Synthesis and antibacterial property of quinolines with potent DNA gyrase activity

Article abstract of DOI:10.1016/j.bmc.2008.11.058

Synthesis of a series of novel tetrahydroquinoline annulated heterocycles has been accomplished by intramolecular imino and bisimino Diels-Alder reaction. These compounds were evaluated for their antibacterial activity. All the synthetic compounds, exhibi

Full text of DOI:10.1016/j.bmc.2008.11.058

Bioorganic & Medicinal Chemistry 17 (2009) 660–666
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Bioorganic & Medicinal Chemistry
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Synthesis and antibacterial property of quinolines with potent DNA
gyrase activity
Ekambaram Ramesh a, Rathna Durga R. S. Manian a, Ragavachary Raghunathan a,
,
*
Shilpakala Sainath b, Malathi Raghunathan b
a Department of Organic Chemistry, Guindy Campus, University of Madras, Chennai 600025, India
b Department of Genetics, Dr. ALMPGIBMS, Taramani Campus, University of Madras, Chennai 600113, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a series of novel tetrahydroquinoline annulated heterocycles has been accomplished by
intramolecular imino and bisimino Diels–Alder reaction. These compounds were evaluated for their anti-
bacterial activity. All the synthetic compounds, exhibited good antibacterial activity against microorgan-
isms of which one of them 7 was found to be as active as the antibiotic ciplofloxacin and is found to have
MIC value of 2.5 mg/mL against Escherichia coli.
Received 25 September 2008
Revised 21 November 2008
Accepted 21 November 2008
Available online 3 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Quinolone
Gyrase
Antibacterial
Bisquinolone
1. Introduction
10, 11 and 12 were synthesized by imino Diels–Alder reaction
(Schemes 3 and 4). All the compounds were characterised by spec-
In recent years the emergence of multi-drug resistant organisms
poses a challenge in the treatment of infectious diseases.1 Hence
there arises a need to synthesise new and effective antimicrobial
drugs to overcome this problem. Over the past decade bacterial
DNA gyrase has drawn much attention as a selected target of potent
antibacterial agents.2 A number of synthetic quinolone antibacterial
agents have been developed and are now widely used in the treat-
ment of infectious diseases.3–7 Quinolones are known to inhibit
DNA gyrase and topoisomerase IV and cause bacterial cell death.
Novobiocin, a naturally occurring antibacterial compound, has been
identified as a potential inhibitor8 of DNA gyrase. In our study we
have synthesized five different types of quinolines (4, 7, 10, 11, 12)
and examined their antibacterial activity against various bacterial
strains considering gyrase as a target. All these compounds were
synthesised by inter and intramolecular imino Diels–Alder reaction
starting from aromatic amines and various carbonyl compounds.
Synthesis of hexahydropyranotetrahydroquinoline derivative 4
was accomplished by a one pot reaction of indane 1,2,3-trione p-
bromo aniline, dihydropyran in the presence of indium trichloride
as a catalyst, using acetonitrile as solvent (Scheme 1). Similarly
uracil annulated quinoline derivative 7 was synthesized by imino
Diels–Alder reaction of N-prenyl pyrrolopyrimidine-6-carbalde-
hyde with p-chloroaniline in the presence of indium trichloride
as catalyst (Scheme 2). In the same way the quinoline derivatives
troscopic and elemental analysis.
2. Results and discussion
Antibacterial activity for the above synthesized compounds (4,
7, 10, 11, 12) were evaluated against six different bacterial strains
1. Staphylcoccus aureus, 2. Escherichia coli, 3. Pseudomonas aerugin-
osa, 4. Klebsiella pneumonia, 5. Salmonella typhimurium, 6. Bacillus
subtilis. Disc diffusion assay was carried out and the zones of inhi-
bition for different concentrations of the synthetic compounds
were measured and the results are given in Table 1. Ciprofloxicin
was used as a reference drug.
From Table 1 it can be seen that all the compounds exhibited
antibacterial activity against various strains. Good inhibitory char-
acteristics are observed for the compounds even for small concen-
tration of 5 lg. Compounds 4, 10, 11, 12 inhibited the growth of
the pathogens particularly P. aeruginosa, while compounds 7, 10,
11 showed good growth inhibition againstK. pneumonia. The inhib-
itory effect was more pronounced for gram ꢀve strains than gram
+ve strains.
2.1. Minimum inhibitory concentration (MIC) of the
compounds using broth micro dilution assay
We further determined the MIC values of the quinoline deriva-
tives against different bacterial strains and these are given in Table
2. The effect of various compounds on the growth of the organisms
* Corresponding author. Tel.: +91 44 22202811; fax: +91 44 22352494.
E-mail address: ragharaghunathan@yahoo.com (R. Raghunathan).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.058

E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
661
NH2
O
H
O
O
O
O
H
O
20 mol% InCl3
CH3CN, rt
O
HN
Br
Br
4
3
1
2
Scheme 1.
O
O
N
NH2
N
N
20 mol% InCl3
CH3CN, rt
O
N
N
CHO
O
N
NH
Cl
7
5
6
Cl
Scheme 2.
nalidixic acid. The other compounds 4, 10 also inhibited DNA gyr-
ase supercoiling, but to a lesser extent when compared to cipro-
floxacin. It is interesting to note that compound 7 consists of an
uracil moiety that might be responsible for the inhibition of gyrase.
NH2
H
H
20 mol% InCl3
CH3CN, rt
N
NH
N
CHO
H
H
Ph
R
R
2.3. Gyrase relaxation assay
Ph
9a, R=H
9b,R=OMe
10 R=H
11 R=OMe
8
To ascertain the effect of the synthetic compounds on the other
activity of gyrase, namely DNA relaxation, different concentrations
of the drugs were incubated with the enzyme and the relaxation of
the supercoiled DNA was evaluated in the absence of ATP. Cipro-
floxacin, novabiocin and nalidixic acid were used for comparison.
Inhibition in DNA relaxation was observed for compounds 7 and
Scheme 3.
was analysed. Uniform amount of inoculum was added to each
tube and after adding the compounds in the mid-log phase, the
turbidity of the medium was measured spectrophotometrically at
A600. The concentration at which the growth of the organisms
was inhibited by 50% and 90% and the range of the concentrations
within which there was no visible growth were determined and
provided in Table 2. From the values it can be seen that significant
inhibition was observed at lower concentrations for the tested
strains, particularly, against E. coli. The average percentage of
growth was found to be 44.1%, 50%, 42.3%, 45.7% and 49% for the
compounds 4, 7, 10, 11, 12, respectively (Fig. 1.1).
12 at a concentration of 1
11, inhibition was not observed even at a higher concentration of
M while for the compound 10 inhibition was found at a concen-
tration greater than 2.5 M. Novabiocin did not inhibit even at a
concentration of 5 M. Nalidixic acid showed inhibition at concen-
tration greater than 2.5 M and ciprofloxacin showed inhibition at
a concentration of 1 M.
lM. In the case of compounds 4 and
5
l
l
l
l
l
2.4. Reverse transcriptase-polymerase chain reaction analysis
Total RNA isolated from the bacterial cells was treated with the
synthetic compounds and RT-PCR was carried out to assess gyrase
gene expression, as outlined in Section 3. The products of RT-PCR
were electrophoresed on a 1% agarose gel keeping GAPDH as the
housekeeping gene. The results showed a very significant decrease
in the intensity of the band of 661 that corresponds to gyrase DNA.
The reduction was higher for compounds 7, 11, compared to that
observed for 4, 10, 12. These results (Fig. 2.8) correlate well with
those obtained from gyrase supercoiling and relaxation assay. Total
RNA without drug treatment was used as control.
2.2. Gyrase supercoiling assay
DNA gyrase supercoiling activity was assessed by measuring
the conversion of relaxed plasmid Pbr322 DNA to the supercoiled
form. The supercoiling assay was performed as described in Section
3 and inhibition of gyrase supercoiling due to the compounds 4, 7,
10 11, 12 was analysed. The results (Figs. 1.2–1.7) showed that
compound 10 inhibited DNA gyrase supercoiling while the com-
pounds 7, 12 were found to inhibit gyrase supercoiling with poten-
cies similar to that of known drugs such as ciprofloxacin, and
20 mol% InCl3
CH3CN, rt
N
CHO
H
HN
N
NH
N
H2N
NH2
Ph Ph
9c
H
H
Ph
12
8
Scheme 4.

662
E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
Table 1
The zone of inhibition for different strains
Compound
Strain
Zone of inhibition (in cm)
5
lg
10
lg
15
lg
20
lg
25 lg
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
0.8
0.8
1
0.8
0.8
0.9
0.9
1.4
1.0
1.0
1
1
1.6
1.3
1.3
0.8
1.2
1.2
2
1.4
1.4
0.9
1.4
1.3
2.3
1.6
1.6
1.0
O
H
O
O
H
HN
Br
—
—
4
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
2.0
1
1.0
2.0
1.0
2.4
1.6
1.4
2.6
1.6
2.6
1.8
1.6
3.0
1.8
0.6
3.0
2
2.0
3.4
2.2
0.7
3.6
2.3
2.3
3.6
2.4
0.9
O
N
N
O
N
NH
—
—
Cl
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
0.9
1.0
1
2.0
1.0
1.2
1.6
1.6
2.6
1.6
0.7
1.3
1.8
1.7
3.0
1.8
0.6
1.4
2.0
2.2
3.4
2.2
0.7
1.7
2.3
2.4
3.6
2.4
0.9
H
H
N
NH
H
Ph
—
H
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
2.0
1.2
2.4
1.0
1.0
2.4
1.4
2.6
1.4
1.6
0.8
2.6
1.6
2.8
1.6
1.8
0.9
3.0
2.2
3.4
2.4
2.2
1.0
3.6
2.3
3.6
2.6
2.4
1.1
H
H
N
NH
H
Ph
—
OMe
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
0.9
1.1
2.0
1.2
1.0
0.7
1.3
1.4
2.4
1.6
1.6
0.9
1.5
1.6
2.6
1.9
1.8
1.0
1.7
2.1
2.8
2.4
2.2
1.2
2.1
2.2
3.0
2.6
2.4
1.3
HN
NH
Ph Ph
H
H
N
N
Ciplofloxicin
Staphylcoccus aureus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumonia
Salmonella typhimurium
Bacillus subtilis
2.2
1.4
1.2
2.4
1.4
0.6
2.4
1.6
1.4
2.6
1.6
0.7
2.6
1.8
1.6
3.0
1.8
0.9
3.0
2.0
2.0
3.4
2.2
1.0
3.6
2.3
2.3
3.6
2.4
1.2
All the five quinoline derivatives showed antimicrobial activ-
ity as indicated by disc diffusion assay. The compounds 7 and
11 showed significant activity against S. aureus, E. coli, K. Pneu-
monia, P. aeruginosa, S. typhi and reduced activity against B.
subtilis.
The MIC values, were determined for the compounds 4, 7,
10, 11, 12 for the above mentioned organisms and the values
of MIC required to inhibit 50% and 90% (MIC50, MIC90) of the
growth are shown in Table 2. Analysis has suggested that
gram-negative bacteria especially Pseudomonas was inhibited
strongly by the five quinoline derivatives. All the quinoline
derivatives showed good activity against members of Enterobac-
teriaceae. The inhibitory action increases with increase in the
concentration of the compounds. Considerable antimicrobial
activity was observed against E. coli. The percentage of growth
was found to be 44.1%, 50%, 42.3%, 45.7% and 49% for the com-
pounds 4, 7, 10, 11 and 12, respectively. The MIC values and
the observed growth inhibition against E. coli has demonstrated
that the compounds 7, 12 might act as potent antibacterial
agents.
The gyrase supercoiling is inhibited by all the quinoline deriva-
tives of which compounds 7 and 12 inhibited gyrase supercoiling
with potencies similar to that of known drugs ciprofloxacin and
nalidxic acid. Gyrase relaxation is noticed for all synthesized quin-
oline derivatives with considerable activity for 11 and 12 at
concentration, For compounds 4, 7 inhibition was not observed
even at a concentration of 5 M and for the compound 10 the inhi-
bition was found at a concentration greater than 2.5 M.
lM
l
l

E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
663
Table 2
(ACME) of 0.25 mm thickness and visualized with iodine. For the
dry experiments, glasswares used were thoroughly dried in a hot
air oven, cooled and assembled under a stream of nitrogen. The or-
ganic extracts of crude products were dried over anhydrous MgSO4.
Solventswerereagentgradeandwerepurifiedaccordingtostandard
procedures. All the chemicals used were commercially available.
The MIC of the compound tested against organisms
Organisms
Compound
Range
MIC (mg/mL)
90%
50%
64
S. aureus
4
7
10
11
8–128
64
—
0.125–8
16–128
0.125–1
32–128
0.125–1
0.250
64
0.5
128
0.5
128
0.5
3.1. Procedure for the synthesis of compound 4
13
128
1
Ciprofloxacin
A mixture of indane 1, 2, 3 trione (1 mmol), 4-bromo aniline
(1 mmol) and 3,4-dihydro-2H-pyran (1 mmol) in the presence of
20 mol % InCl3 in acetonitrile (6 mL) was stirred at room tempera-
ture until the completion of the reaction as evidenced by TLC. The
reaction mixture was quenched with water and extracted with ethyl
acetate (3 ꢁ 10 mL), washed with brine and dried over Na2SO4. After
theremovalof solventunderreduced pressure, thecruderesidue ob-
tained was eluted through column chromatography with petroleum
ether ethyl acetate mixture (7:3) to afford 4. Yield 75%.
E. coli
4
7
10
11
>128
0.03–4
>128
0.06–16
1–>128
0.0075–1
2
0.06
2
0.125
2
0.015
4
0.25
4
0.25
8
13
Ciprofloxacin
0.03
K. pneumonia
P. aeruginosa
S. typhi
4
7
10
11
>125–16
0.015–8
0.5–>128
0.015–16
0.5–128
0.0075–2
0.25
0.125
2
0.25
0.125
0.03
4
2
32
2
2
13
3.2. (4aS,10bS)-9-Bromo-3,4,4a,5,6,10b-hexahydro-5-indane 1,3
dione-2H-pyrano[3,2-c]quinoline 4
Ciprofloxacin
0.5
4
7
10
11
0.5–16
0.06–2
0.06–64
0.06–2
0.25–32
0.015–8
1
8
1
32
1
8
1
0.125
0.5
0.125
1
Red orange powder; mp 224 °C; yield: 69% IR (KBr): 1732 cmꢀ1
;
1H NMR (300 MHz, CDCl3): 1.13–1.42 (m, 4H), 1.73–2.12 (m, 2H),
3.12 (dt, J = 12.2, 3.5 Hz, 1Hb), 4.33 (s, 1H, NH), 5.12 (d, J = 3.5 Hz,
1Ha), 7.44–7.38 (m, 7H),; 13C NMR (75 MHz, CDCl3): 21.3, 23.1,
33.3, 60.7, 62.4, 65.5, 10.6, 111.5, 114.7, 120.3, 124.0, 124.8, 126.8,
130.7, 133.4, 137.4, 142.8, 153.5, 158.9, 177.8, 181.61. Mass m/z:
397.05 (M+). Calculated for C20H16 BrNO3: C, 60.32; H, 4.05; N,
3.52; Br, 20.06. Found: C, 60.45; H, 4.13; N, 3.43.
13
Ciprofloxacin
0.125
4
7
10
11
0.3–5
0.01–0.3
0.3–5
<0.0025–0.15
0.8–40
<0.0025–0.3
1.64
0.09
1.64
0.06
2.51
0.04
2.5
0.15
2.5
0.15
5
0.15
13
Ciprofloxacin
B. subtilis
4
7
10
2.5–5
0.6–1.2
1.2–5
5
0.25
2.5
5
1.2
5
3.3. General procedure for synthesis of compound 7
InCl3 (20 mol %, 44 mg) was added to a mixture of p-chloro ani-
line (1 mmol, 270 mg) and N-alkenyl pyrrolo[2,3-d]pyrimidine-6-
carbaldehyde (500 mg) in acetonitrile (5 mL). The reaction mixture
was stirred at room temperature until the completion of the reac-
tion as indicated by TLC. The mixture was then quenched with
water and extracted with ethyl acetate. The organic layer was
washed with water and dried over MgSO4. The solvent was evapo-
rated in vacuo and the crude product was chromatographed on sil-
ica gel using petroleum ether ethyl acetate mixture (7:3) to afford
compound 7.
11
13
0.15–5
0.6–1.2
<0.0025–0.3
0.30
0.25
0.04
2
5
0.15
Ciprofloxacin
The results are suggestive of down regulation of gyrase gene by
compounds 7 and 12 which is significantly higher than that ob-
tained for 4 and 10. Thus compounds 7 and 12 can be considered
as potent gyrase inhibitors whose activity is similar to the quino-
lones ciprofloxacin in inhibiting gyrase supercoiling and relaxation.
Thus the quinoline derivatives synthesized presently might rapidly
inhibit bacterial deoxyribonucleic acid replication, thereby leading
to cell death.
With the increasing incidence of drug resistance, the develop-
ment of new drugs is essential to successfully combat bacterial dis-
eases and the current analysis has demonstrated the ability of the
synthesised compounds to inhibit DNA gyrase. Thus gyrase inhib-
itors especially modified quinolines, might gain importance as a
new class of highly potent gyrase inhibitors that can serve as lead
compounds for drug development.
3.4. 7a,13b-8,8-Dimethyl pyrrolo[2,3-d]pyrimidine-[2,1-
a]pyrrolo[4,3:2,3]-7a,8,13,13b-tetrahydroquinoline 7
Mp 191 °C, Yield: 92% 1H NMR (MHz, CDCl3): d 1.52 (s, 3H),
1.55 (s, 3H), 3.23 (td, J = 9.7, 10.2 Hz, 1Hb), 3.41 (s, 3H), 3.46
(s, 3H), 3.61 (t, J = 10.2 Hz, 1Hd), 4.10 (dd, J = 7.7, 9.7 Hz, 1Hc),
4.69 (d, J = 11.2 Hz, 1Ha), 5.18 (s, 1H), 6.47 (d J = 8.2 Hz, 1H),
6.84 (s, 1H), 7.12 (d, J = 8.2 Hz, 1H). 13C NMR (MHz, CDCl3): d
25.68, 27.17, 29.22, 30.51, 35.18, 36.64, 55.14, 60.14, 104.14,
107.46, 120.46, 121.27, 123.37, 126.41, 130.79, 132.34, 137.46,
139.49, 154.79, 159.20 ppm. Mass m/z: 384.14 (M+). Calculated
for C20H21ClN4O2: C, 62.42; H, 5.50; N, 14.51. Found: C, 62.56;
H, 5.62; N, 14.38.
3. Materials and experimental methods
All melting points are uncorrected. IR spectra were recorded on a
SHIMADZU FT-IR 8300 instrument. 1H and 13C NMR spectra were re-
corded in CDCl3 using TMS as an internal standard on Bruker 300 at
300 MHz and at 75 MHz, respectively. Mass spectra were recorded
on a JEOL DX 303 HF-mass spectrometer. Most of the chemicals used
were purchased commercially. Column chromatography was per-
formed on silica gel (ACME, 100–200 mesh). Flash column chroma-
tography was performed on silica gel (Sisco, 230–40 mesh).
Routine monitoring of the reactions was made using thin layer chro-
matography developed on glass plates coated with silica gel-G
3.5. Procedure for synthesis of compound 10 and 11
InCl3 (20 mol %, 44 mg) was added to a mixture of aniline
(1 mmol, 270 mg) or p-methoxy aniline and N-cinnamyl pyrrole-
2-carbaldehyde (1 mmol, 500 mg) in acetonitrile (5 mL). The reac-
tion mixture was stirred at room temperature until the completion
of the reaction as indicated by TLC. The work up procedure is same
as mentioned above for 7 afford the product 10 and 11.

664
E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
Figure 1. (a) Growth inhibitory effects of synthetic compounds 4, 7, 10, 11, 12 on E. coli. (b–c) Inhibition of DNA gyrase supercoiling assay by synthetic compounds. Pbr322
topoisomers were analysed by agarose gel electrophoresis. Lane 1–9 increasing concentration of synthetic compounds at 0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 mM, respectively.
3.6. (3bR,9aR)-4,9,9a,10-Tetrahydro-9-phenyl-3bH-
pyrrolizino[1,2-b]quinoline 10
(d, J = 9.2 Hz, 1H), 5.12–5.28 (m, 1Hb), 6.44–7.25 (m, 7H). 13C NMR
(CDCl3): d 42.30, 45.81, 46.60, 63.21, 106.16, 107.67, 113.68,
115.23, 121.34, 125.10, 127.23, 126.36, 125.42, 126.41, 124.91,
131.24, 146.06. Mass m/z: 286.15 (M+). Calculated for C20H18N2: C,
83.88; H, 6.34; N, 9.78. Found: C, 83.99; H, 6.43; N, 9.65.
For compound 10: Liquid, yield: 80%, 1H NMR (CDCl3): d 4.23 (d,
J = 6.1 Hz, 1Ha), 5.63 (dd, J = 14.1, 7.5, 1Hc), 5.63 (t, J = 6.3, 1Hd), 5.07

E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
665
Figure 2. (a–b) Effect of synthetic compounds on gyrase relaxation. Gyrase (60 nM) and supercoiled pBR322 DNA (6 nM) were incubated with increasing concentrations of
drugs and ciprofloxacin, novabiocin and nalixidic acid. Concenration—0, 0.1, 0.25, 0.5, 2.5 mM for 1 h at 37 °C. (c) RT-PCR amplification of the total RNA from the drug (0.5
mM) treated E. coli. Lane M: marker, Lane 1: cells treated with ER1, Lane 2: cells treated with ER2, Lane 3: cells treated with RD1, Lane 4: cells treated with RD2, Lane 5: cells
treated with RD3, Lane 6: negative control (cells without any drug treatment), Lane 7: cells treated with ciprofloxacin. Reverse transcriptase-polymerase chain reaction analysis:
RT-PCR was carried out on the total RNA from the cells treated with the synthesis drugs and known compounds to assess gyrase expression and the products of the RT-PCR
were electrophoresed on a 1% agarose gel. GAPDH was used as the housekeeping gene. The results showed a very significant decrease in the band intensity in the cells treated
with 2, 4. These results (2.8) correlated well the gyrase inhibition assays namely supercoiling and the relaxation assay. There was also decrease in band intensity in the cells
treated with 4, 10, 11. These results were compared with ciprofloxacin, novabiocin and nalidixic acid. Total RNA without any drug treatment was used as control.
3.7. (3bR,9aR)-4,9,9a,10-Tetrahydro-7-methoxy-9-phenyl-3bH-
pyrrolizino[1,2-b]quinoline 11
126.72, 127.42, 128.47, 126.90, 134.15, 145.16. Mass m/z: 316.16
(M+). Calculated for C20H18N2O: C, 79.72; H, 6.37; N, 8.85. Found:
C, 79.83; H, 6.46; N, 8.74.
For compound 11: Liquid, yield: 87%, 1H NMR (CDCl3): d 3.12 (s,
3H), 4.36 (d, J = 5.8 Hz, 1Ha), 5.45 (dd, J = 13.9, 7.2, 1Hc), 5.59 (t,
J = 5.9, 1Hd), 5.23 (d, J = 9.0 Hz, 1H), 5.01–5.23 (m, 1Hb), 6.15–
7.21 (m, 6H). 13C NMR (CDCl3): d 30.14, 41.45, 44.80, 45.55,
62.10, 112.14, 117.63, 112.64, 114.16, 121.13, 125.78, 127.16,
3.8. Procedure for synthesis of compound 12
InCl3 (20 mol %, 44 mg) was added to a mixture of 4,40-meth-
ylenedianiline (1 mmol, 230 mg) and N-cinnamyl pyrrole-2-carb-

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E. Ramesh et al. / Bioorg. Med. Chem. 17 (2009) 660–666
aldehyde (2 mmol, 500 mg) in acetonitrile (5 mL). The reaction
mixture was stirred at room temperature until the completion
of the reaction as indicated by TLC. Similarly the work up
mentioned in the above procedure to afford the product com-
pound 12.
Supercoiling assays were carried out in 15
containing the DNA gyrase assay buffer, 150 ng relaxed pBR322,
and 2–3 L purified DNA gyrase. The mixture was incubated for
lL reaction mixtures
l
3 h at 37 °C for E. coli, the reaction was stopped by the addition
of 50% glycerol containing 0.25% bromophenol blue, and the total
reaction mixture was subjected to electrophoresis on 1% agarose
gel in 1ꢁTBE buffer (Tris/borate/EDTA, pH 8.3). After a run of 3 h
at 90 V, the gel was stained with ethidium bromide (0.7 l
g mLꢀ1
3.9. Bis(4,9,9a,10-Tetrahydro-9-phenyl-3bH-pyrrolizino[1,2-
b]quinolin-7-)methane 12
supercoiling activity was assessed by tracing the brightness of
the bands corresponding to the supercoiling pBR322 DNA, using
a Densylab densitometer (Bio-Rad). One unit (U) of enzyme activity
was defined as the amount of DNA gyrase that converted 150 ng
relaxed pBR322 to the supercoiling form in 30 min at 37 °C for
E. coli.
Yield: 75%; mp 182 °C, 1H NMR (CDCl3): d 3.33 (s, 2H, –CH2–),
4.84 (d, J = 9.9 Hz, 2H), 4.73 (dd, J = 14.8, 7.6 Hz, 2H), 4.84 (t,
J = 6.5 Hz, 2H), 5.12–5.18 (m, 2H), 5.12 (d, J = 8.9 Hz, 2H), 6.33–
7.96 (m, 22H). 13C NMR: d 30.23, 33.13, 41.23, 42.84, 46.23,
98.32, 113.45, 113.23, 120.76, 122.55, 122.78, 123.21, 123.54,
126.18, 127.45, 128.12, 132.84, 131.80, 135.98, 141.60 ppm. Mass
m/z: 584.29 (M+). Calculated for C41H36N4: C, 84.21; H, 6.21; N,
9.58. Found: C, 84.33; H, 6.32; N, 9.42.
4.4. RT-PCR analysis of gyrase gene
Total RNA was isolated from the transformed strains that were
collected at regular intervals upto 24 h, Two microgram of total
RNA was used as template for the synthesis of first strand cDNA
by reverse transcriptase using MMLV. Primers were designed using
primer Express 1.5 software (Applied Biosystems, CA, USA). PCR
4. Methods
4.1. Diffusion method
was performed using 1
containing 2
L 10ꢁ Taq buffer, 1
0.5 L of Taq polymerase, 12.5 L of sterile water and 2 l
l
L of Taq DNA polymerase in 20
l
L reaction
L of dNTPs,
L cDNA
l
l
L of each primer, 1
l
Disc diffusion assay was performed to determine the rate of
inhibition in the growth of bacteria. Inoculum was prepared in
0.85% saline using McFarland standard and spread uniformly on
nutrient agar plates with cotton swabs. Sterile Whatman no. 4 fil-
ter paper discs were placed at equidistance on the agar surface and
drugs of equal volume and varying concentration was placed on
each disc. The zone of inhibition for each concentration was mea-
sured after 24 h incubation at 37 °C.
l
l
template. PCR amplification of gyrase gene was conducted under
the following conditions: an initial cycle of denaturation step at
94 °C for 4 min and final extension step at 72 °C for 7 min. The
RT-PCR products were run on 1.5% agarose gel. Bands were visual-
ized and quantitative data normalized against GAPDH. Densito-
metric analysis of the band was performed using National
Institute of Health (NIH)—Densitometric Image software, available
in the public domain (http://rsb.info.nih.gov/nihimage). The values
are expressed in arbitrary units. This analysis was carried out for
all strains mentioned above.
4.2. MIC of the compounds by broth micro dilution assay
Broth micro dilution was carried out to determine the MIC of
the synthetic compounds as per the protocol of National Com-
mittee of Clinical Laboratory Standards (NCCLS), 1997. A 96 well
sterile microtiter plate with appropriate drug dilution was la-
beled. Antibiotic solution was added to the well. A final inocu-
lum of 105 cfu/mL of the organism was prepared. The test
organism suspension was added to one row and the control
organism was added to the other row. It was incubated at
35.7 °C in air for 18–20 h. The MIC end point was read as the
lowest concentration of antibiotic at which there was no visible
growth.
References and notes
1. (a) Niccolai, D.; Tarsi, L.; Thomas, R. J. Chem. Commun. 1997, 2333; (b) Chu, D. T.
W.; Plattner, J. J.; Katz, L. J. Med. Chem. 1996, 39, 3853.
2. Ferrero, L.; Cameron, B.; Manse, B.; Lagneaux, D.; Crouzet, J.; Famechon, A.;
Blanche, F. Mol. Microbiol. 1994, 13, 641.
3. Rudolph, J.; Theis, H.; Hanke, R.; Endermann, R.; Johannsen, L.; Geschke, F.-U.
J. Med. Chem. 2001, 44, 619.
4. Gray, J. L.; Almstead, J.-I. K.; Gallagher, C. P.; Hu, X. E.; Kim, N. K.; Taylor, C. J.;
Twinem, T. L.; Wallace, C. D.; Ledoussal, B. Bioorg. Med. Chem. Lett. 2003, 13,
2373.
5. Tanitame, A.; Oyamada, Y.; Ofuji, K.; Fujimoto, M.; Iwai, N.; Hiyama, Y.; Suzuki,
K.; Ito, H.; Terauchi, H.; Kawasaki, M.; Nagai, K.; Wachi, M.; Yamagishi, J. J. Med.
Chem. 2004, 47, 3693.
4.3. Gyrase supercoiling assay
6. Oblak, M.; Grdadolnik, S. G.; Kotnik, M.; Jerala, R.; Filopic, M.; Solmajer, T. Bioorg.
Med. Chem. Lett. 2005, 15, 5207.
7. Miyamoto, T.; Matsumoto, J.; Chiba, K.; Egawa, H.; Shibamori, K.; Minamida, A.;
Nishimura, Y.; Okada, H.; Kataoka, M.; Fujita, M.; Hirose, T.; Nakano, J. J. Med.
Chem. 1990, 33, 1645.
The gyrase supercoiling was assessed by measuring the conver-
sion of relaxed plasmid pBR322 DNA to the supercoiled form, as
described previously (bazile et al., Revel-Viravau et al., 1996).
8. Maxwell, A. Mol. Microbiol. 1993, 9, 681.