °C.
Thereafter, the vial was cooled to 20 °C by gas jet cooling system
and excess P(OEt)3 was evaporated in vacuo. The residual solid
was treated with CHCl3 (3 ꢁ 4 mL) whereby the respective title
products (8a–e) were obtained as light yellow solids.
4.1.3.4. Ethyl 1-cyclopropyl-6,8-dimethyl-7-methoxy-4-oxo-1,5-
dihydro-4H-pyrido[30,20:4,5]thieno-[3,2-b]indole-3-carboxylate
(8d). Irradiation time: 1.75 h. Yield 61%; mp 255-257 °C (dec). Anal.
Calcd for C22H22N2O4S (410.49): C, 64.37; H, 5.40; N, 6.82; S, 7.
Found: C, 64.33; H, 5.38; N, 6.78; S, 7.68. IR: mmax (KBr)/cmꢀ1
3288, 2942, 1732, 1594, 1567, 1455, 1346, 1303, 1263, 1150,
1096, 1023, 897 and 805; 1H NMR (300 MHz, DMSO-d6): d 1.18
(m, 2H) and 1.25 (m, 2H) (H2-20/H2-30), 1.28 (t, J = 7.1 Hz, 3H,
CH3CH2O–), 2.31 (s, 3H, C(8)–CH3), 2.48 (s, 3H, C(6)–CH3), 3.66 (s,
3H, –OCH3), 3.73 (m, 1H, H-10), 4.23 (q, J = 7.1 Hz, 2H, –OCH2Me),
7.40 (s, 1H, H-9), 8.29 (s, 1H, H-2), 11.31 (s, 1H, N–H). 13C NMR
4.1.3.1.
[30,20:4,5]thieno[3,2-b]indole-3-carboxylate
time: 1.75 h. Yield 45%; mp 278–280 °C. Anal. Calcd for
19H16N2O3S (352.41): C, 64.76; H, 4.58; N, 7.95. Found: C, 64.43;
Ethyl
1-cyclopropyl-4-oxo-1,5-dihydro-4H-pyrido
(8a). Irradiation
C
H, 4.22; N, 7.97. IR: mmax (KBr)/cmꢀ1 3421, 2985, 1733, 1619,
1545, 1501, 1441, 1381, 1359, 1317, 1263, 1240, 1209, 1170,
1130, 1111, 1041, 1022 and 797; 1H NMR (300 MHz, DMSO-d6):
d 1.19 (m, 2H) and 1.26 (m, 2H) (H2-20/H2-30), 1.28 (t, J = 7.0 Hz,
3H, CH3CH2O–), 3.76 (m, 1H, H-10), 4.23 (q, J = 7.0 Hz, 2H,
–OCH2Me), 7.10 (dd, J = 7.2, 7.8 Hz, 1H, H-8), 7.20 (dd, J = 7.2,
8.0 Hz, 1H, H-7), 7.58 (d, J = 8.0 Hz, 1H, H-6), 7.78 (d, J = 7.8 Hz,
1H, H-9), 8.38 (s, 1H, H-2), 11.88 (s, 1H, N–H). 13C NMR (75 MHz,
DMSO-d6): d 7.6 (C-20/C-30), 14.7 (CH3CH2O–), 36.9 (C-10), 60.4
(–OCH2Me), 107.4 (C-3), 113.5 (C-6), 115.1 (C-9b), 118.6 (C-9),
119.6 (C-4a), 120.0 (C-8), 121.1 (C-9a), 122.7 (C-7), 137.3 (C-4b),
140.6 (C-5a), 145.2 (C-2), 153.0 (C-10a), 164.9 (CO2Et), 169.7 (C-
4). HRMS (ESI): found 353.11007 ([M+H]+), C19H17N2O3S requires
353.11011.
(75 MHz, DMSO-d6):
d
7.5 (C-20/C-30), 11.1 (C(6)–CH3), 14.7
(CH3CH2O–), 17.0 (C(8)–CH3), 36.9 (C-10), 60.5 (-OCH3), 60.7
(–OCH2Me), 108.0 (C-9b), 114.9 (C-3), 116.9 (C-9), 117.5 (C-9a),
123.4 (C-4a), 137.5 (C-4b), 139.6 (C-5a), 141.4 (C-6), 143.8 (C-8),
145.0 (C-2), 152.2 (C-10a), 153.4 (C-7), 165.2 (CO2Et), 169.5 (C-
4). HRMS (ESI): found 411.13792 ([M+H]+), C22H23N2O4S requires
411.13730; found 821.27228 ([2M+H]+), C44H45N4O8S2 requires
821.26733.
4.1.3.5. Ethyl 1-cyclopropyl-7-fluoro-4-oxo-1,5-dihydro-4H-pyr-
ido[30,20:4,5]thieno[3,2-b]indole-3-carboxylate (8e). Irradiation
time: 1.75 h. Yield 54%; mp 305–307 °C (dec). Anal. Calcd for
C
19H15N2FO3S (370.40): C, 61.61; H, 4.08; N, 7.56 S, 8. Found: C,
61.52; H, 3.93; N, 7.49 S, 8. IR: mmax (KBr)/cmꢀ1 3331, 3080,
1721, 1605, 1557, 1481, 1448, 1346, 1303, 1266, 1208, 1161,
1110, 1034, 932, 801 and 699; 1H NMR (300 MHz, DMSO-d6): d
1.19 (m, 2H) and 1.26 (m, 2H) (H2-20/H2-30), 1.28 (t, J = 7.1 Hz,
3H, CH3CH2O–), 3.74 (m, 1H, H-10), 4.22 (q, J = 7.1 Hz, 2H,
4.1.3.2. Ethyl 1-cyclopropyl-7-methyl-4-oxo-1,5-dihydro-4H-pyr-
ido[30,20:4,5]thieno[3,2-b]indole-3-carboxylate (8b). Irradiation
time: 1.75 h. Yield 63%; mp 315–317 °C (dec). Anal. Calcd for
C
20H18N2O3S (366.44): C, 65.56; H, 4.95; N, 7.64. Found: C,
3
65.64; H, 4.74; N, 7.41. IR: mmax (KBr)/cmꢀ1 3378, 2971, 1721,
1601, 1557, 1481, 1445, 1372, 1350, 1310, 1270, 1237, 1208,
1154, 1136, 1107, 1041 and 801; 1H NMR (300 MHz, DMSO-d6):
d 1.19 (m, 2H) and 1.26 (m, 2H) (H2-20/H2-30), 1.28 (t, J = 7.1 Hz,
3H, CH3CH2O–), 2.41 (s, 3H, C(7)–CH3), 3.75 (m, 1H, H-10),
4.23 (q, J = 7.1 Hz, 2H, –OCH2Me), 6.94 (d, J = 8.1 Hz, 1H, H-8),
7.37 (s, 1H, H-6), 7.65 (d, J = 8.1 Hz, 1H, H-9), 8.31 (s, 1H, H-2),
–OCH2Me), 6.96 (ddd, JH–F = 9.7 Hz, J = 8.7, 2.1 Hz, 1H, H-8), 7.29
3
4
(dd, JH–F = 10.1 Hz, J = 2.1 Hz, 1H, H-6), 7.81 (dd, J = 8.7 Hz, JH–F
=
3.1 Hz, 1H, H-9), 8.31 (s, 1H, H-2), 11.97 (s, 1H, N–H). 13C NMR
(75 MHz, DMSO-d6): d 7.5 (C-20/C-30), 14.7 (CH3CH2O–), 36.9 (C-10),
2
60.4 (–OCH2Me), 99.5 (d, JC–F = 26.2 Hz, C-6), 116.6 (C-3), 108.4
(d, JC–F = 24.1 Hz, C-8), 115.0 (C-4a), 107.5 (C-9b), 118.0 (C-9a),
119.8 (d, JC–F = 11.0 Hz, C-9), 137.8 (C-4b), 140.7 (d, JC–F
13.1 Hz, C-5a), 145.3 (C-2), 152.9 (C-10a), 159.6 (d, JC–F = 234 Hz,
C-7), 164.9 (CO2Et), 169.7 (C-4). HRMS (ESI): found 371.08608
([M+H]+), C19H16N2FO3S requires 371.08602; found 393.06802
([M+Na]+), C19H15N2FO3SNa requires 393.06796; found 763.14670
([2M+Na]+), C38H30N4F2O6S2Na requires 763.14670.
2
3
3
=
1
13
11.72 (s, 1H, N–H). C NMR (75 MHz, DMSO-d6): d 7.5 (C-20/C-
30), 14.7 (CH3CH2O–), 22.0 (C(7)–CH3), 36.9 (C-10), 60.4 (-OCH2Me),
109.0 (C-9b), 111.8 (C-3), 113.3 (C-6), 118.3 (C-9), 119.0 (C-9a),
119.2 (C-4a), 121.7 (C-8), 132.1 (C-4b), 141.4 (C-7), 141.5 (C-5a),
145.0 (C-2), 155.2 (C-10a), 166.3 (CO2Et), 173.3 (C-4). HRMS
(ESI): found 367.11107 ([M+H]+), C20H19N2O3S requires
367.11109; found 733.21527 ([2M+H]+), C40H37N4O6S2 requires
733.21490.
4.1.4. General procedure for the synthesis of 1-cyclopropyl-4-
oxo-1,5-dihydro-4H-pyrido[30,20:4,5]thieno[3,2-b]indole-3-
carboxylic acids (9a–e)
A suspension of the corresponding ester (8a–e) (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, the
residual yellow solid product was soaked in methanol (3–5 mL)
and collected by suction filtration.
4.1.3.3. Ethyl 1-cyclopropyl-7-methoxy-4-oxo-1,5-dihydro-4H-pyr-
ido[30,20:4,5]thieno[3,2-b]indole-3-carboxylate (8c). Irradiation
time: 1.75 h. Yield 40%; mp 303–305 °C (dec). Anal. Calcd for
C
20H18N2O4S (382.44): C, 62.81; H, 4.74; N, 7.32; S, 8.38. Found:
C, 62.61; H, 4.58; N, 7.27; S, 8.19. IR: mmax (KBr)/cmꢀ1 3320,
3077, 2982, 2931, 2840, 1728, 1601, 1565, 1499, 1485,
1463, 1419, 1346, 1306, 1274, 1256, 1216, 1154, 1107, 1034,
801, 787 and 696; 1H NMR (300 MHz, DMSO-d6): d 1.17 (m, 2H)
and 1.25 (m, 2H) (H2-20/H2-30), 1.28 (t, J = 7.1 Hz, 3H, CH3CH2O–),
3.75 (m, 1H, H-10), 3.77 (s, 3H, –OCH3), 4.22 (q, J = 7.1 Hz, 2H,
–OCH2Me), 6.77 (dd, J = 8.7, 1.2 Hz, 1H, H-8), 7.10 (d, J = 1.2 Hz,
1H, H-6), 7.66 (d, J = 8.7 Hz, 1H, H-9), 8.29 (s, 1H, H-2), 11.67
4.1.4.1.
thieno[3,2-b]indole-3-carboxylic acid (9a).
1-Cyclopropyl-4-oxo-1,5-dihydro-4H-pyrido[30,20:4,5]
Yield 96%; mp
295–297 °C (dec). Anal. Calcd for C17H12N2O3S (324.36): C, 62.95;
H, 3.73; N, 8.64; S, 9.89. Found: C, 62.65; H, 3.96; N, 8.31; S,
9.83. IR: mmax (KBr)/cmꢀ1 3309, 1706, 1601, 1554, 1477, 1335,
1303, 1034, 805, 736 and 712; 1H NMR (300 MHz, DMSO-d6): d
1.26 (m, 2H) and 1.37 (m, 2H) (H2-20/H2-30), 3.95 (m, 1H, H-10),
7.17 (dd, J = 7.2, 8.0 Hz, 1H, H-8), 7.26 (dd, J = 7.2, 8.1 Hz, 1H,
H-7), 7.62 (d, J = 8.1 Hz, 1H, H-6), 7.89 (d, J = 8.0 Hz, 1H,
H-9), 8.61 (s, 1H, H-2), 12.11 (s, 1H, N–H), 15.51(s, 1H, CO2H).
13C NMR (75 MHz, DMSO-d6): d 7.6 (C-20/C-30), 38.2 (C-10), 109.3
(C-9b), 111.9 (C-3), 113.6 (C-6), 119.1 (C-9), 120.3 (C-8),
120.9 (C-9a), 123.6 (C-7), 124.1 (C-4a), 135.4 (C-4b), 141.0
(C-5a), 145.2 (C-2), 155.7 (C-10a), 166.3 (–CO2H), 173.4 (C-4).
13
(s, 1H, N–H). C NMR (75 MHz, DMSO-d6): d 7.5 (C-20/C-30),
14.7 (CH3CH2O–), 36.9 (C-10), 55.8 (–OCH3), 60.4 (–OCH2Me), 96.9
(C-6), 109.9 (C-8), 114.8 (C-9b), 115.2 (C-3), 115.4 (C-9a), 119.3
(C-9), 120.0 (C-4a), 136.4 (C-4b), 141.7 (C-5a), 144.8 (C-2), 151.8
(C-10a), 156.8 (C-7), 165.0 (CO2Et), 169.6 (C-4). HRMS (ESI): found
405.08817([M+Na]+), C20H18N2O4SNa requires 405.08795; m/z (EI):
382 (M+, 100), 367 (17), 336 (14), 321 (20), 310 (30), 295 (60), 281
(10), 267 (9), 252 (6), 243 (9), 168 (7).

S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
2547
2
HRMS (ESI): found 323.05015 ([MꢀH]ꢀ), C17H11N2O3S requires
(75 MHz, DMSO-d6): d 7.6 (C-20/C-30), 38.2 (C-10), 99.6 (d, JC–F
=
2
323.04959.
26.3 Hz, C-6), 108.8 (d, JC–F = 24.4 Hz, C-8), 109.5 (C-9b), 111.9
(C-3), 116.8 (C-4a), 117.9 (C-9a), 120.6 (d, JC–F = 9.8 Hz, C-9),
136.0 (C-4b), 141.2 (d, JC–F = 12.7 Hz, C-5a), 145.2 (C-2), 155.5
3
3
4.1.4.2. 1-Cyclopropyl-7-methyl-4-oxo-1,5-dihydro-4H-pyrido
1
[30,20:4,5]thieno[3,2-b]indole-3-carboxylic acid (9b).
Yield
(C-10a), 160.0 (d, JC–F = 236 Hz, C-7), 166.2(CO2H), 173.3 (C-4).
95%; mp 350–352 °C (dec). Anal. Calcd for C18H14N2O3S (338.38):
C, 63.89; H, 4.17; N, 8.28; S, 9.48. Found: C, 63.62; H, 4.06; N,
8.15; S, 9.41. IR: mmax (KBr)/cmꢀ1 3320, 3029, 2968, 2957, 1743,
1714, 1608, 1568, 1506, 1481, 1452, 1408, 1372, 1336, 1314,
1223, 1037, 808 and 790; 1H NMR (300 MHz, DMSO-d6): d 1.25
(m, 2H) and 1.36 (m, 2H) (H2-20/H2-30), 2.43 (s, 3H, –CH3), 3.94
(m, 1H, H-10), 6.99 (d, J = 8.1 Hz, 1H, H-8), 7.41 (s, 1H, H-6), 7.75
(d, J = 8.1 Hz, 1H, H-9), 8.60 (s, 1H, H-2), 11.95 (s, 1H, N(5)–H),
HRMS (ESI): found 341.04062 ([MꢀH]ꢀ), C17H10N2FO3S requires
341.04016.
4.2. Biology
4.2.1. Antimicrobial activity
The in vitro antimicrobial assay was carried out against several
bacteria and fungi by using the twofold serial broth dilution meth-
od.32 Antibacterial activity was tested towards Gram positive bac-
teria (B. cereus ATCC 11778, B. megaterium ATCC 19213, B. subtilis
ATCC 6633, B. thuringiensis var. kurstaki BGSC 4D1, S. aureus ATCC
6538, S. epidermidis ATCC 12228, quinolone- and penicillin-resis-
tant clinical isolates of both S. aureus and S. epidermidis) and 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 both A. baumannii and
E. coli strains). Antifungal activity was obtained by means of
yeasts (S. cerevisiae ATCC 9763, C. tropicalis ATCC 1369) and mould
(A. niger ATCC 6275). The minimum inhibitory concentrations (MIC,
13
15.54 (s, 1H, CO2H). C NMR (75 MHz, DMSO-d6): d 7.6 (C-20/
C-30), 22.0 (–CH3), 38.2 (C-10), 109.0 (C-9b), 111.8 (C-3), 113.4
(C-6), 118.8 (C-9), 119.0 (C-9a), 119.2 (C-4a), 122.1(C-8), 133.1
(C-4b), 141.4 (C-7), 141.5 (C-5a), 144.9 (C-2), 155.2 (C-10a),
166.3 (CO2H), 173.3 (C-4). HRMS (ESI): found 337.06569 ([MꢀH]ꢀ),
C18H13N2O3S requires 337.06524.
4.1.4.3. 1-Cyclopropyl-7-methoxy-4-oxo-1,5-dihydro-4H-pyrido
[30,20:4,5]thieno[3,2-b]indo-le-3-carboxylic acid (9c).
Yield
96%; mp 317–319 °C (dec). Anal. Calcd for C18H14N2O4S (354.38):
C, 61.01; H, 3.98; N, 7.90; S. 9.05. Found: C, 60.77; H, 3.76; N,
7.86; S. 9.04. IR: mmax (KBr)/cmꢀ1 3333, 3039, 2977, 2951, 1750,
1719, 1601, 1566, 1511, 1478, 1448, 1396, 1377, 1341, 1305,
1233, 1042, 799 and 781; 1H NMR (300 MHz, DMSO-d6): d 1.25
(m, 2H) and 1.36 (m, 2H) (H2-20/H2-30), 3.79 (s, 3H, –OCH3), 3.93
(m, 1H, H-10), 6.81 (d, J = 8.1 Hz, 1H, H-8), 7.11 (s, 1H, H-6), 7.77
(d, J = 8.1 Hz, 1H, H-9), 8.75 (s, 1H, H-2), 11.92 (s, 1H, N-H), 15.58
l
g/mL) were determined as the lowest concentration for each com-
pound at which no growth was observed. Ciprofloxacin, levofloxa-
cin, moxifloxacin and gemifloxacin were screened as reference
antibacterial fluoroquinolones, miconazole as standard antifungal
drug.
Dimethyl sulfoxide solutions of the test compounds were pre-
pared and then diluted in the applicable media in a concentration
13
(s, 1H, CO2H). C NMR (75 MHz, DMSO-d6): d 7.6 (C-20/C-30),
38.2 (C-10), 55.8 (–OCH3), 96.8 (C-6), 109.7 (C-9b), 110.4 (C-8),
111.7 (C-3), 115.2 (C-9a), 117.8 (C-4a), 119.9 (C-9), 134.5 (C-4b),
142.2 (C-5a), 144.6 (C-2), 154.5 (C-10a), 157.3 (C-7), 166.4
(–CO2H), 173.2 (C-4). HRMS (ESI): found 353.07756 ([MꢀH]ꢀ),
range from 100 lg/mL to 0.0000015 lg/mL. Haemophilus Test
Medium, Mueller Hinton Broth and Sabouraud Dextrose Broth
were used as specific culture media for the growth of H. influenzae,
other bacteria and fungi, respectively. Tubes were inoculated with
a suspension of the different bacteria (5x105 CFU/mL) and fungi
(1ꢁ103 CFU/mL). At the end of an incubation period of 24 h at
37 °C (bacteria) or 48 h at 30 °C (fungi), the minimum inhibitory
concentrations were detected. In addition, for each microorganism,
drug-free tubes and tubes containing only dimethyl sulfoxide were
tested as growth and solvent controls, respectively. Organism-free
tubes were kept as sterility controls.
C18H13N2O4S requires 353.07743.
4.1.4.4. 1-Cyclopropyl-6,8-dimethyl-7-methoxy-4-oxo-1,5-dihy-
dro-4H-pyrido[30,20:4,5]thieno[3,2-b]indole-3-carboxylic
acid
(9d). Yield 94%; mp 320–322 °C (dec). Anal. Calcd for C20H18N2O4S
(382.44) requires C, 62.81; H, 4.74; N, 7.33; S, 8.38. Found:
C, 62.78; H, 4.74; N, 7.26; S, 8.24. IR: mmax (KBr)/cmꢀ1 3397,
2935, 1706, 1608, 1554, 1463, 1408, 1328, 1303, 1256,
1154, 1088, 1030, 998, 852, 801 and 744; 1H NMR (300 MHz,
DMSO-d6): d 1.24 (m, 2H) and 1.35 (m, 2H) (H2-20/H2-30), 2.32
(s, 3H, C(8)–CH3), 2.50 (s, 3H, C(6)–CH3), 3.68 (s, 3H, –OCH3),
3.92 (m, 1H, H-10), 7.51 (s, 1H, H-9), 8.58 (s, 1H, H-2), 11.58
(s, 1H, N-H), 15.72(s, 1H, CO2H). 13C NMR (75 MHz, DMSO-d6): d
7.6 (C-20/C-30), 11.3 (C(6)–CH3), 17.0 (C(8)–CH3), 38.2 (C-10), 60.5
(–OCH3), 109.9 (C-9b), 111.7 (C-3), 117.4 (C-9), 118.0 (C-9a),
124.0 (C-4a), 135.6 (C-4b), 140.2 (C-5a), 140.3 (C-6), 140.6 (C-8),
144.7 (C-2), 154.0 (C-7), 155.0 (C-10a), 166.4 (CO2H), 173.2 (C-4).
HRMS (ESI): found 381.09207 ([MꢀH]ꢀ), C20H17N2O4S requires
381.09145.
Three replicates were done with each microorganism and at
least three independent experiments were performed.
4.2.2. DNA gyrase and topoisomerase IV inhibition
Test compounds were evaluated for their inhibitory activity
against quinolone target enzymes by using E. coli DNA gyrase
and topoisomerase IV (Inspiralis, Norwich, UK) and were compared
to reference drugs ciprofloxacin and moxifloxacin.
To detect the inhibition of DNA gyrase activity, supercoiling as-
say was carried out with relaxed pBR322 DNA as a substrate. Sev-
eral amounts of each compound were incubated at 37 °C with
35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM dithio-
threitol, 1.8 mM spermidine, 1 mM ATP, 6.5% w/v glycerol,
4.1.4.5.
1-Cyclopropyl-7-fluoro-4-oxo-1,5-dihydro-4H-pyrido
Yield
0.1 mg/mL albumin, 16 ng/lL relaxed pBR322 DNA and 0.026 U/lL
[30,20:4,5]thieno[3,2-b]indole-3-carboxylic acid (9e).
of gyrase protein. After 30 min the reactions were stopped by
the addition of a chloroform/isoamyl alcohol 24:1 solution and
bromophenol blue dye. The blue aqua phase was then loaded on
1.0% w/v agarose gel and analyzed by electrophoresis.
The inhibition of topoisomerase IV activity was detected in the
decatenation assay using kinetoplast DNA as a substrate. Com-
pounds were treated in different amounts with reaction mixtures
containing 40 mM HEPES-KOH pH 7.6, 100 mM potassium gluta-
96%; mp 335–337 °C (dec). Anal. Calcd for C17H11N2FO3S (342.35)
requires C, 59.64; H, 3.24; N, 8.18; S, 9.37. Found: C, 59.44; H,
3.21; N, 8.06; S, 9.28. IR: mmax (KBr)/cmꢀ1 3331, 3048, 1714,
1612, 1583, 1565, 1499, 1481, 1339, 1310, 1267, 1114, 1038
and 794; 1H NMR (300 MHz, DMSO-d6): d 1.26 (m, 2H) and 1.37
(m, 2H) (H2-20/H2-30), 3.94 (m, 1H, H-10), 7.02 (ddd, J = 2.0, J = 8.8,
3
3
Hz, JH–F = 9.8 Hz, 1H, H-8), 7.33 (dd, J = 2.0 Hz, JH–F = 9.9 Hz, 1H,
4
H-6), 7.93 (dd, J = 8.8 Hz, JH–F = 3.3 Hz, 1H, H-9), 8.60 (s, 1H,
mate, 10 mM Mg(OAc)2, 10 mM dithiothreitol, 1 mM ATP, 50
lg/mL
H-2), 12.20 (s, 1H, N(5)-H), 15.45 (s, 1H, CO2H). 13C NMR
albumin, 6.6 ng/ L kDNA and 0.016 U/ L topoisomerase IV protein.
l
l

2548
S. A. Al-Trawneh et al. / Bioorg. Med. Chem. 19 (2011) 2541–2548
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activity observed for the enzymes in drug-free controls.
Representative results of at least three independent experi-
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4.2.3. Cell proliferation assay
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MCF-7 breast cancer cells and A549 NSCLC cell lines were ob-
tained from ATCC and were cultured in RPMI; human-derm fibro-
blasts (HuDe) were obtained from Istituto Zooprofilattico
Sperimentale della Lombardia e dell’Emilia Romagna, Italy, and
were cultured in DMEM. All media were supplemented with
2 mM glutamine and 10% Fetal Bovin Serum (FBS, Gibco Life Tech-
nologies) and cells were maintained under standard cell culture
conditions at 37 °C in a water-saturated atmosphere of 5% CO2 in
air.
Cells were treated with the indicated compounds at concentra-
tions ranging from 0.1 to 20 lM for 72 h. Cell viability was assessed
with tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT), obtained from Sigma (Dorset, UK),
and assayed as previously described.33 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). Concentrations
that inhibit 50% (IC50) (e.g., the point at which viability is 50%)
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.
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Acknowledgment
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We wish to thank the Higher Council for Science and Technol-
ogy (Amman, Jordan) for financial support.
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