Synthesis, antimycobacterial and antibacterial activity of ciprofloxacin derivatives containing a N-substituted benzyl moiety

Article abstract of DOI:10.1016/j.bmcl.2012.07.040

We report herein the design and synthesis of a series of novel ciprofloxacin (CPFX) derivatives with remarkable improvement in lipophilicity by introducing a substituted benzyl moiety to the N atom on the C-7 piperazine ring of CPFX. Antimycobacterial and

Full text of DOI:10.1016/j.bmcl.2012.07.040

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5971–5975
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, antimycobacterial and antibacterial activity of ciprofloxacin
derivatives containing a N-substituted benzyl moiety
Shuo Wang a, Xue-Dong Jia a, Ming-Liang Liu a, , Yu Lu b, , Hui-Yuan Guo a
⇑
⇑
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
b Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Beijing 101149, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the design and synthesis of a series of novel ciprofloxacin (CPFX) derivatives with
remarkable improvement in lipophilicity by introducing a substituted benzyl moiety to the N atom on
the C-7 piperazine ring of CPFX. Antimycobacterial and antibacterial activity of the newly synthesized
compounds was evaluated. Results reveal that compound 4f has good in vitro activity against all of the
Received 6 May 2012
Revised 7 July 2012
Accepted 10 July 2012
Available online 17 July 2012
tested Gram-positive strains including MRSA and MRSE (MICs: 0.06–32
more potent than or comparable to the parent drug CPFX (MICs: 0.25–128
teria P. aeruginosa (MICs: 0.5–4 g/mL) and M. tuberculosis H37Rv ATCC 27294 (MIC: 1
Ó 2012 Elsevier Ltd. All rights reserved.
l
g/mL) which is two to eightfold
g/mL), Gram-negative bac-
g/mL).
l
Keywords:
Ciprofloxacin derivatives
Synthesis
l
l
Antimycobacterial activity
Antibacterial activity
Fluoroquinolones (FQs), one of the important classes of weap-
ons in our antibacterial arsenal,1 are used mainly for the treatment
of respiratory tract infections (RTI), urinary tract infections (UTI),
sexually transmitted diseases (STD), gastrointestinal and abdomi-
nal infections, skin and soft tissue infections, and infections of
the bone and joints.2 These antibacterial agents act by inhibiting
two type II bacterial topoisomerase enzymes, DNA gyrase (sub-
units encoded by gyrA and gyrB) and topoisomerase IV (subunits
encoded by parC and parE). Cell death is caused by trapping the
topoisomerase protein–DNA complex thus disrupting normal
DNA replication, inducing oxidative damage, and triggering cell-
death mechanisms.3,4
On the other hand, DNA gyrase is considered to be the sole
topoisomerase drug target of FQs in Mycobacterium tuberculosis
(MTB) because of no evidence of the topoisomerase IV parC and
parE gene homologs in the genome of MTB.5 Resistance to FQs re-
mains relatively low in clinical isolates of MTB currently, and there
are no reports of cross-resistance or antagonism with other classes
of anti-tuberculosis (anti-TB) drugs.6,7 Ciprofloxacin (CPFX, Scheme
1), ofloxacin and sparfloxacin were recommended as second-line
agents for the treatment of TB mainly in cases involving resistance
or intolerance to first-line anti-TB therapy by WHO in 1996.8 More-
over, moxifloxacin and gatifloxacin possessing a particularly strong
in vitro and in vivo activity against MTB9,10 are currently being fur-
ther evaluated as anti-TB drugs.
However, increasing bacterial resistance to FQs due to the high
level of use and to some degree of abuse, has put enormous
pressure on the public health systems. For example, a significant
increase in resistance of Escherichia coli and Pseudomonas aerugin-
osa as well as MTB to CPFX, the most consumed antibacterial agent
worldwide,11 was noted recently. It was reported that the overall
resistance rate of Gram-negative and -positive isolates to CPFX in-
creased from 40.8% (2000) to 51.7% (2004) at Xingshan County
Hospital, China,12 and overall resistance of MTB to CPFX increased
from 6.0% (1989–1994) to 26.8% (1995–2000) at the Makati Med-
ical Center, Makati City, Philippines.13 Therefore, there is an urgent
need for the discovery and development of effective novel FQs to
confer desirable biological and pharmacological properties. Clearly,
a more practical strategy is to modify the structures of existing FQs
to increase potency and overcome resistance.
Structure–activity relationship (SAR) studies of FQs have indi-
cated that the basic group at the C-7 position, the only an area that
substitution of bulky functional group is permitted, greatly influ-
ences their antibacterial potency, spectrum and safety.14,15 It is
generally believed that the action of FQs increases with an increase
in lipophilicity.16 A large number of existing FQ derivatives were
synthesized by introduction of an additional functional moiety
on the N atom of the C-7 side chain to increase the lipophilicity,
and some of which were found to have stronger antibacterial or
anti-MTB activity than the corresponding parent FQs.17,18 More-
over, several cephalosporin derivatives possessing a substituted
benzyloxime moiety in the C-7 position, such as GR69153,19
⇑
Corresponding authors. Tel./fax: +86 10 63036965 (M.-L.L.); tel.: +86 10
89509357; fax: +86 10 80505770 (Y.L.).
E-mail addresses: lmllyx@yahoo.com.cn (M.-L. Liu), luyu4876@hotmail.com
(Y. Lu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.040

5972
S. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5971–5975
O
F
COOH
O
N
N
N
F
COOH
HN
K2CO3
DMF
N
.
CPFX HCl
R
N
OH
Br
CHO
4a-p
PBr3
R
NaBH4
MeOH
R
R
CH2Cl2
3a-p
2a-o
1a-e
k: R = 3',4'-methylenedioxyl
l: R = 2',3',4'-trimethoxyl
a
2',5'-dimethoxyl
: R =
f: R = 4'-methoxyl
3',5'-dimethoxyl
b: R = 4',5'-dimethoxyl-2'-nitro
c: R = 4',5'-methylenedioxyl-2'-nitro
d: R = 2'-bromo-4',5'-dimethoxyl
e: R = 3',4'-ethylenedioxyl
g
: R =
m
3',4',5'-trimethoxyl
: R =
h: R = 2',3'-dimethoxyl
i: R = 3',4'-dimethoxyl
n: R = 4'-benzyloxyl
o: R = 4'-benzyloxyl-3'-methoxyl
p: R = hydrogen
j
2'-chloro-3',4'-dimethoxyl
: R =
Scheme 1. Synthesis of novel ciprofloxacin derivatives 4a–p.
LB1052220 and RU-59863,21 show superior antibacterial activity to
the corresponding methyloxime analogs against some pathogens.
These research results intensified our interest, it was decided to
design and synthesize a series of novel CPFX derivatives by intro-
duction of a lipophilic substituted benzyl moiety to the N atom
on the C-7 piperazine ring of CPFX in this study, and evaluate their
anti-MTB and antibacterial activity. Our primary object was to
optimize the potency of CPFX against MTB and clinically important
pathogens including MRSA.
bond is observed between the carboxylic and carbonyl groups
(Fig. 1).24
Lipophilicity of the new synthesized derivatives 4a–p and the
parent CPFX is expressed in the term of their ClogP values which
were calculated with Chem office 2010 software. As shown in Table
1, a remarkable improvement in the lipophilicity of the derivatives
4a–p as evidenced by ClogP values (0.87–3.26) which are much
more than that of CPFX (ꢀ0.73) (statistically significant at
p < 0.001 using t test) (Table 1).
Detailed synthetic pathways to novel CPFX derivatives 4a–p by
one-pot method are depicted in Scheme 1. Reduction of various
benzaldehydes 1a–e with sodium borohydride in methanol gave
the phenylmethanols 2a–e, and then nucleophilic substitution of
2a–e and commercially available compounds 2f–o with phospho-
rus tribromide in dichloromethane yielded the corresponding
(bromomethyl) benzenes 3a–o. Finally, 3a–o and commercially
available 3p were condensed directly with CPFX in the presence
of potassium carbonate to produce the target compounds 4a–p fol-
lowed well-established literature procedures.22,23 In addition, we
were also successful in preparing X-ray quality single crystals of
target compound 4k obtained by slow evaporation of a dichloro-
methane-methanol solution. In the crystal structure, the pipera-
zine ring and benzo[d][1,3]dioxole ring adopt chair and planar
conformations, respectively. An intramolecular O–H. . .O hydrogen
The target compounds 4a–p were initially evaluated for their
in vitro activity against MTB H37Rv ATCC 27294 using the Micro-
plate Alamar Blue Assay (MABA).25,26 The minimum inhibitory
concentration (MIC) is defined as the lowest concentration effect-
ing a reduction in fluorescence of P90% relative to the mean of
replicate bacterium-only controls and MICs of the compounds
along with CPFX for comparison are presented in Table 1.
The data reveal that the target compounds 4a–p have potential
activity against MTB H37Rv ATCC 27294 (MICs: 1?32
although they are less active than the parent drug CPFX (MIC:
0.5 g/mL). Among of them, compounds 4f and 4p bearing the
lg/mL),
l
simplest substituent (R = methoxyl) and without substituents
(R = hydrogen) on the benzene ring, respectively, display the high-
est activity (MICs: 1
have also useful activity (MICs: 2
l
g/mL), and compounds 4c, 4e, 4g, 4h, 4j, 4k
g/mL) against this strain.
l
Figure 1. X-ray structure of compound 4k.

S. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5971–5975
5973
Table 1
Structures, lipophilicity and antimycobacterial activity of compounds 4a–p
O
N
F
COOH
3'
4'
R
5'
2'
N
N
1'
6'
4a-p
Compd.
R
ClogPa
mpb (°C)
MIC (l
g/mL) MTBc
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
CPFX
20,50-Dimethoxyl
1.58
1.17
1.48
2.16
1.50
1.49
1.58
1.23
1.23
1.78
1.54
0.87
0.87
3.26
3.00
1.57
ꢀ0.73
232
>280
215
>32
>32
2
>32
2
1
2
2
16
2
40,50-Dimethoxyl-20-nitro
40,50-Methylenedioxyl-20-nitro
20-Bromo-40,50-dimethoxyl
30,40-Ethylenedioxyl
40-Methoxyl
254–255
242
248–249
199
177–178
172–173
187–188
238
30,50-Dimethoxyl
20,30-Dimethoxyl
30,40-Dimethoxyl
20-Chloro-30,40-dimethoxyl
30,40-Methylenedioxyl
20,30,40-Trimethoxyl
30,40,50-Trimethoxyl
40-Benzyloxyl
2
197
185
213
105
16
32
32
16
1
40-Benzyloxyl-30-methoxyl
hydrogen
237–238
0.5
CPFX, ciprofloxacin.
a
The ClogP is calculated by Chem office 2010 software.
Melting points are uncorrected.
MTB: MTB H37Rv ATCC 27294.
b
c
The target compounds 4a–p were evaluated for their in vitro
Generally, the target compounds 4a–p29 have considerable
antibacterial activity against the tested Gram-positive strains. For
example, they show good potency in inhibiting the growth of
antibacterial activity against representative Gram-positive and
Gram-negative strains using standard techniques.27 Minimum
inhibitory concentration (MIC)28 is defined as the concentration
of the compound required to give complete inhibition of bacterial
growth and MICs of the synthesized compounds along with CPFX
for comparison are reported in Tables 2 and 3.
methicillin-sensitive S. aureus (MSSA) (MICs:0.03–4
Among of them, the activity of compounds 4a, 4e, 4f, 4h, 4i, 4k,
4l against methicillin-resistant S. aureus (MRSA) (MICs: 4–64 g/
mL), and compounds 4e and 4f against S. epidermidis including
lg/mL).
l
Table 2
In vitro antibacterial activity of CPFX derivatives 4a–p against Gram-positive strains
Strains
MIC(l
g/mL)
4a
4b
4c
0.5
0.5
1
4d
4e
0.125
0.25
0.25
0.5
4f
0.06
0.125
0.25
0.25
4g
4h
4i
4j
4k
0.25
0.5
1
4l
4m
0.25
0.25
0.5
4n
4o
4p
0.25
0.25
0.25
0.25
CPFX
0.25
0.5
0.5
0.5
S.a.
0.5
0.5
0.5
1
1
2
4
4
4
1
1
0.5
1
0.06
0.03
0.03
0.125
0.125
0.125
0.125
0.25
0.06
0.5
0.5
0.5
4
0.25
0.5
2
0.5
2
MSSA .1
MSSA .2
MSSA .3
MRSA.1
MRSA.2
MRSA.3
MSSE.1
MSSE.2
MRSE.1
MRSE .2
E.f.1
E.f.2
E.f.3
E.fa.1
E.fa.2
S.p.1
S.p.2
1
2
1
0.125
0.25
0.125
1
0.5
1
0.25
0.5
1
32
64 32
64 32
16 32
32 16
32 16
16
16
4
0.5
8
8
8
4
32
8
1
128
>128
64
16
32
16
0.25
8
8
32
8
>128
32
4
32
2
32
32
64
32
16
64
16
64
128
32
32
>128
>128
>128
1
32
128
64
32
64
32
16
64
32
128
16
0.5
8
8
16
8
64
16
1
16
2
32
32
16
1
64
16
4
8
8
32
8
>128
64
4
64
2
32
128
64
1
32
32
32
8
64
32
4
32
2
64
16
2
16
16
16
16
32
16
8
128
128
1
32
16
32
8
64
32
8
32
8
64
16
32
16
16
32
8
>128
>128
4
0.5
8
2
1
4
0.25
0.5
16
16
16
16
128
16
16
16
8
16 16
16 16
16 32
16 32
128 64
16 32
128
>128
128
128
>128
>128
16
>128
8
>128
16
64
64
16
>128
64
8
32
8
64
32
32
32
8
64
64
4
32
4
64
8
32 16
0.5
>128
4
16
16
16
16
16
16
16
>128 32
>128 32
8
2
8
2
64 16
>128 32
16
1
16
8
8
16
32
16
32
8
2
8
1
64
128 16
>128 32
64
32
Abbreviations: S.a., Staphylococcus aureus ATCC 2592; MSSA.1, methicillin-sensitive Staphylococcus aureus 10-11; MSSA.2, methicillin-sensitive Staphylococcus aureus 10-13;
MSSA.3, methicillin-sensitive Staphylococcus aureus 10-14; MRSA.1, methicillin-resistant Staphylococcus aureus 10-11; MRSA.2, methicillin-resistant Staphylococcus aureus 10-
13; MRSA.3, methicillin-resistant Staphylococcus aureus 10-15; MSSE.1, methicillin-sensitive Staphylococcus epidermidis 10-11; MSSE.2, methicillin-sensitive Staphylococcus
epidermidis 10-15; MRSE.1, methicillin-resistant Staphylococcus epidermidis 10-10; MRSE.2, methicillin-resistant Staphylococcus epidermidis 10-13; E.f.1, Enterococcus faecalis
10-5; E.f.2, Enterococcus faecalis 10-6; E.f.3, Enterococcus faecalis 10-7; E.fa.1, Enterococcus faecium 10-6; E.fa.2, Enterococcus faecium 10-9; S.p.1, Streptococcus pneumonia 10-1;
S.p.2, Streptococcus pneumonia 10-6: CPFX, ciprofloxacin.

5974
S. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5971–5975
methicillin-resistant S. epidermidis (MRSE) (MICs: 0.5–16
better than or comparable to CPFX. In particular, the most active
compound 4f (MICs: 0.06–32 g/mL) in this study was found to
be two to eightfold more potent than or comparable to CPFX (MICs:
0.25–128 g/mL) against all of the tested Gram-positive strains.
lg/mL) is
l
l
On the other hand, the target compounds 4a–p are generally less
active than the parent CPFX against the tested Gram-negative
strains with few exceptions. It is noted that compounds 4c and 4f
possess good potency against all of the six clinical strains of P. aeru-
ginosa (MICs: 0.5–4 lg/mL). However, all of 4a–p, like CPFX, have
no virtual activity against the extended-spectrum b-lactamase
(ESBLs)-producing E. coli and Klebsiella pneumonia due partly to
resistance of these ESBLs-producing strains inherently to FQs.
Variations (R) on the benzene ring of the benzyl moiety in this
study include mono-/bi-/tri-methoxyl, benzyloxyl, 30,40-methylen-
edioxyl/ethylenedioxyl, nitro, chlorine, bromine and hydrogen
substitution (Table 1). The activity imparted to CPFX derivatives
by R groups against Gram-positive strains was in the order: meth-
oxyl (4f) > H (4p) > benzyloxyl (4n) for mono-substitution; 20,30-
dimethoxyl
(4h) > 30,40-dimethoxyl
(4i) > 20,50-dimethoxyl
(4a) > 30,50-dimethoxyl (4g) for dimethoxyl substitution. It is also
interesting to note that introduction of an additional group on
the 30,40-dimethoxylbenzene ring of CPFX derivative (4i) seems to
be detrimental to the activity and decreased activity caused by
the introduction of an electron-withdrawing group is more
remarkable than an electron-donating one. It can be exemplified
by the relative contribution of related R groups to activity as fol-
lows:
30,40-dimethoxyl > 20,30,40-trimethoxyl ꢁ 30,40,50-trimeth-
oxyl > 20-chloro-30,40-dimethoxyl > 20-bromo-40,50-dimeth-
oxyl > 40,50-dimethoxyl-20-nitro.
In summary, a series of novel CPFX derivatives with remarkable
improvement in lipophilicity, as compared to the parent drug
CPFX, were designed, synthesized and evaluated for their in vitro
anti-MTB and antibacterial activity. Our results reveal that the
most active compound 4f has good Gram-positive activity (MICs:
0.06–32
comparable to the parent CPFX (MICs:0.25–128
activity against Gram-negative bacteria P. aeruginosa (MICs: 0.5–
g/mL) and MTB H37Rv ATCC 27294 (MIC: 1 g/mL). However,
l
g/mL) which is two to eightfold more potent than or
l
g/mL), useful
4
l
l
the target compounds 4a–p are generally less active than CPFX
against MTB H37Rv ATCC 27294 and Gram-negative strains. It sug-
gests that merely an increase in lipophilicity of the tested com-
pounds does not result in enhanced antimycobacterial and
antibacterial activity.
Acknowledgment
The work was supported by the National S&T Major Special
Project on Major New Drug Innovation (No. 2012ZX09301002-
001-017).
References and notes
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15.20 (1H, s). ESI–MS: m/z 527 (M+H+). HRMS (ESI, m/z): C26H27FN4O7 (M+H+).
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(2H, m), 2.49–2.60 (4H, m), 3.28–3.03 (6H, m), 3.78–3.83 (3H, m), 7.25 (2H, s),
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Found: 452.1980. Compound 4g, yield: 37.42% (from 2g), light yellow solid. 1
H
NMR (400 MHz, CDCl3+D2O) d: 1.16–1.18 (2H, m), 1.30–1.31 (2H, m), 2.58–
2.60 (4H, m), 3.32–3.35 (4H, m), 3.49 (2H, s), 3.73–3.74 (6H, m), 3.78–3.81 (1H,
m), 6.39–6.40 (1H, m), 6.50–6.51 (2H, m), 7.56–7.57 (1H, d, J = 8 Hz), 7.88–7.91
(1H, d, J = 12 Hz), 8.65 (1H, s), 15.21 (1H, s). ESI–MS: m/z 482 (M+H+). HRMS
(ESI, m/z): C26H28FN3O5 (M+H+). Calcd: 482.2082; Found: 482.2085. Compound
4h, yield: 23.91% (from 2h), off white solid. 1H NMR (400 MHz, CDCl3 + D2O) d:
1.16–1.17 (2H, m), 1.29–1.31 (2H, m), 2.60–2.62 (4H, m), 3.30–3.32 (4H, m),
3.55–3.56 (3H, m), 3.74 (3H, s), 3.79 (3H, s), 6.64–7.06 (3H, m), 7.55–7.56 (1H,
d, J = 4 Hz), 7.88–7.91 (1H, d, J = 12 Hz), 8.65 (1H, s), 15.21 (1H, s). ESI–MS: m/z
482 (M+H+). HRMS (ESI, m/z): C26H28FN3O5 (M+H+). Calcd: 482.2083; Found:
482.2085. Compound 4i, yield: 43.66% (from 2i), light yellow solid. 1H NMR
(400 MHz, CDCl3 + D2O) d: 1.16–1.18 (2H, m), 1.28–1.31 (2H, m), 2.58–2.59
(4H, m), 3.28–3.33 (4H, m), 3.49 (2H, s), 3.73 (6H, s), 3.79–3.97 (1H, m), 6.79–
6.91 (3H, m), 7.55–7.57 (1H, d, J = 8 Hz), 7.88–7.9 1(1H, m), 8.65 (1H, s), 15.21
(1H, s). ESI–MS: m/z 482 (M+H+). HRMS (ESI, m/z): C26H28FN3O5 (M+H+). Calcd:
482.2080; Found: 482.2085. Compound 4j, yield: 33.98% (from 2j), light yellow
solid. 1H NMR (400 Hz, DMSO-/d6 + D2O) d: 1.17–1.19 (2H, m), 1.29–1.31 (2H,
m), 2.64–2.65 (4H, m), 3.30–3.32 (4H, m), 3.59 (2H, s), 3.74–3.82 (7H, m), 7.03–
7.05 (1H, d, J = 8 Hz), 7.20–7.22 (1H, d, J = 8 Hz), 7.56–7.58 (1H, d, J = 8 Hz),
7.88–7.92 (1H, d, J = 12 Hz), 8.65 (1H, s), 15.21 (1H, s). ESI–MS: m/z 516 (M+H+).
HRMS (ESI, m/z): C26H27ClFN3O5 (M+H+). Calcd: 516.1695; Found: 516.1696.
Compound 4k, yield: 34.41% (from 2k), light yellow solid. 1H NMR(400 MHz,
DMSO-d6 + D2O) d: 1.16–1.18 (2H, m), 1.28–1.31 (2H, m), 2.57–2.58 (4H, m),
3.30–3.33 (4H, m), 3.47 (2H, s), 3.78–3.82 (1H, m), 5.99 (2H, s), 6.78–6.90 (3H,
m), 7.55–7.57 (1H, d, J = 8 Hz), 7.88–7.91 (1H, d, J = 12 Hz), 8.649 (1H, s), 15.21
(1H, s). ESI–MS: m/z 466 (M+H+). HRMS (ESI, m/z): C25H24FN3O5 (M+H+). Calcd:
466.1769; Found: 466.1772. Compound 4l, yield: 31.31% (from 2l), light yellow
solid. 1H NMR(400 MHz, CDCl3 + D2O) d: 1.16–1.18 (2H, m), 1.29–1.31 (2H, m),
2.59–2.60 (4H, m), 3.30–3.31 (5H, m), 3.48 (2H, s), 3.74 (3H, s), 3.78 (3H, s),
3.80(3H, m), 6.77–6.79 (1H, d, J = 8 Hz), 7.00–7.02 (1H, d, J = 8 Hz), 7.54–7.56
(1H, d, J = 8 Hz), 7.87–7.91 (1H, d, J = 12 Hz), 8.65(1H, s), 15.21 (1H, s). ESI–MS:
m/z 512 (M+H+). HRMS (ESI, m/z): C27H30FN3O6 (M+H+). Calcd: 512.2100;
Found: 512.2191. Compound 4m, yield: 41.10% (from 2m), light yellow solid.
1H NMR (400 MHz, CDCl3 + D2O) d: 1.17–1.19 (2H, m), 1.30–1.31 (2H, m), 2.60
(4H, m), 3.26–3.36 (5H, m), 3.49 (2H, s), 3.64 (3H, s), 3.77 (6H, s), 6.64 (2H, s),
7.56–7.58 (1H, d, J = 8 Hz), 7.88–7.92 (1H, d, J = 16 Hz), 8.65 (1H, s), 15.21 (1H,
s). ESI–MS: m/z 512 (M+H+). HRMS (ESI, m/z): C27H30FN3O6 (M+H+). Calcd:
512.2190; Found: 512.2191. Compound 4n, yield: 48.39% (from 2n), light
yellow solid. 1H NMR(400 MHz, CDCl3 + D2O) d: 1.19–1.21 (2H, m), 1.34–1.36
(2H, m), 2.67 (4H, s), 3.34 (4H, s), 3.50–3.54 (3H, m), 5.07 (2H, s), 6.97 (1H, s),
7.26–7.28 (4H, m), 7.33–7.45 (5H, m), 8.00–8.03 (1H, d, J = 12 Hz), 8.76 (1H, s),
15.02 (1H, s). ESI–MS: m/z 512 (M+H+). HRMS (ESI, m/z): C31H30FN3O4 (M+H+).
Calcd: 528.2313; Found: 528.2293. Compound 4o, yield: 52.06% (from 2o),
yellow solid. 1H NMR (400 MHz, DMSO-d6 + D2O) d: 1.16–1.17 (2H, m), 1.29–
1.31 (2H, m), 2.58 (4H, s), 3.15–3.16 (1H, m), 3.30–3.33 (3H, m), 3.48 (2H, s),
3.77–3.80 (4H, m), 5.05 (2H, s), 6.81–6.83 (1H, m), 6.95–7.00 (2H, m), 7.32–
7.44 (5H, m), 7.55–7.57 (1H, d, J = 8 Hz), 7.88–7.91 (1H, d, J = 12 Hz), 8.65 (1H,
s), 15.21 (1H, s). ESI–MS: m/z 558 (M+H+). HRMS (ESI, m/z): C32H32FN3O5
(M+H+). Calcd: 558.2395; Found: 558.2398. Compound 4p, yield: 52.26% (from
2p), yellow solid. 1H NMR (400 MHz, CDCl3 + D2O) d: 1.16–1.21 (2H, m), 1.34–
1.38 (2H, m), 2.73 (4H, m), 3.39–3.52 (4H, m), 3.53–3.55 (1H, m), 3.65 (2H, s),
7.26–7.37 (6H, m), 7.96–8.00 (1H, d, J = 16 Hz), 8.74 (1H, s), 15.00 (1H, s). ESI–
MS: m/z 558 (M+H+). HRMS (ESI, m/z): C24H24FN3O3 (M+H+). Calcd: 422.1877;
Found: 422.1874.
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27. MICs were determined as described by the NCCLS (see: National Committee for
Clinical Laboratory Standards, Performance Standards for Antimicrobial
Susceptibility Testing: 11th Informational Supplement, vol. 21, National
Committee for Clinical Laboratory Standards, Wayne, PA, 2001, M100-S11.
The MIC was defined as the lowest concentration of each compound resulting
in inhibition of visible growth of bacteria after incubation at 35 °C for 18–24 h.
28. To a stirring solution of 2,5-dimethoxyl benzaldehyde 1a (0.67 g, 4 mmol)
dissolved in anhydrous methanol (30 mL) was added NaBH4 (0.84 g, 22 mmol)
in batches at 0 °C over a period of 0.5 h. The reaction mixture was stirred at the
same temperature for 2 h. After removal of the methanol under reduced
pressure, the residue was diluted with methylene chloride (30 mL), washed
with distilled water (3 ꢂ 10 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give crude product 2a which was then
dissolved in anhydrous methylene chloride (50 mL) and cooled to 0–5 °C by ice
bath. To this solution was added dropwise phosphorus tribromide (0.5 mL,
5 mmol) over a period of 15 min and stirred for 0.5 h at the same temperature,
and then washed with saturated brine (3 ꢂ 15 mL), dried over anhydrous
sodium sulfate, and concentrated under reduced pressure to give crude
compound 3a. To
a solution of 3a dissolved in N,N-dimethyl formamide
(20 mL) was added anhydrous potassium carbonate (1.10 g, 8 mmol) and 1-
cyclopropyl-6-fluoro-7-(piperazin-1-yl)-1,4-dihydroquinoline-4-oxo-3-
carboxylic acid hydrochloride (CPFXꢃHCl, 0.73 g, 2 mmol). The reaction mixture
was stirred at 40 °C for 12 h, and then diluted with methylene chloride (50 mL),
washed with distilled water (3 ꢂ 50 mL), dried over anhydrous sodium sulfate
and concentrated under reduced pressure. The residue obtained was purified
by column chromatography (silica gel) eluted with petroleum ether and ethyl
acetate (v:v = 10:1) to afford the target compound 4a . Yield: 62.38 % (from 1a),
off-white solid.1H NMR (400 MHz, CDCl3 + D2O) d: 1.17–1.19 (2H, m),1.30–1.31
(2H, m) , 2.62–2.66 (4H, m), 3.34–3.35 (4H, m,), 3.54 (2H, s), 3.70 (3H, s), 3.73
(3H, s), 3.80–3.81 (1H, m), 6.78–6.81 (1H, m), 6.90–6.95 (2H, m), 7.56–7.58
(1H, d, J = 8 Hz), 7.88–7.90 (1H, d, J = 8 Hz), 8.65 (1H, s), 15.22 (1H, s). ESI–MS:
m/z 482 (M+H+). HRMS–ESI: m/z C26H28FN3O5 Calcd: 482.2085; Found:
482.2085.
29. The other target compounds 4b–p were obtained as off-white solids in a
similar manner as for the preparation of 4a. Compound 4b, yield: 23.74% (from
2b), yellow solid. 1H NMR(400 MHz, CDCl3 + D2O) d: 1.17 (2H, m), 1.30–1.31
(2H, m), 2.54–2.65 (4H, m), 3.28–3.34 (4H, m), 3.57 (2H, s), 3.76–3.81 (7H, m),
7.08–7.13 (2H, d, J = 20 Hz), 7.56–7.58 (1H, d, J = 8 Hz), 7.90 (1H, s), 8.65 (1H, s),