Synthesis and in vitro antibacterial activity of fluoroquinolone derivatives containing 3-(N′-alkoxycarbamimidoyl)-4-(alkoxyimino) pyrrolidines

Article abstract of DOI:10.1016/j.ejmech.2010.08.050

A series of novel 7-[3-(N′-alkoxycarbamimidoyl)-4-(alkoxyimino) pyrrolidin-1-yl] fluoroquinolone derivatives were designed, synthesized and characterized by ¹H NMR, MS and HRMS. These fluoroquinolones were screened for their in vitro antibacter

Full text of DOI:10.1016/j.ejmech.2010.08.050

European Journal of Medicinal Chemistry 45 (2010) 5498e5506
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro antibacterial activity of fluoroquinolone derivatives
containing 3-(N0-alkoxycarbamimidoyl)-4-(alkoxyimino) pyrrolidines
1
1
*
Qiang Guo , Lian-Shun Feng , Ming-Liang Liu , Yi-Bin Zhang, Yun Chai, Kai Lv, Hui-Yuan Guo, Li-You Han
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel 7-[3-(N0-alkoxycarbamimidoyl)-4-(alkoxyimino)pyrrolidin-1-yl] fluoroquinolone
derivatives were designed, synthesized and characterized by 1H NMR, MS and HRMS. These fluo-
roquinolones were screened for their in vitro antibacterial activity. Most of them exhibit good potency in
Received 13 June 2010
Received in revised form
20 August 2010
Accepted 23 August 2010
Available online 15 September 2010
inhibiting the growth of Staphylococcus aureus and Staphylococcus epidermidis (MIC: 0.06e4.00
The activity of compounds 33 and 43 against S. aureus including MRSA and S. epidermidis including MRSE
(MIC: 0.06e0.125 g/mL) is more than or comparable to the reference drugs levofloxacin and gemi-
mg/mL).
m
floxacin. In addition, compound 33 is 32 and 16e32 fold more potent than both the reference drugs
against Enterococcus faecium 08-7 and Klebsiella pneumoniae 09-22, respectively.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Fluoroquinolones
Synthesis
Antibacterial activity
1. Introduction
As part of an ongoing program to develop new fluoroquinolones,
we have focused our attention on introducing new functional
Quinolones have become a major class of antibacterial agents
mainly used to fight both community-acquired and serious
hospital-acquired infections. These antibiotics act by binding two
type II bacterial topoisomerase enzymes, DNA gyrase and top-
oisomerase IV [1,2]. Although most of them currently in the market
or under development are generally characterized by a broad
antimicrobial spectrum, their activity against clinically important
Gram-positive cocci including Staphylococci, Streptococci and
Enterococci is relatively moderate. This has not only limited qui-
nolones usage in infections caused by these organisms, but also is
believed to be one of the reasons for the rapidly developing qui-
nolone resistance. Thus, recent efforts have been directed toward
the synthesis of new quinolones. This can provide improved Gram-
positive antibacterial activity, while retaining the good Gram-
negative activity of early fluoroquinolones, such as ciprofloxacin
and ofloxacin [3e6].
groups to the pyrrolidine ring. Previous work on pyrrolidine
analogs suggested the importance of the oxime functional group
with respect to biological activity. For example, gemifloxacin
(GMFX, Fig. 1) shows much more antibacterial activity than its
desoximino analog [10]. In this study, an additional oxime group
was introduced to the aminomethyl moiety of the 3-aminomethyl-
4-(methoxyimino)pyrrolidine, the side chain compound at C-7
position of GMFX. A series of fluoroquinolone compounds con-
taining pyrrolidinyl substitution at the C-7 position were designed
and synthesized. These derivatives are structurally novel, having
an alkoxycarbamimidoyl and an alkoxyimino group at the 3- and
4-position of the pyrrolidine ring, respectively. Our primary
objective was to optimize the potency of these compounds against
Gram-positive and Gram-negative organisms.
2. Chemistry
Structureeactivity relationship (SAR) studies of quinolone
antibacterial agents showed that the basic group at the C-7 position
is the most adaptable site for chemical change and an area that
greatly influences their potency, spectrum and safety [7,8]. In
general, 5- and 6-membered nitrogen heterocycles have been
proven to be the optimal substituents [9].
Addition reaction of ethylglycinate hydrochloride 1 with acry-
lonitrile in the presence of sodium hydroxide gave the secondary
amine 2, which was subsequently treated with di-tert-butox-
ycarbonyl dicarbonate (Boc2O) to produce Boc-protected cyano
ester 3. The compound 3 was cyclized to the cyano pyrolidone
4 in a good yield by refluxing with sodium hydride in toluene.
The cyano ketone 4 was reacted with O-alkylhydroxyamines to
give the cyano oximes 5a,b, the latters were subsequently
reacted with hydroxylamine or O-alkylhydroxyamines to yield the
* Corresponding author.
E-mail address: lmllyx@yahoo.com.cn (M.-L. Liu).
These authors contributed equally to the work.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.050

Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
5499
Fig. 1. Structure of Gemifloxacin.
alkoxycarbamimidoyl oximes 6aef, which upon deprotection
afforded the new pyrrolidine derivatives 7aef by pumping
hydrogen chloride gas [10,11] (Scheme 1).
The coupling of the pyrrolidine derivatives 7aef with various
compounds containing quinolone and naphthyridone cores was
carried out according to well-established literature procedures
(Scheme 2) [12e14]. In the case of quinolones 9e18, condensation
of 7aef with 8a,b was performed in the presence of triethylamine.
However, boric chelates 8ceh were required to increase reactivity
as regard quinolones 19e49.
Scheme 2. Synthesis of novel fluoroquinolones 9e49. Reagents and conditions:
(i) 7aef Et3N, acetonitrile; (ii) NaOH, and then HCl.
In addition, since the oxime group can exist in the E- or
Z-configuration, it was necessary to determine the geometry of
each oxime compound. It was a pity that we were not successful in
preparing X-ray-quality single crystals of compounds 19e49, but
we were able to obtain X-ray data for compound 7d. As expected,
the geometries of both two alkoxyimino groups on the five-
membered piperidine ring adopting an envelope conformation are
the Z-configurations confirmed by single crystal X-ray diffraction
analysis [15].
3. Antibacterial activity
The synthesized compounds were screened for their in vitro
antibacterial activity against representative Gram-positive and
Gram-negative bacteria by means of standard twofold serial dilu-
tion method using agar media [16] for determination of MIC. Lev-
ofloxacin (LVFX) and GMFX were used as control drugs.
4. Results and discussion
4.1. Chemistry
4.2. Antibacterial activity
The synthesis of new pyrrolidine derivatives 7aef and novel
fluoroquinolones 9e49 was performed is outlined in Schemes 1
and 2, respectively. The chemical structures of them were charac-
terized by 1H NMR, MS and HRMS and their data are presented in
the experimental section. The structures and physical data of the
target compounds 9e49 are reported in Table 1.
The novel fluoroquinolones 9e49 were tested for in vitro anti-
bacterial activity against fourteen Gram-positive and eleven Gram-
negative strains and LVFX and GMFX were used as reference drugs.
The data generated from this study (Table 2) showed that most
of the target compounds exhibit good potency in inhibiting the
growth of Staphylococci aureus including MRSA and Staphylococci
Scheme 1. Synthesis of new pyrrolidine derivatives 7aef. Reagents and conditions: (i) acrylonitrile, NaOH, menthol; (ii) (Boc)2O, methanol; (iii) NaH, toluene; (iv) R1ONH2$ HCl,
KOH, methanol; (v) R2ONH2$ HCl, Na2CO3, methanol; (vi) HCl (gas), methanol.

5500
Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
Table 1 (continued)
Table 1
Structures and physical data of novel fluoroquinolones 9e49.
Compd.
X
R
R1
R2
H
Yield (%)
64.5
Mp (ꢀC)
O
F
COOH
O
O
O
O
O
O
29
Me
245e249
X
N
R
N
R2ON
H2N
30
31
32
33
34
Et
H
74.8
68.5
67.8
74.4
71.3
140e143
162e164
176e178
153e155
150e153
R1ON
Compd.
X
N
R
R1
R2
H
Yield (%)
Mp (ꢀC)
Me
Et
Me
Me
Et
9
Me
35.8
224e225
10
11
12
N
N
N
Et
H
45.5
43.0
48.6
242e243
176e178
169e172
Me
Et
Me
Et
Me
Me
Et
35
36
37
38
39
40
CF
CF
CF
CF
CF
CF
Et
Et
Et
Et
Et
Et
Me
Et
Me
Et
Me
Et
H
H
Me
Me
Et
73.2
60.4
64.5
71.5
74.4
69.5
202e205
163e164
152e153
155e156
147e150
144e146
13
14
N
N
Me
Et
Et
Et
51.2
50.5
186e187
152e153
Et
41
42
43
CF
CF
CF
Me
Et
Me
Me
Et
26.0
28.5
20.4
135e137
126e129
140e143
15
16
17
18
N
N
N
N
2,4-F2-C6H3
2,4-F2-C6H3
2,4-F2-C6H3
2,4-F2-C6H3
Me
Et
Me
Et
Me
Me
Et
52.8
34.5
47.6
57.2
139e142
111e112
122e125
130e134
Et
19
20
21
COMe
COMe
COMe
Me
Et
H
78.0
73.2
70.5
106e109
137e139
115e118
Me
44
45
46
47
48
49
CF
CF
CF
CF
CF
CF
2-F-C2H4
2-F-C2H4
2-F-C2H4
2-F-C2H4
2-F-C2H4
2-F-C2H4
Me
Et
Me
Et
Me
Et
H
H
Me
Me
Et
72.5
66.4
74.0
70.6
63.2
65.0
146e149
150e151
142e144
154e156
149e152
151e152
H
Me
Me
Et
22
23
24
COMe
COMe
COMe
Et
Me
Et
73.2
76.0
68.5
119e122
114e115
102e105
epidermidis including MRSE (MIC: 0.06e4
compounds 33 and 43 against S. aureus and S. epidermidis (MIC:
0.06e0.125 g/mL) is more than or comparable to LVFX and GMFX,
and compound 33 is 32 and 16e32 fold more potent than both the
reference drugs against Enterococcus faecium 08-7 and Klebsiella
pneumoniae 09-22, respectively. In addition, the activity (MIC:
mg/mL). The activity of
Me
Et
m
Et
0.25 mg/mL) of compounds 10 and 38 against two strains of Strep-
tococcus pneumoniae is 8e16 fold more potent than LVFX. However,
overall the target compounds exhibit less activity than the refer-
ence drugs against Gram-negative strains. These results suggested
that introduction of another oxime group into the aminomethyl
moiety of the pyrrolidine ring causes reduced antibacterial activity
against Gram-negative strains.
25
26
27
COCHF2
COCHF2
COCHF2
Me
Et
H
30.5
28.5
24.0
162e164
155e158
145e148
H
Et
Me
5. Conclusion
The forty-one fluoroquinolone compounds (9e49) were synthe-
sized by condensations of 3-(N0-alkoxycarbamimidoyl)-4-(alkox-
yimino)pyrrolidinewithvarious quinolone andnaphthyridone cores.
28
COCHF2
Et
Et
24.6
145e147

Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
5501
The in vitro antibacterial activity was examined using representative
Gram-positive and Gram-negative strains, and results showed most
of the target compounds exhibit good potency in inhibiting the
growth of S. aureus and S. epidermidis. The activity of compounds 33
and 43 against S. aureus including MRSA and S. epidermidis including
MRSE is more than or comparable to LVFX and GMFX. But all the
target compounds show less activity than the reference drugs against
Gram-negative strains. The reduced activity might be due to the
conjugated effect between the introduced new oxime and the amino
group possessing a lone pair of electrons on the nitrogen, and
hindering the amino group from participating in hydrogen bonding
with DNA gyrase.
6.1.2. N-tert-Butoxycarbonyl-3-cyano-4-oxopyrrolidine (4)
To a refluxing suspension of 80% sodium hydride (7.5 g, 0.25 mol)
in dry toluene (80 mL) was added dropwise a solution of the cyano
ester 3 (40.5 g, 0.15 mol) dissolved in dry toluene (80 mL) over
a period of 0.5 h. The reaction mixture was stirred for 1 h at the same
temperature, cooled to room temperature and then water (200 mL)
was added slowly. The aqueous layer was separated and adjusted to
pH 7 with acetic acid (10%). The solid obtained was filtered, washed
twice with water and dried in vacuo to give the title compound 4
(22.2 g, 70.5%) as a white solid, mp: 160e161 ꢀC 1H NMR (CDCl3,
400 MHz)
d (ppm): 1.47 (9H, s, Boc-9H), 3.64e4.06 (4H, m, pyrroli-
dine), 4.34e4.38 (1H, m, pyrrolidine). MS (ESI, m/z): 209 (M ꢁ H)ꢁ.
The activity of the quinolone and naphthyridone nuclei in this
study is in the order: 1-cyclopropyl-8-fluoroquinolone > 1-(2,4-
difluorophenyl)-1,8-naphthyridone z levofloxacin nuclei z 1-cyclo
propyl-8-methoxylquinolone > 1-cyclopropyl-8- difluoromethoxy
quinolone > 1-ethyl-8-fluoroquinolone > 1-(2-fluoroethyl)-8-fluoro
quinolone > 1-cyclopropyl-1,8- naphthyridone. The relative contri-
bution of the substituents at C-7 position to antibacterial activity is as
follows:3-(N0-ethoxycarbamimidoyl)-4-(methoxyimino)pyrrolidine >
6.1.3. General procedure for the synthesis of N-tert-butoxycarbonyl-
3-cyano- 4-(alkyloxyimino)pyrrolidines 5a,b
To a mixture of O-alkylhydroxyamine hydrochloride (140.0mmol)
and potassium hydroxide (5.6 g, 100.0 mmol) in methanol (200 mL)
was added dropwise a solution of the cyano pyrolidone 4 (15.0 g,
70 mmol) dissolved in methanol (50 mL) while stirring at 55e60 ꢀC
over 25 min. The reaction mixture was stirred for 2 h at the same
temperature and concentrated under reduced pressure. The residue
was diluted with water (100 mL) and extracted with methylene
chloride (3 ꢂ 50 mL). The combined extracts were washed with
saturated brine, dried over anhydrous sodium sulfate and concen-
trated under reduced pressure. The crude product was purified by
column chromatography (silica gel) eluted with petroleum ether and
ethyl acetate (v: v ¼ 5:1) to afford the title compounds 5a,b as
colorless oils.
3-(N0-methoxycarbamimidoyl)-4-(methoxyimino)pyrrolidine
>
3-(ethoxyimino)-4-(N0- methoxycarbamimidoyl)pyrrolidine > 3-
(N0-hydroxycarbamimidoyl)-4-(methoxyimino) pyrrolidine
z
3-
3-(ethoxyimino)-4-(N0-hydroxycarbamimidoyl)pyrrolidine
(N0-ethoxycarbamimidoyl)-4- (ethoxyimino)pyrrolidine.
>
6. Experimental protocol
6.1. Chemistry
6.1.3.1. Compound 5a. Yield: 43.0%; 1H NMR (400 MHz, CDCl3),
d
(ppm): 1.46 (9H, s, Boc-9H), 3.68e3.90 (3H, m, pyrrolidine), 3.97
All chemical reagents and solvents used in this study were
purchased from Beihua Fine Chemicals Company (Beijing, China).
Melting points were determined in open glass capillaries and are
uncorrected. 1H NMR spectra were recorded on a Varian Mercury-
400 spectrometer using tetramethylsilane as internal standard.
Electrospray ionization (ESI) mass spectra and high resolution mass
spectra (HRMS) were obtained on an MDSSCIEX Q-Tap mass spec-
trometer and AccuTOF CS JMS-T100CS (JEOL) mass spectrometer,
respectively. Merck silica gel ART5554 60F254 plates were used for
analytical TLC. Column chromatography was carried out on silica
gel HG/T2354-92 made in Haiyang Chemical Company (Qingdao,
China).
(3H, s, OCH3), 4.06e4.18 (2H, m, pyrrolidine). MS (ESI, m/z): 262
(M þ Na)þ.
6.1.3.2. Compound 5b. Yield: 54.5%; 1H NMR (400 MHz, CDCl3)
d
(ppm): 1.29 (3H, t, J ¼ 6.8 Hz, OCH2CH3), 1.47 (9H, s, Boc-9H),
3.69e3.73 (1H, m, pyrrolidine), 3.82e3.89 (1H, m, pyrrolidine),
3.90e4.07 (3H, m, pyrrolidine), 4.22 (2H, q, J ¼ 6.8 Hz, OCH2CH3).
MS (ESI, m/z): 276 (M þ Na)þ.
6.1.4. General procedure for the synthesis of N-tert-butoxycarbonyl-
3-(N0- alkoxycarbamimidoyl)-4-(alkoxyimino)pyrrolidines 6aef
To
a mixture of hydroxylamine or O-alkylhydroxyamine
hydrochloride (100.0 mmol) and anhydrous sodium carbonate
(12.70 g, 120.0 mmol) in methanol (150 mL) was added the cyano
oximes 5a,b (83.6 mmol) while stirring at 55 ꢀC. The reaction
mixture was stirred for 18e50 h at the same temperature and
filtered. The filtrate was concentrated under reduced pressure, the
residue was diluted with water (100 mL) and extracted with
methylene chloride (3 ꢂ 50 mL). The combined extracts were
washed with saturated brine, dried over anhydrous sodium sulfate
and concentrated under reduced pressure. The crude product was
purified by column chromatography (silica gel) eluted with petro-
leum ether and ethyl acetate (v: v ¼ 3:1) to afford the title
compounds 6aef.
6.1.1. N-tert-Butoxycarbonyl-2-(2-cyanoethyl)aminoacetic acid
ethyl ester (3)
A mixture of ethylglycinate hydrochloride 1 (69.7 g, 0.50 mol),
sodium hydroxide (20.0 g, 0.50 mol) and methanol (300 mL) was
stirred for 0.5 h, and then acrylonitrile (29.2 g, 0.55 mol) was added
dropwise over a period of 40 min at room temperature. The reac-
tion mixture was heated to refluxing and stirred for 3 h to give the
secondary amine 2, which was pure enough to be used for the next
step without further purification.
To the reaction mixture containing the amine 2 was added
(Boc)2O (109.0 g, 0.50 mol) at room temperature, stirred at
40e50 ꢀC for 1 h and filtered. The filtrate was concentrated under
reduced pressure. The residue was diluted with ethyl acetate
(200 mL), washed with water and then saturated saline, dried over
anhydrous sodium sulfate and filtered. The filtrate was concen-
trated under reduced pressure to afford the title compound 3
(103.6 g, 80.9%) as a colorless oil. 1H NMR (400 MHz, CDCl3)
6.1.4.1. Compound 6a. Yield: 54.9%; 1H NMR (400 MHz, CDCl3)
d
(ppm): 1.46 (9H, s, Boc-9H), 3.64e3.85 (3H, m, pyrrolidine), 3.91
(3H, s, OCH3), 4.05e4.14 (2H, m, pyrrolidine). MS (ESI, m/z): 273
(M þ H)þ.
d
(ppm): 1.23e1.28 (3H, m, OCH2CH3), 1.46 (9H, s, Boc-9H),
6.1.4.2. Compound 6b. Yield: 57.5%; 1H NMR (400 MHz, CDCl3)
2.64e2.69 (2H, m, OCH2CH3), 3.53e3.57 (2H, m, NCH2CH2CN),
3.75e4.00 (2H, m, NCH2CO), 4.03e4.05 (2H, m, NCH2CH2CN). MS
(ESI, m/z): 279 (M þ Na)þ.
d
(ppm): 1.25 (3H, t, J ¼ 6.4 Hz, OCH2CH3), 1.46 (9H, s, Boc-9H),
3.53e3.86 (3H, m, pyrrolidine), 4.05e4.20 (2H, m, pyrrolidine), 4.16
(2H, q, J ¼ 6.4 Hz, OCH2CH3). MS (ESI, m/z): 287 (M þ H)þ.

5502
Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
Table 2
Antibacterial activity of novel fluoroquinolones 9e49.
Compd. Strains MIC (
S.a.1
mg/mL)
S.a.2
128
S.a.3
S.e.1
S.e.2
S.e.3
S.e.4
128
S.e.5
128
S.p.1
S.p.2
E.fm.1
E.fm.2
9
1
1
0.25
1
0.25
2
8
0.25
0.25
0.25
0.06
0.25
0.25
0.125
0.25
0.25
1
0.25
1
128
2
1
2
2
2
0.25
1
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
>128
64
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
>128
64
>128
>128
>128
128
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
LVFX
GMFX
0.25
0.25
0.5
2
2
0.25
0.25
0.25
0.125
0.25
8
0.25
0.25
0.25
0.06
0.25
0.25
0.25
0.125
0.125
1
0.25
0.25
0.25
0.125
0.25
0.25
0.25
>128
128
>128
>128
16
0.25
0.25
2
0.25
0.25
0.25
0.25
0.25
0.25
0.25
>128
>128
0.25
32
>128
128
4
>128
8
32
>128
>128
0.5
0.25
0.25
0.25
0.5
0.125
2
0.25
0.25
0.25
64
0.25
0.5
0.25
0.125
0.125
1
0.5
1
0.5
0.125
0.125
0.25
0.25
0.25
0.25
0.125
0.06
0.25
0.25
0.125
0.5
0.25
0.5
0.125
0.5
32
>128
128
4
1
8
4
32
64
4
8
64
32
0.06
0.5
0.5
0.25
0.5
0.5
0.5
4
32
32
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.125
0.25
0.25
0.5
0.25
1
0.125
0.25
0.25
0.25
0.25
0.25
0.5
0.25
0.25
0.25
1
0.25
4
2
1
64
64
0.25
1
0.25
64
0.125
0.25
0.25
2
0.25
0.25
0.25
0.25
0.06
64
0.25
1
0.25
0.25
0.25
0.25
1
0.25
4
8
1
0.125
1
64
64
0.25
0.25
>128
64
>128
16
16
64
>128
>128
>128
0.125
0.25
0.125
0.25
0.125
1
0.25
1
0.25
1
0.125
8
0.125
0.125
0.06
1
0.5
0.125
2
4
0.25
0.25
0.25
0.125
0.06
0.25
0.5
0.25
0.25
0.06
0.25
0.25
0.25
0.25
0.06
8
0.25
0.125
0.25
0.125
0.25
0.25
0.25
0.25
0.5
0.06
8
0.25
0.125
0.5
0.25
0.25
0.25
>128
16
>128
8
64
1
16
1
0.25
16
>128
16
32
32
1
4
4
0.25
0.25
0.25
0.125
64
0.25
1
0.25
8
0.25
0.5
0.25
0.25
0.06
0.25
0.25
8
>128
0.06
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
1
1
2
8
0.25
0.25
0.25
0.25
0.125
1
1
>128
>128
1
0.25
0.5
>128
0.25
0.125
0.125
0.5
>128
>128
>128
>128
>128
>128
>128
0.25
0.25
0.06
1
0.5
0.125
4
0.25
4
0.125
0.25
0.25
0.125
0.06
0.125
0.25
0.06
0.25
0.125
0.06
1
0.25
0.5
0.06
0.5
0.125
0.25
0.06
0.5
0.25
0.25
0.06
1
4
0.25
1
0.25
>128
>128
>128
>128
>128
>128
>128
2
>128
>128
>128
0.5
0.25
>128
0.25
>128
4
2
0.5
0.5
0.5
0.5
0.5
2
>128
0.125
0.25
0.125
4
0.25
0.06
0.125
0.125
0.25
4
0.25
0.06
0.25
0.25
0.5
0.25
0.25
0.25
8
2
0.25
>128
>128
0.25
2
0.06
0.125
0.25
4
0.25
0.125
8
8
4
8
1
8
2
0.25
4
0.06
0.125
0.125
0.125
16
2
Abbreviations: S. a.1, S. aureus ATCC259223; S. a.2, methicillin-resistant S. aureus 08-1; S. a.3, methicillin-sensitive S. aureus 08-2; S. e.1, methicillin-resistant S. epi-
dermidis 09-2; S. e.2, methicillin-resistant S. epidermidis 09-3; S. e.3, methicillin-resistant S. epidermidis 09-4; S. e.4, methicillin-sensitive S. epidermidis 09-3; S. e.5,
methicillin-sensitive S. epidermidis 09-6; S. p.1, S. pneumoniae 08-2; S. p.2, S. pneumoniae 08-4; E. fm.1, E. faecium 08-2; E. fm.2, E. faecium 08-7; E. fa.1, E. faecalis 08-10;
E. fa.2, E. faecalis 08-12; E.co.1, E. coli ATCC25922; E.co.2, E. coli 08-21; E.co.3, E. coli 08-22; K.p.1, K. pneumoniae 09-21; K.p.2, K. pneumoniae 09-22; K.p.3, K. pneumoniae
09-23; P.a.1, P. aeruginosa ATCC27853; P.a.2, P. aeruginosa 09-32; P.a.3, P. aeruginosa 09-33; P.a.4, P. aeruginosa 09-34; P.a.5 P. aeruginosa 09-35.
6.1.4.3. Compound 6c. Yield: 54.9%; 1H NMR (400 MHz, CDCl3)
(ppm): 1.46 (9H, s, Boc-9H), 3.74e3.80 (4H, m, pyrrolidine and
OCH2CH3), 1.39 (9H, s, Boc-9H), 3.30e3.56 (3H, m, pyrrolidine), 3.81
(2H, q, J ¼ 6.8 Hz, OCH2CH3), 3.93e3.99 (2H, m, pyrrolidine), 4.03
(2H, q, J ¼ 6.8 Hz, OCH2CH3). MS (ESI, m/z): 315 (M þ H)þ.
d
OCH3), 3.89 (3H, s, OCH3), 3.90e3.91 (1H, m, pyrrolidine), 4.04e4.13
(3H, m, pyrrolidine). MS (ESI, m/z): 287 (M þ H)þ.
6.1.5. General procedure for the synthesis of 3-(N0-
6.1.4.4. Compound 6d. Yield: 53.2%; 1H NMR (400 MHz, CDCl3)
alkoxycarbamimidoyl)- 4-(alkoxyimino)pyrrolidines 7aef
To a stirring solution of alkoxycarbamimidoyl oximes 6aef
(18.4 mmol) dissolved in methanol (50 mL) was pumped dry
hydrogen chloride gas at room temperature for 0.5 h. The reac-
tion mixture was allowed to stir for another 0.5 h at the same
temperature. The resulting solid was collected by suction, and
dried in vacuo to give the title compounds 7aef as off-white
solids.
d
(ppm): 1.26 (3H, t, J ¼ 6.8 Hz, OCH2CH3), 1.46 (9H, s, Boc-9H),
3.58e3.62 (1H, m, pyrrolidine), 3.76 (3H, s, OCH3), 4.03e4.19 (4H,
m, pyrrolidine and OCH2CH3). MS (ESI, m/z): 301 (M þ H)þ.
6.1.4.5. Compound 6e. Yield: 49.6%; 1H NMR (400 MHz, CDCl3)
d
(ppm): 1.25 (3H, t, J ¼ 6.8 Hz, OCH2CH3), 1.46 (9H, s, Boc-9H),
3.61e3.65 (1H, m, pyrrolidine), 3.77e3.82 (1H, m, pyrrolidine), 3.90
(3H, s, OCH3), 3.92e3.99 (1H, m, pyrrolidine), 4.03 (2H, q, J ¼ 6.8 Hz,
OCH2CH3), 4.10e4.18 (2H, m, pyrrolidine). MS (ESI, m/z): 323
(M þ Na)þ.
6.1.5.1. Compound 7a. Yield: 70.9%; mp: 124e126 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d (ppm): 3.40e3.79 (2H, m, pyrrolidine), 3.84
6.1.4.6. Compound 6f. Yield: 56.2%; 1H NMR (400 MHz, CDCl3)
(3H, s, OCH3), 3.99e4.28 (3H, m, pyrrolidine). MS (ESI, m/z): 173
(M þ H)þ.
d
(ppm): 1.08 (3H, t, J ¼ 6.8 Hz, OCH2CH3), 1.16 (3H, t, J ¼ 6.8 Hz,

Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
5503
Compd. Strains MIC (mg/mL)
E.fa.1
E.fa.2
4
0.25
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
0.5
128
64
>128
>128
32
E.co.1
E.co.2
E.co.3
K.p.1
K.p.2
K.p.3
P.a.1
P.a.2
P.a.3
P.a.4
P.a.5
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
0.25
>128
64
>128
>128
32
>128
>128
>128
>128
128
>128
128
>128
>128
128
128
128
>128
16
>128
128
128
>128
128
16
128
128
16
128
128
>128
128
128
128
128
128
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
0.25
>128
16
>128
>128
>128
>128
>128
64
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
32
>128
1
>128
>128
>128
>128
>128
>128
>128
32
4
8
8
8
>128
>128
64
>128
64
64
>128
128
128
32
>128
64
>128
128
64
>128
>128
128
>128
>128
128
32
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
1
64
16
128
1
32
32
128
1
4
2
4
8
32
64
8
32
64
16
16
0.5
1
0.5
>128
>128
128
>128
>128
128
32
64
128
>128
>128
64
>128
128
64
>128
128
64
8
>128
16
64
>128
128
>128
>128
>128
>128
>128
64
>128
>128
32
16
32
64
64
128
>128
>128
64
>128
128
64
>128
64
64
>128
128
16
128
128
8
128
32
32
>128
>128
64
>128
>128
64
>128
>128
64
>128
>128
64
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
>128
>128
1
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
>128
>128
>128
>128
>128
>128
1
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
2
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
4
8
0.5
>128
16
64
>128
16
64
128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
1
>128
1
1
1
>128
>128
>128
>128
>128
>128
8
>128
128
128
>128
>128
4
>128
>128
>128
>128
>128
8
8
1
>128
0.25
0.25
2
64
8
16
16
>128
>128
4
>128
>128
128
>128
>128
8
>128
>128
16
>128
>128
16
>128
4
4
64
128
32
64
64
>128
>128
>128
8
>128
>128
128
>128
2
>128
16
>128
>128
128
>128
>128
>128
16
>128
>128
>128
0.125
4
0.5
2
0.25
0.5
16
16
16
2
16
8
8
0.5
2
0.5
6.1.5.2. Compound 7b. Yield: 83.3%; mp: 95e96 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
3.39e3.88 (3H, m, pyrrolidine), 3.96e4.34 (4H, m, pyrrolidine and
OCH2CH3). MS (ESI, m/z): 187 (M þ H)þ.
6.1.6. General procedure for the synthesis of fluoroquinolone
derivatives 9e18
d
(ppm): 1.19 (3H, t, J ¼ 6.8 Hz, OCH2CH3),
A mixture of 1 8a,b (1.0 mmol), 7aef (1.2 mmol), dry triethyl-
amine (8.0 mmol) and dry acetonitrile (20 mL) was stirred at room
temperature under an atmosphere of nitrogen for 3e8 h. The
resulting solid was collected by suction, and dried in vacuo to give
the title compounds 9e18 as off-white solids.
6.1.5.3. Compound 7c. Yield: 74.5%; mp: 168e170 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d (ppm): 3.62 (3H, s, OCH3), 3.63e3.80 (3H, m,
pyrrolidine), 3.83 (3H, s, OCH3), 3.89e3.95 (2H, m, pyrrolidine). MS
(ESI, m/z): 187 (M þ H)þ.
6.1.6.1. Compound 9. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.06e1.19 (4H, m, 2 ꢂ cyclopropylCH2), 3.70e3.88 (6H, m, pyrroli-
dine and OCH3), 4.14e4.55 (3H, m, pyrrolidine and cyclopropylCH),
8.05 (1H, d, J ¼ 12.8 Hz, C5-H), 8.59 (1H, s, C2-H). MS (ESI, m/z): 419
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C18H19FKN6O5 (M þ K)þ:
457.10380; Found: 457.10850.
6.1.5.4. Compound 7d. Yield: 92.4%; mp: 163e165 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 1.14e1.20 (3H, m, OCH2CH3), 3.65
(3H, s, OCH3), 3.68e3.79 (5H, m, pyrrolidine), 4.03e4.09 (2H, m,
OCH2CH3). MS (ESI, m/z): 201 (M þ H)þ.
6.1.5.5. Compound 7e. Yield: 86.4%; mp: 102e103 ꢀC; 1H NMR
6.1.6.2. Compound 10. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
(400 MHz, DMSO-d6)
3.66e3.94 (10H, m, pyrrolidine, OCH3, and OCH2CH3). MS (ESI,
m/z): 201 (M þ H)þ.
d
(ppm): 1.16 (3H, t, J ¼ 6.8 Hz, OCH2CH3),
1.07e1.25 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.70e3.73
(3H, m, pyrrolidine), 4.07e4.16 (3H, m, pyrrolidine and cyclo-
propylCH), 4.22e4.24 (2H, m, OCH2CH3), 8.04 (1H, d, J ¼ 12.8 Hz, C5-
H), 8.59 (1H, s, C2-H). MS (ESI, m/z): 433 (M þ H)þ. HRMS-ESI (m/z):
6.1.5.6. Compound 7f. Yield: 80.6%; mp: 135e137 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
3.42e3.60 (3H, m,
2 ꢂ OCH2CH3), 4.04e4.11 (2H, m, pyrrolidine). MS (ESI, m/z): 215
(M þ H)þ.
Calcd. For C19H21FN6NaO5 (M
455.14852.
þ
Na)þ: 455.14551; Found:
d
(ppm): 1.12e1.22 (6H, m, 2 ꢂ OCH2CH3),
pyrrolidine), 3.82e3.95 (4H, m,
6.1.6.3. Compound 11. 1H NMR (400 MHz, DMSO-d6)
1.06e1.19 (4H, m, cyclopropylCH2), 3.71e3.73 (2H, m,
d (ppm):
2
ꢂ

5504
Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
pyrrolidine and OCH3), 3.85 (3H, s, OCH3), 4.08e4.23 (2H, m, pyr-
rolidine), 4.47e4.57 (2H, m, pyrrolidine and cyclopropylCH), 8.04
(1H, d, J ¼ 12.4 Hz, C5-H), 8.58 (1H, s, C2-H). MS (ESI, m/z): 433
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C19H22FN6O5 (M þ H)þ:
433.15357; Found: 433.16039.
concentrated under reduced pressure. The residue was dissolved in
sodium hydroxide solution (2%, 20 mL) and stirred for 1e4 h at
room temperature. The reaction mixture was adjusted to pH 7 with
hydrochloric acid (6 N). The resulting solid was collected by suction,
and dried in vacuo to give the title compounds 19e49 as off-white
solids.
6.1.6.4. Compound 12. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.08e1.23 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.02e3.09
(3H, m, pyrrolidine), 3.57 (3H, s, OCH3), 3.69e3.75 (2H, m, pyrro-
lidine), 4.05e4.10 (3H, m, OCH2CH3 and cyclopropylCH), 8.06 (1H, d,
J ¼ 12.8 Hz, C5-H), 8.59 (1H, s, C2-H). MS (ESI, m/z): 447 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C20H24FN6O5 (M þ H)þ: 447.17922;
Found: 447.17756.
6.1.7.1. Compound 19. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
0.97e1.15 (4H, m, 2 ꢂ cyclopropylCH2), 3.61 (3H, s, OCH3), 3.82 (3H,
s, OCH3), 3.93e3.94 (2H, m, pyrrolidine), 4.13e4.16 (2H, m, pyrro-
lidine and cyclopropylCH), 4.22e4.38 (2H, m, pyrrolidine), 7.71 (1H,
d, J ¼ 13.2 Hz, C5-H), 8.67 (1H, s, C2-H). MS (ESI, m/z): 448 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C20H23FN5O6 (M þ H)þ: 448.16324;
Found: 448.15990.
6.1.6.5. Compound 13. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.02e1.19 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.70e3.73
(2H, m, pyrrolidine), 3.78e3.83 (2H, m, pyrrolidine), 3.85 (3H, s,
OCH3), 4.14e4.22 (2H, m, OCH2CH3), 4.48e4.68 (2H, m, pyrrolidine
and cyclopropylCH), 8.06 (1H, d, J ¼ 12.4 Hz, C5-H), 8.59 (1H, s, C2-
H). MS (ESI, m/z): 447 (M þ H)þ. HRMS-ESI (m/z): Calcd. For
C20H24FN6O5 (M þ H)þ: 447.17922; Found: 447.17642.
6.1.7.2. Compound 20. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.01e1.23 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.61 (3H, s,
OCH3), 3.82e3.86 (2H, m, pyrrolidine), 3.93e4.07 (4H, m, pyrroli-
dine and cyclopropylCH), 4.09e4.18 (2H, m, OCH2CH3), 7.68 (1H, d,
J ¼ 12.0 Hz, C5-H), 8.71 (1H, s, C2-H). MS (ESI, m/z): 462 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C21H25FN5O6 (M þ H)þ: 462.17889;
Found: 462.17778.
6.1.6.6. Compound 14. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.03e1.23 (10H, m, 2 ꢂ cyclopropylCH2 and 2 ꢂ OCH2CH3), 3.71e3.79
(4H, m, pyrrolidine), 4.05e4.21 (4H, m, 2 ꢂ OCH2CH3), 4.48e4.58
(2H, m, pyrrolidine and cyclopropylCH), 8.06 (1H, d, J ¼ 12.8 Hz, C5-
H), 8.59 (1H, s, C2-H). MS (ESI, m/z): 461 (M þ H)þ. HRMS-ESI (m/z):
Calcd. For C21H25FN6NaO5 (M þ Na)þ: 483.17681; Found: 483.17450.
6.1.7.3. Compound 21. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.01e1.16 (4H, m, 2 ꢂ cyclopropylCH2), 3.59 (3H, s, OCH3), 3.67 (3H,
s, OCH3), 3.71e3.90 (2H, m, pyrrolidine), 3.95 (3H, s, OCH3),
4.08e4.31 (4H, m, pyrrolidine and cyclopropylCH), 7.72
(1H, d, J ¼ 13.2 Hz, C5-H), 8.68 (1H, s, C2-H). MS (ESI, m/z): 462
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C21H25FN5O6 (M þ H)þ:
462.17889; Found: 462.17516.
6.1.6.7. Compound 15. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.30
(3H, s, OCH3), 3.61e3.77 (5H, m, pyrrolidine), 3.80 (3H, s, OCH3),
7.32e7.36 (1H, m, Ph-H), 7.58e7.62 (1H, m, Ph-H), 7.77e7.83 (1H,
m, Ph-H), 8.12 (1H, d, J ¼ 12.4 Hz, C5-H), 8.83 (1H, s, C2-H). MS (ESI,
m/z): 505 (M þ H)þ. HRMS-ESI (m/z): Calcd. For C22H20F3N6O5
(M þ H)þ: 505.14473; Found: 505.14464.
6.1.7.4. Compound 22. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.01e1.11 (4H, m, 2 ꢂ cyclopropylCH2), 1.17 (3H, t, J ¼ 6.4 Hz,
OCH2CH3), 3.56 (3H, s, OCH3), 3.64 (3H, s, OCH3), 3.89e4.14
(6H, m, pyrrolidine and cyclopropylCH), 4.29 (2H, q, J ¼ 6.4 Hz,
OCH2CH3), 7.72 (1H, d, J ¼ 13.2 Hz, C5-H), 8.67 (1H, s, C2-H). MS
6.1.6.8. Compound 16. 1H NMR (400 MHz, DMSO-d6)
d
(ppm):
(ESI, m/z): 476 (M
þ
H)þ. HRMS-ESI (m/z): Calcd. For
1.16e1.19 (3H, m, OCH2CH3), 3.46e3.61 (4H, m, pyrrolidine and
OCH2CH3), 4.03e4.05 (3H, m, pyrrolidine), 7.32e7.33 (1H, m, Ph-H),
7.56e7.61 (1H, m, Ph-H), 7.77e7.83 (1H, m, Ph-H), 8.13 (1H, d,
J ¼ 12.4 Hz, C5-H), 8.85 (1H, s, C2-H). MS (ESI, m/z): 519 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C23H22F3N6O5 (M þ H)þ: 519.16038;
Found: 519.15930.
C22H26FN5NaO6 (M þ Na)þ: 498.17648; Found: 498.17577.
6.1.7.5. Compound 23. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
0.98e1.22 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.56e3.60
(2H, m, pyrrolidine), 3.63 (3H, s, OCH3), 3.81 (3H, s, OCH3),
3.88e4.00 (2H, m, pyrrolidine and cyclopropylCH), 4.12e4.16 (2H,
m, OCH2CH3), 4.29e4.31 (2H, m, pyrrolidine), 7.72 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.67 (1H, s, C2-H). MS (ESI, m/z): 476 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C22H26FN5O6 (M þ H)þ: 476.19454;
Found: 476.19422.
6.1.6.9. Compound 17. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.06e1.26 (3H, m, OCH2CH3), 3.06e3.10 (2H, m, pyrrolidine),
3.25e3.39 (3H, m, pyrrolidine), 3.59e3.77 (2H, m, OCH2CH3), 3.80
(3H, s, OCH3), 7.33e7.35 (1H, m, Ph-H), 7.56e7.60 (1H, m, Ph-H),
7.62e7.83 (1H, m, Ph-H), 8.13 (1H, d, J ¼ 12.4 Hz, C5-H), 8.84 (1H, s,
C2-H). MS (ESI, m/z): 519 (M þ H)þ. HRMS-ESI (m/z): Calcd. For
C23H22F3N6O5(M þ H)þ: 519.16038; Found: 519.15660.
6.1.7.6. Compound 24. 1H NMR (400 MHz, DMSO-d6)
0.95e1.02 (4H, m, 2 ꢂ cyclopropylCH2), 1.07 (3H, t, J ¼ 6.8 Hz,
OCH2CH3), 1.20 (3H, t, 6.8 Hz, OCH2CH3), 3.56e3.60
d (ppm):
J
¼
(2H, m, pyrrolidine), 3.71e3.81 (4H, m, pyrrolidine and
OCH2CH3), 3.83e3.96 (2H, m, pyrrolidine and cyclopropylCH),
4.26 (2H, q, J ¼ 6.8 Hz, OCH2CH3), 7.72 (1H, d, J ¼ 13.6 Hz, C5-
H), 8.67 (1H, s, C2-H). MS (ESI, m/z): 490 (M þ H)þ. HRMS-ESI
(m/z): Calcd. For C23H29FN5O6 (M þ H)þ: 490.21019; Found:
490.20826.
6.1.6.10. Compound 18. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
0.96e1.19 (6H, m, 2 ꢂ OCH2CH3), 3.62e3.86 (5H, m, pyrrolidine),
4.00e4.07 (4H, m, 2 ꢂ OCH2CH3), 7.33e7.36 (1H, m, Ph-H), 7.57e7.60
(1H, m, Ph-H), 7.79e7.81 (1H, m, Ph-H), 8.13 (1H, d, J ¼ 12.8 Hz, C5-H),
8.85 (1H, s, C2-H). MS (ESI, m/z): 533 (M þ H)þ. HRMS-ESI (m/z):
Calcd. For C24H24F3N6O5(M þ H)þ: 533.17603; Found: 533.17486.
6.1.7.7. Compound 25. 1H NMR (400 MHz, DMSO-d6 þ D2O)
d (ppm):
6.1.7. General procedure for the synthesis of fluoroquinolone
derivatives 19e49
A mixture of 8ceh (1.0 mmol), 7aef (1.2 mmol), dry triethyl-
amine (8.0 mmol) and dry acetonitrile (20 mL) was stirred for
6e10 h at room temperature under an atmosphere of nitrogen.
After completion of the condensation, the reaction mixture was
0.77e0.89 (4H, m, 2 ꢂ cyclopropylCH2), 3.74e3.81 (3H, m, pyrroli-
dine), 3.84 (3H, s, OCH3), 3.85e4.08 (3H, m, pyrrolidine and cyclo-
propylCH), 6.93 (1H, t, J ¼ 73.2 Hz, OCHF2), 7.82 (1H, d, J ¼ 13.2 Hz,
C5-H), 8.65 (1H, s, C2-H). MS (ESI, m/z): 484 (M þ H)þ. HRMS-ESI
(m/z): Calcd. For C20H21F3N5O6 (M þ H)þ: 484.14439; Found:
484.14924.

Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
5505
6.1.7.8. Compound 26. 1H NMR (400 MHz, DMSO-d6)
d
(ppm):
(1H, s, C2-H). MS (ESI, m/z): 462 (M þ H)þ. HRMS-ESI (m/z): Calcd.
For C21H25FN5O6 (M þ H)þ: 462.17889, Found: 462.17804.
0.98e1.09 (4H, m, 2 ꢂ cyclopropylCH2), 1.22 (3H, t, J ¼ 6.4 Hz,
OCH2CH3), 3.30e3.60 (1H, m, pyrrolidine), 3.87 (2H, q, J ¼ 6.4 Hz,
OCH2CH3), 3.95e4.14 (5H, m, pyrrolidine and cyclopropylCH), 6.86
(1H, d, J ¼ 60.4 Hz, OCHF2), 7.87 (1H, d, J ¼ 13.6 Hz, C5-H), 8.78 (1H,
s, C2-H). MS (ESI, m/z): 498 (M þ H)þ. HRMS-ESI (m/z): Calcd. For
C21H23F3N5O6 (M þ H)þ: 498.16004; Found: 498.15794.
6.1.7.16. Compound 34. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.07e1.22 (6H, m, 2 ꢂ OCH2CH3), 1.41 (3H, d, J ¼ 8.0 Hz,
CH3), 3.76e3.81 (2H, m, pyrrolidine), 3.83e3.84 (1H, m, pyrroli-
dine), 3.92e3.99 (1H, m, OCH2CHN), 4.01e4.09 (2H, m,
OCH2CH3), 4.23e4.61 (2H, m, OCH2CH3), 4.89e4.91 (2H, m, pyrro-
lidine), 5.58e5.65 (2H, m, OCH2CHN), 7.58 (1H, d, J ¼ 13.2 Hz, C5-H),
8.94 (1H, s, C2-H). MS (ESI, m/z): 476 (M þ H)þ. HRMS-ESI (m/z):
Calcd. For C22H27FN5O6 (M þ H)þ: 476.19454; Found: 476.19159.
6.1.7.9. Compound 27. 1H NMR (400 MHz, DMSO-d6)
1.02e1.22 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.51e3.56
(2H, m, pyrrolidine), 3.57 (3H, s, OCH3), 3.85e3.96 (3H, m, pyr-
rolidine and OCH2CH3), 4.02e4.11 (3H, m, pyrrolidine and cyclo-
d (ppm):
propylCH), 6.93 (1H, d,
J
¼
74.0 Hz, OCHF2), 7.89 (1H, d,
6.1.7.17. Compound 35. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.42
J ¼ 13.2 Hz, C5-H), 8.78 (1H, s, C2-H). MS (ESI, m/z): 512 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C22H25F3N5O6 (M þ H)þ: 512.17569;
Found: 512.17774.
(3H, m, CH2CH3), 3.56e3.59 (1H, m, pyrrolidine), 3.85 (3H, s, OCH3),
4.01e4.03 (4H, m, pyrrolidine), 4.55e4.62 (2H, m, CH2CH3), 7.82
(1H, d, J ¼ 13.6 Hz, C5-H), 9.03 (1H, s, C2-H). MS (ESI, m/z): 424
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C18H19F2N5NaO5 (M þ Na)þ:
446.12519; Found: 446.12402.
6.1.7.10. Compound 28. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.01e1.22 (10H, m, 2 ꢂ cyclopropylCH2 and 2 ꢂ OCH2CH3),
3.55e3.57 (1H, m, pyrrolidine), 3.74e3.97 (5H, m, 2 ꢂ OCH2CH3,
and cyclopropylCH), 4.02e4.11 (4H, m, pyrrolidine), 6.92
(1H, d, J ¼ 73.0 Hz, OCHF2), 7.88 (1H, d, J ¼ 12.8 Hz, C5-H), 8.75 (1H,
s, C2-H). MS (ESI, m/z): 526 (M þ H)þ. HRMS-ESI (m/z): Calcd. For
C23H27F3N5O6 (M þ H)þ: 526.19134; Found: 526.19147.
6.1.7.18. Compound 36. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.17e1.23 (3H, m, CH2CH3), 1.42 (3H, t, J ¼ 6.8 Hz, OCH2CH3),
3.56e3.89 (1H, m, pyrrolidine), 4.01e4.03 (2H, m, CH2CH3), 4.10
(2H, q, J ¼ 6.8 Hz, OCH2CH3), 4.38e4.45 (2H, m, pyrrolidine),
4.54e4.57 (2H, m, pyrrolidine), 7.81 (1H, d, J ¼ 12.4 Hz, C5-H), 8.88
(1H, s, C2-H). MS (ESI, m/z): 438 (M þ H)þ. HRMS-ESI (m/z): Calcd.
For C19H21F2N5O5 (M þ H)þ: 438.15890; Found: 438.16112.
6.1.7.11. Compound 29. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.43e1.44 (3H, m, CH3), 3.30 (3H, s, OCH3), 3.77e3.81 (2H, m, pyr-
rolidine), 3.92e3.98 (1H, m, pyrrolidine), 4.25e4.34 (2H, m, pyr-
rolidine), 4.41e4.60 (1H, m, OCH2CHN), 5.34e5.48 (2H, m,
OCH2CHN), 7.57 (1H, d, J ¼ 13.6 Hz, C5-H), 8.90 (1H, s, C2-H). MS (ESI,
m/z): 434 (M þ H)þ. HRMS-ESI (m/z): Calcd. For C19H20FN5NaO6
(M þ Na)þ: 456.12953; Found: 456.12632.
6.1.7.19. Compound 37. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.53
(3H, t, J ¼ 6.8 Hz, CH2CH3), 3.58 (3H, s, OCH3), 3.60e3.63 (1H, m,
pyrrolidine), 3.83 (3H, s, OCH3), 4.06e4.14 (2H, m, pyrrolidine), 4.49
(2H, q, J ¼ 6.8 Hz, CH2CH3), 4.81e4.83 (2H, m, pyrrolidine), 7.88 (1H,
d, J ¼ 13.6 Hz, C5-H), 9.35 (1H, s, C2-H). MS (ESI, m/z): 438 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C19H22F2N5O5 (M þ H)þ: 438.15890;
Found: 438.16256.
6.1.7.12. Compound 30. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.20
(3H, t, J ¼ 6.4 Hz, OCH2CH3), 1.41e1.43 (3H, m, CH3), 4.06 (2H, q,
J ¼ 6.4 Hz, OCH2CH3), 4.21e4.32 (3H, m, pyrrolidine), 4.37e4.40
(1H, m, OCH2CHN), 4.44e4.58 (1H, m, pyrrolidine), 4.83e4.84 (1H,
m, pyrrolidine), 5.33e5.46 (2H, m, OCH2CHN), 7.54 (1H, d,
J ¼ 13.2 Hz, C5-H), 8.84 (1H, s, C2-H). MS (ESI, m/z): 448 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C20H22FN5NaO6 (M þ Na)þ: 470.14518;
Found: 470.14774.
6.1.7.20. Compound 38. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.19
(3H, t, J ¼ 6.8 Hz, CH2CH3), 1.42 (3H, t, J ¼ 7.2 Hz, OCH2CH3),
3.52e3.55 (1H, m, pyrrolidine), 3.58 (3H, s, OCH3), 3.98e4.01
(2H, m, pyrrolidine), 4.02 (2H, q, J ¼ 6.8 Hz, CH2CH3), 4.37 (2H, q,
J ¼ 7.2 Hz, OCH2CH3), 4.43e4.55 (2H, m, pyrrolidine), 7.82 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.87 (1H, s, C2-H). MS (ESI, m/z): 452 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C20H23F2N5NaO5 (M þ Na)þ: 474.15649;
Found: 474.15579.
6.1.7.13. Compound 31. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.43
(3H, d, J ¼ 7.2 Hz, CH3), 3.46e3.50 (1H, m, pyrrolidine), 3.59
(3H, s, OCH3), 3.71e3.77 (1H, m, pyrrolidine), 3.81 (3H, s, OCH3),
3.92e3.94 (1H, m, OCH2CHN), 4.24e4.62 (2H, m, pyrrolidine),
4.89e4.91 (1H, m, pyrrolidine), 5.66e5.74 (2H, m, OCH2CHN), 7.58
(1H, d, J ¼ 13.2 Hz, C5-H), 8.94 (1H, s, C2-H). MS (ESI, m/z): 448
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C20H23FN5O6 (M þ H)þ:
448.16324; Found: 448.15153.
6.1.7.21. Compound 39. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.12
(3H, t, J ¼ 7.2 Hz, OCH2CH3), 1.42 (3H, t, J ¼ 6.8 Hz, CH2CH3),
3.54e3.58 (1H, m, pyrrolidine), 3.79e3.84 (2H, m, CH2CH3), 3.86
(3H, s, OCH3), 3.98e4.01 (2H, m, pyrrolidine), 4.40 (2H, t, J ¼ 7.2 Hz,
OCH2CH3), 4.55e4.57 (2H, m, pyrrolidine), 7.82 (1H, d, J ¼ 13.6 Hz,
C5-H), 8.89 (1H, s, C2-H). MS (ESI, m/z): 452 (M þ H)þ. HRMS-ESI (m/
z): Calcd. For C20H24F2N5O5 (M þ H)þ: 452.17455; Found: 452.17331.
6.1.7.14. Compound 32. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.18 (3H, d, J ¼ 6.0 Hz, CH3), 1.43 (3H, t, J ¼ 7.6 Hz, OCH2CH3),
3.28 (3H, s, OCH3), 3.46e3.50 (2H, m, pyrrolidine), 3.92e3.93
(1H, m, OCH2CHN), 4.00 (2H, q, J ¼ 7.6 Hz, OCH2CH3), 4.20e4.45
(3H, m, pyrrolidine), 5.64e5.72 (2H, m, OCH2CHN), 7.58 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.93 (1H, s, C2-H). MS (ESI, m/z): 462 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C21H25FN5O6 (M þ H)þ: 462.17889,
Found: 462.18083.
6.1.7.22. Compound 40. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.18
(3H, t, J ¼ 7.2 Hz, CH2CH3), 1.20 (3H, t, J ¼ 7.2 Hz, OCH2CH3), 1.53
(3H, t, J ¼ 7.2 Hz, OCH2CH3), 3.58e3.62 (1H, m, pyrrolidine),
3.82 (2H, q, J ¼ 7.2 Hz, OCH2CH3), 4.06e4.13 (5H, m, OCH2CH3 and
pyrrolidine), 4.49e4.50 (2H, m, pyrrolidine), 4.81e4.83 (2H, m,
pyrrolidine), 7.88 (1H, d, J ¼ 13.6 Hz, C5-H), 9.35 (1H, s, C2-H). MS
(ESI, m/z): 466 (M þ H)þ. HRMS-ESI (m/z): Calcd. For C21H26F2N5O5
(M þ H)þ: 466.19020; Found: 466.18862.
6.1.7.15. Compound 33. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.10
(3H, t, J ¼ 7.2 Hz, OCH2CH3), 1.41e1.48 (3H, m, CH3), 3.47e3.50 (1H,
m, pyrrolidine), 3.73e3.79 (1H, m, OCH2CHN), 3.83e3.85 (2H, m,
pyrrolidine), 3.98e4.33 (4H, m, pyrrolidine and OCH2CH3),
5.60e5.67 (2H, m, OCH2CHN), 7.58 (1H, d, J ¼ 13.2 Hz, C5-H), 8.94
6.1.7.23. Compound 41. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.06e1.27 (4H, m, 2 ꢂ cyclopropylCH2), 3.58 (3H, s, OCH3),
3.66e3.79 (1H, m, cyclopropylCH), 3.83 (3H, s, OCH3), 3.99e4.14
(3H, m, pyrrolidine), 4.33e4.45 (2H, m, pyrrolidine), 7.78 (1H, d,

5506
Q. Guo et al. / European Journal of Medicinal Chemistry 45 (2010) 5498e5506
J ¼ 13.6 Hz, C5-H), 8.56 (1H, s, C2-H). MS (ESI, m/z): 450 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C20H22F2N5O5 (M þ H)þ: 450.15890;
Found: 450.16007.
(m/z): Calcd. For C20H23F3N5O5 (M þ H)þ: 470.16513; Found:
470.16317.
6.1.7.31. Compound 49. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.07
6.1.7.24. Compound 42. 1H NMR (400 MHz, DMSO-d6)
d
(ppm):
(3H, t, J ¼ 6.8 Hz, OCH2CH3), 1.17 (3H, t, J ¼ 6.8 Hz, OCH2CH3), 3.84
(2H, q, J ¼ 6.8 Hz, OCH2CH3), 3.99e4.03 (5H, m, pyrrolidine), 4.06
(2H, q, J ¼ 6.8 Hz, OCH2CH3), 4.82e4.96 (4H, m, CH2CH2F), 7.83 (1H,
d, J ¼ 13.2 Hz, C5-H), 8.83 (1H, s, C2-H). MS (ESI, m/z): 484 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C21H25F3N5O5 (M þ H)þ: 484.18078;
Found: 484.18128.
1.07e1.27 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.31e3.35
(3H, m, pyrrolidine and cyclopropylCH), 3.59 (3H, s, OCH3),
3.67e4.15 (5H, m, pyrrolidine and OCH2CH3), 7.67 (1H, d, J ¼ 11.2 Hz,
C5-H), 8.56 (1H, s, C2-H). MS (ESI, m/z): 464 (M þ H)þ. HRMS-ESI
(m/z): Calcd. For C21H24F2N5O5 (M þ H)þ: 464.17455; Found:
464.17695.
6.2. Antibacterial activity
6.1.7.25. Compound 43. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.06e1.17 (7H, m, 2 ꢂ cyclopropylCH2 and OCH2CH3), 3.55e3.59
(1H, m, cyclopropylCH), 3.75e3.84 (2H, m, pyrrolidine), 3.85 (3H, s,
OCH3), 4.00e4.44 (5H, m, pyrrolidine and OCH2CH3), 7.77 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.64 (1H, s, C2-H). MS (ESI, m/z): 464 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C21H24F2N5O5 (M þ H)þ: 464.17455;
Found: 464.17134.
All compounds were screened for their in vitro antibacterial
activity against representative Gram-positive and Gram-negative
strains, by means of standard twofold serial dilution method using
agar media [16]. Minimum inhibitory concentration (MIC) is
defined as the minimum concentration of the compound required
to give complete inhibition of bacterial growth after incubation at
35 ꢀC for 18e24 h.
6.1.7.26. Compound 44. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
3.06e3.09 (1H, m, pyrrolidine), 3.56e3.59 (1H, m, pyrrolidine), 3.82
(3H, s, OCH3), 4.00e4.02 (3H, m, pyrrolidine), 4.81e4.96 (4H, m,
CH2CH2F), 7.83 (1H, d, J ¼ 12.8 Hz, C5-H), 8.99 (1H, s, C2-H). MS (ESI,
m/z): 442 (M þ H)þ. HRMS-ESI (m/z): Calcd. For C18H19F3N5O5
(M þ H)þ: 442.13383; Found: 442.13352.
Acknowledgments
The work was supported by the National S&T Major Special
Project on Major New Drug Innovation (No. 2009ZX09301-003).
6.1.7.27. Compound 45. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
References
1.17e1.27 (3H, m, OCH2CH3), 4.01e4.13 (6H, m, OCH2CH3 and pyr-
rolidine), 4.81e4.94 (5H, m, pyrrolidine and CH2CH2F), 7.82 (1H, d,
J ¼ 13.6 Hz, C5-H), 8.76 (1H, s, C2-H). MS (ESI, m/z): 456 (M þ H)þ.
HRMS-ESI (m/z): Calcd. For C19H21F3N5O5 (M þ H)þ: 456.14948;
Found: 456.14549.
[1] K. Drlica, M. Malik, R.J. Kerns, X.L. Zhao, Antimicrob. Agents Chemother. 52 (2008)
385e392.
[2] D.J. Dwyer, M.A. Kohanski, B. Hayete, J.J. Collins, Mol. Syst. Biol. 3 (2007)
142e156.
[3] D.R. Choi, J.H. Shin, J. Yang, S.Y. Yoon, Y.H. Yung, Bioorg. Med. Chem. Lett. 14
(2004) 1273e1277.
6.1.7.28. Compound 46. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
[4] C.Y. Hong, Y.K. Kim, Y.H. Lee, J.H. Kwak, Bioorg. Med. Chem. Lett. 8 (1998)
221e226.
[5] V. Cecchetti, A. Fravolini, M.C. Lorenzini, O. Tabarrini, P. Terni, T. Xin, J. Med.
Chem. 39 (1996) 436e445.
[6] L.J.V. Piddock, Antimicrob. Agents Chemother. 38 (1994) 163e169.
[7] A. Bryskier, J.F. Chantot, Drugs 49 (Suppl. 2) (1995) 16e28.
[8] H. Koga, A. Itoh, S. Murayama, S. Suzue, T. Irikura, J. Med. Chem. 23 (1980)
1358e1363.
[9] J.M. Domagala, J. Antimicrob. Chemother. 33 (1994) 685e706.
[10] C.Y. Hong, Y.K. Kim, J.H. Chang, S.H. Kim, H. Choi, D.H. Nam, Y.Z. Kim,
J.H. Kwak, J. Med. Chem. 40 (1997) 3584e3593.
3.30e3.56 (1H, m, pyrrolidine), 3.58 (3H, s, OCH3), 3.82 (3H, s,
OCH3), 3.97e4.01 (4H, m, pyrrolidine), 4.81e4.94 (4H, m, CH2CH2F),
7.83 (1H, d, J ¼ 13.6 Hz, C5-H), 8.82 (1H, s, C2-H). MS (ESI, m/z): 456
(M þ H)þ. HRMS-ESI (m/z): Calcd. For C19H20F3N5NaO5 (M þ Na)þ:
478.13142; Found: 478.13194.
6.1.7.29. Compound 47. 1H NMR (400 MHz, DMSO-d6)
d (ppm): 1.17
(3H, t, J ¼ 5.6 Hz, OCH2CH3), 3.56 (3H, s, OCH3), 3.97e4.10 (4H,
m, pyrrolidine), 4.37 (2H, q, J ¼ 5.6 Hz, OCH2CH3), 4.82e4.96
(5H, m, pyrrolidine and CH2CH2F), 7.84 (1H, d, J ¼ 13.6 Hz, C5-H),
8.83 (1H, s, C2-H). MS (ESI, m/z): 470 (M þ H)þ. HRMS-ESI (m/z):
Calcd. For C20H23F3N5O5 (M þ H)þ: 470.16513; Found: 470.16048.
[11] Z.L. Wan, Y. Chai, M.L. Liu, H.Y. Guo, Chin. J. Med. Chem. 19 (2009) 109e111.
[12] J.X. Wang, Q. Guo, Y. Chai, L.S. Feng, H.Y. Guo, M.L. Liu, Chin. Chem. Lett. 21
(2010) 55e58.
[13] Y.B. Zhang, L.S. Feng, X.F. You, Q. Guo, H.Y. Guo, M.L. Liu, Arch. Pharm. Chem.
Life Sci. 343 (2010) 143e151.
[14] Y. Chai, Z.L. Wan, B. Wang, H.Y. Guo, M.L. Liu, Eur. J. Med. Chem. 44 (2009)
4063e4069.
[15] Q. Guo, L.Y. Sun, H.Y. Guo, M.L. Liu, Acta Crystallogr. E. E65 (2009) o580.
[16] 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 35ꢀC for 18-24 h.
6.1.7.30. Compound 48. 1H NMR (400 MHz, DMSO-d6)
d (ppm):
1.10e1.14 (3H, m, OCH2CH3), 3.73e3.80 (2H, m, pyrrolidine), 3.82
(3H, s, OCH3), 3.94e3.99 (2H, m, OCH2CH3), 4.31e4.42 (3H, m,
pyrrolidine), 4.80e4.85 (4H, m, CH2CH2F), 7.81 (1H, d, J ¼ 13.6 Hz,
C5-H), 8.76 (1H, s, C2-H). MS (ESI, m/z): 470 (M þ H)þ. HRMS-ESI