Design, Synthesis, and invitro Antibacterial Activity of Fluoroquinolone Derivatives Containing a Chiral 3-(Alkoxyimino)-2-(aminomethyl)azetidine Moiety

Article abstract of DOI:10.1002/cmdc.201200210

A series of novel (R)/(S)-7-(3-alkoxyimino-2-aminomethyl-1-azetidinyl)fluoroquinolone derivatives were synthesized and evaluated for their invitro antibacterial activity against representative strains. Our results reveal that 12 of the target compounds generally show better activity (MIC: <0.008-0.5μgmL^-1) against the tested Gram-positive strains including MRSA and MRSE than levofloxacin (LVFX, MIC: 0.125-8μgmL^-1). Their activity is similar to that of gemifloxacin (GMFX, MIC: <0.008-4μgmL^-1). However, they are generally less active than the two reference drugs against Gram-negative strains. Moreover, against clinical strains of S.aureus including MRSA and S.epidermidis including MRSE, the MIC₅₀ values (0.06-16μgmL^-1) and MIC₉₀ values (0.5-32μgmL^-1) of compounds 16w, y, and z are 2-8- and 2-16-fold less than LVFX, respectively, and 16w (MIC₉₀ range: 0.5-4μgmL^-1) was also found to be more active than GMFX (MIC₉₀ range: 1-8μgmL^-1).

Full text of DOI:10.1002/cmdc.201200210

MED
DOI: 10.1002/cmdc.201200210
Design, Synthesis, and in vitro Antibacterial Activity of
Fluoroquinolone Derivatives Containing a Chiral
3-(Alkoxyimino)-2-(aminomethyl)azetidine Moiety
Kai Lv,[a] Yexin Sun,[a] Lanyin Sun,[a] Zengquan Wei,[a] Huiyuan Guo,[a] Jinwei Wu,[b] and
Mingliang Liu*[a]
A series of novel (R)/(S)-7-(3-alkoxyimino-2-aminomethyl-1-aze-
tidinyl)fluoroquinolone derivatives were synthesized and evalu-
ated for their in vitro antibacterial activity against representa-
tive strains. Our results reveal that 12 of the target compounds
4 mgmLÀ1). However, they are generally less active than the
two reference drugs against Gram-negative strains. Moreover,
against clinical strains of S. aureus including MRSA and S. epi-
dermidis including MRSE, the MIC50 values (0.06–16 mgmLÀ1
)
generally show better activity (MIC: <0.008–0.5 mgmLÀ1
)
and MIC90 values (0.5–32 mgmLÀ1) of compounds 16w, y, and z
are 2–8- and 2–16-fold less than LVFX, respectively, and 16w
(MIC90 range: 0.5–4 mgmLÀ1) was also found to be more active
than GMFX (MIC90 range: 1–8 mgmLÀ1).
against the tested Gram-positive strains including MRSA and
MRSE than levofloxacin (LVFX, MIC: 0.125–8 mgmLÀ1). Their ac-
tivity is similar to that of gemifloxacin (GMFX, MIC: <0.008–
Introduction
Quinolones have become a major class of antibacterial agents
that are used mainly to fight both community-acquired and se-
rious hospital-acquired infections. These antibiotics act by
binding two type II bacterial topoisomerases, DNA gyrase (top-
oisomerase II) and topoisomerase IV.[1,2]
Since the discovery of norfloxacin by Koga et al. in 1980,[3]
most research concerning quinolone antibacterials has focused
on the basic group at the C7 position, which greatly influences
the potency, spectrum, and safety of these compounds. So far,
all of the marketed fluoroquinolones, including ciprofloxacin,
moxifloxacin, levofloxacin (LVFX), and gemifloxacin (GMFX)
(Figure 1) possess a five- or six-membered nitrogen heterocycle
at the C7 position.[4,5] There are significantly fewer published
reports of azetidinyl-based quinolones than of pyrrolidinyl-, pi-
perazinyl-, and piperidinyl-based analogues.
3341, Figure 1), bearing a 3-(hydroxy)azetidine moiety, displays
excellent activity against Gram-positive pathogens including
both methicillin-susceptible S. aureus (MSSA) and -resistant
S. aureus (MRSA),[8] and phase III clinical studies began in April
2008.[9] WQ-3813 (Figure 1), with a C7 3-(isopropylamino)azeti-
dine ring structurally related to delafloxacin, has the potential
to be developed into a treatment for respiratory infections in
both community and hospital settings[9] owing to its activity
against major respiratory pathogens including multidrug- and
quinolone-resistant isolates.
Fujita et al. reported that the antibacterial activity of 2-ami-
nomethyl-1-azetidinyl-based quinolones is superior to that of
the corresponding 3-aminomethyl-1-pyrolidinyl- and piperazin-
1-yl-based analogues.[10] In addition, previous work on pyrroli-
dinyl quinolones emphasized the importance of the oxime
functional group with respect to biological activity. For exam-
ple, GMFX shows much more antibacterial activity than its des-
methyloximino analogue.[5]
Recently, a number of quinolone derivatives containing an
azetidine moiety, a rather common and stable functional
group frequently found in antimicrobial agents such as b-lac-
tams and polyoximic acids,[6,7] were synthesized, some of
which were found to have potent in vitro and in vivo antimi-
crobial activity. It was reported that delafloxacin (ABT-492, RX-
Inspired by the above research results, we considered the in-
troduction of an alkyloxime moiety to 2-(aminomethyl)azeti-
dine to construct structurally unique compounds, 3-(alkoxyimi-
no)-2-(aminomethyl)azetidines, as side chains at the 7-position
of quinolones (Figure 2). Moreover, considering that our side
chains have a chiral center at the 2-position of the rings, prep-
[a] Dr. K. Lv,+ Y. Sun,+ L. Sun, Z. Wei, Prof. H. Guo, Prof. M. Liu
Institute of Medicinal Biotechnology
Chinese Academy of Medical Sciences, 100050 Beijing (China)
E-mail: lmllyx@yahoo.com.cn
[b] J. Wu
Zhejiang Starry Pharmaceutical Co. Ltd., 317300 Xianju (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200210.
Figure 1. Structures of gemifloxacin, delafloxacin, and WQ-3813.
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(R)-6 was treated with isobutyl chloroformate in the presence
of N-methylmorpholine (NMM) to give a mixed anhydride,
which was then treated with diazomethane to afford diazoke-
tone (S)/(R)-7 in 68–71% yield. Cyclization of (S)/(R)-7 via an in-
tramolecular rhodium-catalyzed NÀH insertion reaction in di-
chloromethane at À788C produced the key intermediate (S)/
(R)-8.[14,15] Interestingly, we successfully obtained compounds
(S)/(R)-6 and -7 with recrystallization methods instead of
column chromatographic separation techniques, as in the re-
ported procedures.[13–15]
Figure 2. Design of the new azetidine side chain.
aration of both enantiomers is desirable, based on the concept
that biologically active molecules interact with the chiral bind-
ing site of the target enzyme, and hence the chirality of
a given molecule has a significant impact on its biological ac-
tivity.[10] Herein we describe the design and synthesis of (R)/(S)-
The azetidine-3-one (S)/(R)-8 was smoothly converted into
the oximes (R)-9a–h/(S)-9g,h by condensation with the above
alkylhydroxylamine derivatives 3a–e obtained as shown in
Scheme 1 and commercially available O-alkylhydroxylamine hy-
drochlorides in the presence of pyridine.[16] Removal of the
TBDPS protecting group of (R)-9a–h/(S)-9g,h by tetra-n-buty-
lammonium fluoride (TBAF) in THF afforded the oxime alcohols
(R)-10a–h/(S)-10g,h, which were then converted into the cor-
responding bromides by nucleophilic substitution with tetra-
bromomethane in dichloromethane/triphenylphosphine, in 70–
80% yield.[17] However, Gabriel reaction of the resulting bro-
mides and phthalimide potassium in DMF met with no success,
we were unable to obtain the desired products (S)-11 a–h/(R)-
11 g,h, but instead, 2-methyleneazetidine derivatives via elimi-
nation reaction, unexpectedly.
3-(alkoxyimino)-2-(aminomethyl)azetidine
derivatives
and
a series of novel quinolone compounds containing these cyclic
amines at the C7 position. Our primary objective was to opti-
mize the potency of these quinolones against Gram-positive
and Gram-negative organisms.
Results and Discussion
Chemistry
Detailed synthetic pathways to new 3-(alkyloximino)-2-(amino-
methyl)azetidines (S)-14a–h/(R)-14g,h and novel quinolone
derivatives 16a–z are shown below in Schemes 2 and 3, re-
spectively.
Subsequently, Mitsunobu reaction of (R)-10a–h/(S)-10g,h
with phthalimide in diethyl azodicarboxylate (DEAD)/PPh3/THF
was used to furnish condensates (S)-11 a–h/(R)-11 g,h,[18,19]
which, upon hydrazinolysis in ethanol, gave primary amines
(S)-12a–h/(R)-12g,h. At this stage, however, we had to protect
the amino group with Boc for purification. The bis-Boc protect-
ing groups of (S)-13a–h/(R)-13g,h were then removed by
pumping dry hydrogen chloride gas in dichloromethane to
afford new azetidine dihydrochlorides (S)-14a–h/(R)-14g,h
(Scheme 2).
Commercially unavailable O-alkylhydroxylamines 3a–e were
first prepared according to Scheme 1. N-Hydroxyphthalimide
1 was treated with bromoalkanes (RBr) in N,N-dimethylforma-
mide (DMF) in the presence of 18-crown-6 and potassium car-
Finally, the target compounds 16a–w were obtained by cou-
pling the new azetidine derivatives (S)-14a–h/(R)-14g,h with
various compounds containing naphthyridone and quinolone
cores in the presence of triethylamine. However, for 16s–z,
boric chelates 15c–f were required to increase reactivity
(Scheme 3).[20] All of the compounds synthesized were thor-
oughly characterized by analytical techniques.
Because the oxime group is present in the E or Z configura-
tion, it was necessary to determine the geometries of all the
oxime target compounds 16a–z. Unfortunately we were
unable to prepare X-ray-quality single crystals of any oxime in-
termediate or product. Nonetheless, we speculate that the
oxime group of the target compounds is present in a mixed
configuration of E and Z isomers due to signal doublings of
Scheme 1. Synthesis of O-alkylhydroxylamine hydrochlorides 3a–e. Reagents
and conditions: a) RBr, 18-crown-6, K2CO3, DMF, 808C, 6 h, 56–71%;
b) NH2NH2·H2O, CH2Cl2, MeOH, RT, 3 h; c) HCl, EtOH, 1 h, RT, 46–61% (for two
steps).
bonate at 808C, and the resulting condensates 2a–e were sub-
sequently treated with hydrazine hydrate in dichloromethane/
methanol to produce the desired compounds 3a–e by follow-
ing well-established procedures.[11]
1
the azetidine ring of the compounds observed in the H NMR
spectra. An approximation of the isomer ratio (Table 1) is possi-
ble only based on the 1H NMR data of the hydrogen atom
linked to the chiral center of the azetidine ring, which are ap-
parently separate for most of the compounds 16a–z. In addi-
tion, further separation of compounds 16a–z into E and Z
oxime components by chromatographic techniques is currently
in progress.
Treatment of l/d-serine (S)/(R)-4 with di-tert-butyl dicarbon-
ate (Boc2O) in dioxane gave Boc-protected compound (S)/(R)-
5,[12] and the hydroxy group of (S)/(R)-5 was subsequently pro-
tected by reaction with tert-butyldiphenylchlorsilane (TBDPSCl)
in DMF in the presence of imidazole to yield serine derivative
(S)/(R)-6, in an overall yield of 73–74% for the two steps.[13] (S)/
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Against Gram-positive strains
such as MSSA, MRSA, methicillin-
susceptible S. epidermidis (MSSE),
methicillin-resistant S. epidermi-
dis (MRSE), and Streptococcus
pneumoniae, compounds 16a, b,
d, g, i, k, l, n, u, w, x, and z show
strong antibacterial activity (MIC
<0.008–0.5 mgmLÀ1), which is
generally better than LVFX (MIC
0.125–8 mgmLÀ1) and similar to
GMFX (MIC <0.008–4 mgmLÀ1).
Among them, compounds 16a,
b, i, l, u, and z (MIC <0.008–
0.06 mgmLÀ1) were found to be
four- to 1024-fold more active
against MSSE and MRSE than the
two reference drugs (MIC 0.25–
8 mgmLÀ1).
Compounds 16w, y, and z,
with better Gram-positive activi-
ty, were chosen for further evalu-
ation of their in vitro activity
against the clinical strains of
MSSA, MRSA, MSSE, and MRSE.
The MIC50 and MIC90 ranges for
these compounds, along with
those of LVFX and GMFX for
comparison, are listed in Table 4.
Against the four species of bac-
teria tested, the MIC50 (0.06–
16 mgmLÀ1) and MIC90 values
(0.5–32 mgmLÀ1) of 16w, y, and z
are 2–8- and 2–16-fold less than
Scheme 2. Synthesis of 3-(alkyloximino)-2-(aminomethyl)azetidines (S)-14a-h/(R)-14g,h. Reagents and conditions:
a) NaOH, (Boc)2O, dioxane, RT, 12 h, then KHSO4; b) TBDPSCl, imidazole, DMF, RT, 7 h, 73–74% (for two steps);
c) iBuOCOCl, NMM, CH2N2, Et2O, 8 h, RT, 68–71%; d) Rh2(OAc)4, CH2Cl2, À788C, 12 h, 46–53%; e) R1ONH2·HCl, pyri-
dine, EtOH, 608C, 5 h; f) TBAF, THF, RT, 10 h, 41–60% (for two steps); g) phthalimide, Ph3P, DEAD, THF, RT, 48–71%;
h) NH2NH2·H2O, EtOH, reflux, 3 h; i) (Boc)2O, MeOH, RT, 12 h, 53–82% (for two steps); j) HCl(g), CH2Cl2, 1 h, RT, 47–
67%.
for LVFX (MIC50 0.25–32 mgmLÀ1
,
MIC90 8–64 mgmLÀ1), respectively,
and compound 16w was also
found to have superior activity
(MIC90 0.5–4 mgmLÀ1) to that of
GMFX (MIC90 1–8 mgmLÀ1).
Scheme 3. Synthesis of the target compounds 16a–z. Reagents and conditions: a) Et3N, MeCN, 508C, 5 h, 18–35%;
b) 1. Et3N, MeCN, 508C, 5 h; 2. 5% NaOH, 508C, 12 h, 13–27%. See Table 1 for structures.
In the case of Gram-positive
strains, the activity imparted to
the 1-cyclopropyl-1,8-naphthyri-
done ring by the alkyl group of
Antibacterial activity
the oxime moiety is in the order: cyclobutyl ꢀ cyclopentyl ꢁ
ethyl ꢀ cyclopropylmethyl > methyl ꢀ benzyl > 3’,4’-methyle-
nedioxylbenzyl. However, different results were obtained in
comparisons of the activity of the other naphthyridone or qui-
nolone ring systems (Table 2).
The target compounds 16a–z were evaluated for their in vitro
antibacterial activity against representative Gram-positive and
Gram-negative strains using standard techniques.[21] Minimum
inhibitory concentration (MIC) is defined as the compound
concentration required to give complete inhibition of bacterial
growth, and MIC values of the synthesized compounds, along
with those of the reference drugs LVFX and GMFX for compari-
son, are listed in Tables 2, 3, and 4. These data suggest that all
of the target compounds 16a–z have considerable activity
against the Gram-positive strains (MICs < 0.008–8 mgmLÀ1), al-
though they are generally less active than LVFX and GMFX
against the Gram-negative strains, with a few exceptions.
Notably, the chirality at C2 of the azetidinyl group of the
target compounds influences activity. For example, the S enan-
tiomers 16g, i, q, and u show better activity against Gram-pos-
itive strains than the corresponding R enantiomers 16h, j, r,
and v (Table 2), and similar results were also obtained in com-
parisons of the Gram-negative activity (Table 3). This suggests
that the S enantiomers may fit better into the binding sites of
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Chiral Fluoroquinolone Derivatives
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bacterial activity; compounds 16w, y, and z were then chosen
for further evaluation of their activity against clinical isolates.
Twelve of the target compounds generally show better activity
(MIC <0.008–0.5 mgmLÀ1) against Gram-positive strains includ-
ing MRSA and MRSE than LVFX (MIC 0.125–8 mgmLÀ1), and ac-
tivity similar to GMFX (MIC <0.008–4 mgmLÀ1), although they
are generally less active than the two reference drugs against
the Gram-negative strains. Against clinical strains of MSSA,
MRSA, MSSE, and MRSE, compounds 16w, y, and z are more
active than LVFX, and 16w (MIC90 0.5–4 mgmLÀ1) was also
found to be more active than GMFX (MIC90 1–8 mgmLÀ1).
Table 1. Structures and physical data of compounds 16a–z.
[a]
Compd R1
R2
Y
*
Ratio[b] mp [8C][c]
16a
16b
16c
cyclobutyl
cyclopentyl
cyclohexyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
N
N
N
N
S
49:51 181–183
60:40 157–158
85:15 214–216
35:65 148–151
S
S
S
16d cyclopropylmethyl
16e
cyclopropyl
N
S
45:55 135–137
16 f
16g
16h
16i
16j
16k
benzyl
methyl
methyl
ethyl
ethyl
cyclobutyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
2,4-difluoro-
phenyl
2,4-difluoro-
phenyl
N
N
N
N
N
N
S
S
R
S
R
S
37:63 137–138
32:68 151–153
45:55 171–173
50:50 170–172
20:80 168–170
Experimental Section
Synthesis
Melting points were determined in open capillaries and are uncor-
rected. 1H NMR (400, 500, or 600 MHz) and 13C NMR (125 or
150 MHz) spectra were recorded at 258C on Varian Mercury spec-
trometers. Chemical shifts (d) are given in ppm relative to tetrame-
thylsilane or the respective NMR solvent. Electrospray ionization
(ESI) mass spectra and high-resolution mass spectra (HRMS) were
obtained on a MDSSCIEX Q-Tap mass spectrometer. The reagents
were all of analytical grade or chemically pure. TLC was performed
on silica gel plates (Merck, ART5554 60 F254).
–
148–150
16l
cyclopentyl
N
N
S
S
50:50 178–180
48:52 173–174
16m cyclopropylmethyl 2,4-difluoro-
phenyl
2,4-difluoro-
phenyl
16n
N
N
S
S
20:80 170–172
16o
16p
16q
16r
benzyl
methyl
ethyl
2,4-difluoro-
phenyl
2,4-difluoro-
phenyl
2,4-difluoro-
phenyl
2,4-difluoro-
phenyl
–
174–176
45:55 193–194
182–184
General procedure for the synthesis of 2a–e: To a stirring solu-
tion of 2-(hydroxy)isoindoline-1,3-dione (1, 48.94 g, 300 mmol) in
DMF (500 mL) was added K2CO3 (82.93 g, 600 mmol), alkyl bro-
mides (1200 mmol), and 18-crown-6 (15.85 g, 60 mmol) at room
temperature. The reaction mixture was stirred at 808C for 6 h.
Cooled to room temperature, the mixture was poured into distilled
H2O (1200 mL). The precipitate was filtered, washed with petrole-
um ether (PE) and dried in vacuo to yield the title compounds
2a–e.
N
N
N
S
S
R
–
ethyl
48:52 183–185
C-
OMe
16s
16t
cyclopropyl
cyclopropyl
S
S
51:49 120–121
51:49 118–120
methyl
ethyl
C-
OMe
C-
OMe
C-
General procedure for the synthesis of 3a–e: To a stirring solu-
tion of 2a–e (40 mmol) in CH2Cl2 (300 mL) and MeOH (30 mL) was
added 80% NH2NH2·H2O (5.0 mL, 80 mmol) at room temperature.
The reaction mixture was stirred at the same temperature for 3 h
and then filtered. The filtrate was washed with 10% NH3(aq). The or-
ganic layer was dried over anhydrous MgSO4 and then filtered. The
filtrate was concentrated under reduced pressure. The residue was
dissolved in EtOH (60 mL), concentrated HCl (10 mL) was added,
and the mixture was stirred for 1 h at room temperature. The mix-
ture was concentrated under reduced pressure, and the crude
product was recrystallized from Et2O (100 mL) to afford the title
compounds 3a–e.
16u
16v
cyclopropyl
cyclopropyl
S
43:57 108–110
37:63 110–112
ethyl
R
OMe
16w
16x
S
S
46:64 154–155
20:80 204–206
methyl
methyl
16y
16z
cyclopropyl
ethyl
C-F
C-F
S
S
52:48 145–147
47:53 167–169
[a] Configuration. [b] Isomer ratios based on the 1H NMR spectroscopic
data of the hydrogen atom linked to the chiral center of the azetidine
ring. [c] Melting points are uncorrected.
General procedure for the synthesis of (S)/(R)-6: To a solution of
l/d-serine ((S)/(R)-4, 42.04 g, 400 mmol) in 25% NaOH (180 mL)
was added a solution of (Boc)2O (130.95 g, 600 mmol) in dioxane
(250 mL) at 08C. The reaction mixture was stirred for 12 h at room
temperature, and the dioxane was removed under reduced pres-
sure. The residue was diluted with ice water (1000 mL), adjusted to
pH 2–3 with KHSO4, and extracted with EtOAc. The combined ex-
tracts were dried over anhydrous MgSO4 and then filtered. The fil-
trate was concentrated under reduced pressure to afford the crude
product (S)/(R)-5, which was used directly in the next step without
further purification.
the target enzymes, causing more potent enzyme inhibition
and hence more potent antibacterial activity.
Conclusions
In summary, a series of (R)/(S)-7-(3-alkoxyimino-2-aminomethyl-
1-azetidinyl)fluoroquinolone derivatives were designed, synthe-
To a solution of the above crude (S)/(R)-5 and imidazole (108.93 g,
1600 mmol) in DMF (200 mL) was added dropwise tert-butylchloro-
diphenylsilane (137.44 g, 500 mmol) over a period of 1 h at room
1
sized, and characterized by H NMR, MS, HRMS, and 13C NMR.
These derivatives were initially evaluated for their in vitro anti-
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M. Liu et al.
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Table 2. In vitro antibacterial activity of compounds 16a–z against Gram-positive strains.
Compd
Strains MIC [mgmLÀ1
S.a.2[b] MSSA1[c] MSSA2[d] MRSA1[e] MRSA2[f] MSSE1[g] MSSE2[h] MRSE1[i] MRSE2[j] S.p.1[k] S.p.2[l] S.p.3[m] S.p.4[n] S.p.5[o]
]
S.a.1[a]
16a
16b
16c
16d
16e
16 f
16g
16h
16i
0.06
0.125
0.25
0.06
0.5
0.125
0.25
0.06
0.03
0.015
0.25
0.125
0.25
0.125
0.5
0.015
0.25
0.125
0.25
0.25
0.125
2
0.015
0.06
0.06
0.125
0.25
0.25
1
0.125
0.5
0.25
0.5
8
0.5
4
0.5
0.5
0.5
0.5
1
0.06
0.5
0.5
0.25
1
0.25
1
0.25
0.5
8
0.25
8
0.5
0.06
0.25
0.25
0.125
0.25
1
0.125
0.25
0.5
0.25
0.5
0.125
0.25
4
0.125
4
0.125
0.06
0.25
0.125
0.03
0.25
0.5
0.25
0.5
0.06
0.25
8
0.5
0.5
1
0.25
1
0.25
0.5
8
0.5
8
0.5
0.06
0.25
0.5
0.125
0.25
1
0.5
0.5
1
0.25
1
0.25
0.5
8
0.5
8
0.5
0.06
0.25
0.5
0.125
0.25
1
<0.008 <0.008 <0.008 <0.008
<0.008 <0.008 <0.008 <0.008
0.06
0.5
0.5
0.25
1
0.25
0.25
2
0.03
0.03
0.5
0.25
0.5
0.5
0.5
4
0.06
0.25
0.125
0.25
0.5
0.25
0.5
0.125
0.25
4
0.125
4
0.06
0.5
0.5
0.25
1
0.25
0.25
4
0.25
1
0.125 0.125
0.06
0.25
0.25
0.125
0.25
1
0.03
0.03
0.5
0.25
1
0.25
0.5
8
0.5
0.25
1
0.5
0.5
8
0.5
0.25
1
0.5
0.5
8
0.015
<0.008 <0.008
0.125
8
0.125
8
0.015
0.125
8
0.125
8
<0.008 <0.008 <0.008 <0.008
0.25
0.5
0.06
0.25
16j
16k
16l
0.25
0.125
0.06
1
0.25
0.125
0.06
1
0.25
0.125
0.06
1
0.25
0.125
0.06
1
0.125 0.125 0.125
0.06
0.25
0.25
0.125
0.125
0.5
0.5
0.5
0.125
0.25
4
0.06
1
0.25
0.5
1
0.5
1
0.5
0.25
0.06
1
0.06
0.25
0.125
0.03
0.25
0.5
0.25
0.5
0.06
0.25
4
0.06
1
0.25
1
1
1
16m
16n
16o
16p
16q
16r
16s
16t
16u
16v
16w
16x
16y
16z
0.25
1
0.25
1
0.5
1
0.5
1
1
1
1
1
1
1
<0.008 <0.008
0.5
0.5
1
0.5
1
1
2
0.5
2
0.5
2
1
2
1
0.5
0.5
0.25
0.25
4
2
0.5
0.25
0.25
8
0.06
0.25
0.25
0.06
0.03
0.25
0.5
0.25
0.25
8
0.06
0.25
0.25
0.06
0.03
0.25
0.5
0.25
0.06
2
0.25
0.25
4
0.06
0.25
0.125
0.25
0.125
0.25
8
0.06
0.25
0.06
0.06
0.03
0.25
0.25
0.06
2
0.25
0.06
2
4
4
0.06
0.125
0.06
0.125
2
0.03
4
8
0.06
0.125
0.06
0.125
2
0.03
4
8
0.06
0.125
0.06
0.03
0.03
0.125
0.5
0.5
0.5
0.5
0.06
0.5
0.5
0.125
0.125
0.06
0.25
0.25
0.125
0.125
0.06
0.25
0.25
0.125 0.125 0.125
0.125 0.125
0.06
0.06
0.25
0.125 0.125
0.125 0.125
0.06
0.06
0.25
0.06
0.03
0.03
0.06
0.25
0.25
0.06
0.25
0.25
GMFX[p] <0.008 0.015
LVFX[q]
0.125 0.25
0.125
[a] S. aureus ATCC25923. [b] S. aureus ATCC43300. [c] Methicillin-sensitive S. aureus 12-1. [d] Methicillin-sensitive S. aureus 12-4. [e] Methicillin-resistant
S. aureus 12-3. [f] Methicillin-resistant S. aureus 12-15. [g] Methicillin-sensitive S. epidermidis 12-2. [h] Methicillin-sensitive S. epidermidis 12-22. [i] Methicillin-
resistant S. epidermidis 12-2. [j] Methicillin-resistant S. epidermidis 12-33. [k] Streptococcus pneumoniae 12-1. [l] S. pneumoniae 12-2. [m] S. pneumoniae 12-3.
[n] S. pneumoniae 12-4. [o] S. pneumoniae 12-5. [p] Gemifloxacin. [q] Levofloxacin.
temperature. The reaction mixture was stirred for 7 h at the same
temperature, diluted with distilled H2O (300 mL), and adjusted to
pH 2–3 with 3n citric acid. The mixture was extracted with EtOAc,
and the combined extracts were washed with saturated brine,
dried over anhydrous Na2SO4, and filtered. The filtrate was concen-
trated under reduced pressure. The crude product was recrystal-
lized from a mixed solvent of EtOAc and PE (1:5, v/v) to afford the
title compound (S)/(R)-6.
phy (silica gel), eluting with PE and EtOAc (8:1, v/v) to give the title
compound (S)/(R)-8.
General procedure for the synthesis of (R)-9a–h/(S)-9g–h: To
a stirring solution of (S)/(R)-8 (21.95 g, 50 mmol) in EtOH was
added pyridine and the appropriate O-alkylhydroxylamine hydro-
chloride (60 mmol). The reaction mixture was stirred at 608C for
5 h and concentrated under reduced pressure. The residue was
treated with EtOAc and washed successively with H2O and saturat-
ed saline, dried over anhydrous MgSO4, and filtered. The filtrate
was concentrated under reduced pressure. The crude products (R)-
9a–d, h and (S)-9g were used directly in the next step, whereas
(R)-9e–g and (S)-9h were purified by column chromatography
(silica gel), eluting with PE and EtOAc (10:1, v/v).
General procedure for the synthesis of (S)/(R)-7: To a stirring solu-
tion of (S)/(R)-6 (17.72 g, 40 mmol) and isobutyl chloroformate
(6.83 g, 50 mmol) in anhydrous THF (100 mL) was added dropwise
NMM (5.06 g, 50 mmol) over a period of 10 min at À258C. The re-
action mixture was stirred for 30 min at the same temperature,
and a solution of freshly prepared diazomethane in Et2O (150 mL,
100 mmol) was added dropwise over a period of 1 h. The mixture
was stirred for 8 h at room temperature and concentrated under
reduced pressure. The residue was treated with PE (300 mL) at
À308C and filtered. The precipitate was washed with PE and dried
in vacuo to yield the title compound (S)/(R)-7.
General procedure for the synthesis of (R)-10a–h/(S)-10g,h: To
a stirring solution of (R)-9a–h/(S)-9g,h (40 mmol) in THF (300 mL)
was added TBAF (60 mmol, 60 mL 1.0n solution in THF) at room
temperature. The reaction mixture was stirred for 10 h at the same
temperature, and concentrated under reduced pressure. The resi-
due was treated with EtOAc, and washed successively with H2O
and saturated saline, dried over anhydrous MgSO4, and filtered.
The filtrate was concentrated under reduced pressure. The crude
products were purified by column chromatography (silica gel),
eluting with PE and EtOAc (3:2, v/v) to afford the title compounds
(R)-10a–h/(S)-10g,h.
General procedure for the synthesis of (S)/(R)-8: To a solution of
(S)/(R)-7 (18.68 g, 40 mmol) in anhydrous CH2Cl2 was added
Rh2(OAc)4 (0.18 g, 0.4 mmol) at À788C. The reaction mixture was
stirred for 4 h at the same temperature and overnight at room
temperature, then filtered. The filtrate was concentrated under re-
duced pressure. The residue was purified by column chromatogra-
1234
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ChemMedChem 2012, 7, 1230 – 1236

Chiral Fluoroquinolone Derivatives
MED
Table 3. In vitro antibacterial activity of compounds 16a–z against Gram-negative strains.
Compd
Strains MIC [mgmLÀ1
E.co.2[g] E.co.3[h] E.co.4[i] E.co.5[j] E.co.6[k] K.p.1[l] K.p.2[m] K.p.3[n] K.p.4[o]
]
P.a.1[a] P.a.2[b] P.a.3[c] P.a.4[d] P.a.5[e] E.co.1[f]
16a
16b
16c
16d
16e
16 f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
8
4
16
2
8
4
2
32
4
64
8
16
8
8
2
8
4
8
16
16
8
4
8
16
16
32
16
8
16
8
16
16
32
8
16
16
8
4
8
16
16
8
4
8
0.125
0.25
1
0.03
0.5
0.125
0.03
1
<0.008
1
4
4
16
4
4
8
1
16
1
16
16
8
128
>128
>128
128
>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
128
>128
>128
>128
64
>128
64
16
>128
>128
>128
>128
>128
0.06
>128
>128
128
64
128
128
64
>128
>128
16
32
16
4
2
16
4
1
8
32
16
8
32
8
>128
2
16
>128
8
>128
8
64
16
2
32
0.5
16
32
16
1
0.25
0.5
4
0.125
8
1
0.25
0.25
0.5
2
0.125
1
1
0.25
4
0.015
4
2
4
2
4
4
0.06
1
2
4
0.5
2
8
0.25
1
1
0.25
0.06
0.125
8
16
32
8
16
16
8
8
16
32
8
16
16
8
8
2
16
0.5
32
16
16
16
8
16
16
64
16
16
16
16
16
16
8
16
16
32
16
16
16
8
16
16
64
16
16
16
16
16
16
16
4
32
8
16
16
8
>128
64
4
64
8
32
8
0.015
4
2
4
8
4
4
0.06
1
2
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
64
>128
>128
64
64
128
64
32
64
32
32
64
8
32
64
4
32
8
>128
4
16
16
16
2
64
32
>128
16
32
64
8
64
128
32
32
8
128
2
32
16
16
2
2
2
1
2
8
8
8
16
32
8
16
16
16
16
16
8
4
4
16
2
8
16
16
1
8
8
16
4
4
32
0.5
1
16
0.5
4
8
4
8
8
64
1
2
2
0.015
0.5
1
2
0.25
0.5
8
16
16
16
16
4
4
16
8
16s
16t
4
1
8
8
16u
16v
16w
16x
16y
16z
GMFX[p]
LVFX[q]
>128
128
64
>128
128
64
4
4
0.125
0.125
0.25
0.06
0.015
0.03
0.5
1
1
0.25
0.06
0.125
16
16
16
16
8
16
16
16
16
8
4
2
1
1
4
1
16
16
8
64
32
8
0.125
0.5
16
16
2
0.5
4
4
[a] Pseudomonas aeruginosa ATCC27853. [b] P. aeruginosa 12-1. [c] P. aeruginosa 12-2. [d] P. aeruginosa 12-4. [e] P. aeruginosa 12-5. [f] E. coli ATCC25922.
[g] E. coli 12-1. [h] E. coli 12-3. [i] Extended-spectrum b-lactamase-producing (ESBL+) E. coli 12-1. [j] ESBL+ E. coli 12-3. [k] ESBL+ E. coli 12-4. [l] Klebsiella
pneumoniae 12-1. [m] K. pneumoniae 12-2. [n] ESBL+ K. pneumoniae 12-2. [o] ESBL+ K. pneumoniae 12-4. [p] Gemifloxacin. [q] Levofloxacin.
General procedure for the synthesis of (S)-11a–h/(R)-11g–h: To
Table 4. Comparative in vitro antibacterial activity of 16w, 16y, and 16z
a
mixture of (R)-10a-h/(S)-10g,h (40 mmol), PPh3 (20.98 g,
against clinical isolates.
80 mmol) and phthalimide (11.76 g, 80 mmol) in anhydrous THF
(300 mL) was added dropwise diethyl azodicarboxylate (13.93 g,
80 mmol) at room temperature over a period of 10 min. The reac-
tion mixture was stirred for 12 h at the same temperature and con-
centrated under reduced pressure. The residue was treated with
EtOAc, washed successively with H2O and saturated saline, dried
over anhydrous MgSO4, and filtered. The filtrate was concentrated
under reduced pressure, and the residue was treated with PE at
08C. The precipitate was filtered, and the filtrate was concentrated
under reduced pressure. The crude products (S)-11 a and (R)-11 g,h
were used directly in the next step, whereas (S)-11 b–h were puri-
fied by column chromatography (silica gel), eluting with PE and
EtOAc (3:1, v/v).
Microorganism
Compd
MIC [mgmLÀ1
]
Range
MIC50
MIC90
MSSA[a]
MRSA[b]
MSSE[c]
MRSE[d]
16w
16y
16z
GMFX[e]
LVFX[f]
16w
16y
0.06–2
0.06–8
0.03–16
0.03–4
0.125–32
0.06–8
0.25–16
0.06–32
0.03–8
0.25–64
0.5
0.125–2
0.06–2
0.06–1
0.25–8
0.06–0.5
0.06–4
0.03–8
0.015–4
0.125–8
0.06
0.125
0.06
0.03
0.25
2
8
16
8
32
0.5
0.5
0.125
0.25
0.5
0.5
2
2
0.5
4
2
8
16
4
32
4
16
32
8
64
0.5
2
2
1
16z
GMFX
LVFX
16w
16y
General procedure for the synthesis of (S)-13a–h/(R)-13g–h: A
mixture of (S)-11 a–h/(R)-11 g–h (20 mmol) and 80% NH2NH2·H2O
(1.88 mL, 30 mmol) in EtOH (150 mL) was heated at reflux and
stirred for 3 h at the same temperature. The precipitate was fil-
tered, and the filtrate was concentrated under reduced pressure.
The residue was treated with EtOAc, and washed successively with
H2O and saturated saline, dried over anhydrous MgSO4, and fil-
tered. The filtrate was concentrated under reduced pressure to
give the crude products (S)-12a–h/(R)-12g–h as light-yellow oils,
which were used directly in the next step.
16z
GMFX
LVFX
16w
16y
16z
GMFX
LVFX
8
0.5
4
8
1
8
[a] Methicillin-sensitive S. aureus; 10 isolates. [b] Methicillin-resistant
S. aureus; 10 isolates. [c] Methicillin-sensitive S. epidermidis; 10 isolates.
[d] Methicillin-resistant S. epidermidis; 10 isolates. [e] Gemifloxacin. [f] Lev-
ofloxacin.
To a mixture of the above crude (S)-12a–h/(R)-12g,h in EtOH
(50 mL) was added (Boc)2O (6.55 g, 30 mmol) at room temperature.
ChemMedChem 2012, 7, 1230 – 1236
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www.chemmedchem.org
1235

M. Liu et al.
MED
The reaction mixture was stirred overnight at the same tempera-
ture, and concentrated under reduced pressure. The crude product
(S)-13a was used directly in the next step, whereas (S)-13b–h and
(R)-13g,h were purified by column chromatography (silica gel),
eluting with PE and EtOAc (3:1, v/v).
Acknowledgements
This work was supported by the National S&T Major Special Proj-
ect on Major New Drug Innovation (number 2012ZX09301002-
001-017).
General procedure for the synthesis of (S)-14a–h/(R)-14g,h: To
a stirring solution of (S)-13a–h/(R)-13g,h (10 mmol) in CH2Cl2
(30 mL) was pumped dried HCl(g) for 1 h at 0–58C. The reaction
mixture was allowed to stir for another 30 min at room tempera-
ture, and the precipitate was collected by suction to give the title
compounds (S)-14a–h/(R)-14g,h.
Keywords: antibiotics · chirality · drug design · medicinal
chemistry · quinolones
[1] K. Drlica, M. Malik, R. J. Kerns, X. Zhao, Antimicrob. Agents Chemother.
2008, 52, 385–392.
General procedure for the synthesis of the target compounds
16a–r: To a solution of (S)-14a–h/(R)-14g,h (1 mmol) and Et3N
(3 mmol) in anhydrous MeCN (10 mL) was added 15a,b (0.8 mmol)
at room temperature. The reaction mixture was stirred for 5 h at
508C, and concentrated under reduced pressure. The residue was
dissolved in 20% AcOH (10 mL), stirred for 0.5 h at 508C, and fil-
tered. The filtrate was adjusted to pH 6.5–7.5 by 15% NaOH and
extracted by CH2Cl2. The combined extracts were dried over anhy-
drous MgSO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography (silica gel), elut-
ing with CH2Cl2 and MeOH (10:1, v/v) to afford the title compounds
16a–r.
[2] R. E. Hancock, Mol. Syst. Biol. 2007, 3, 142.
[3] H. Koga, A. Itoh, S. Murayama, S. Suzue, T. Irikura, J. Med. Chem. 1980,
23, 1358–1363.
[4] Y. Chai, M. L. Liu, K. Lv, L. S. Feng, S. J. Li, L. Y. Sun, S. Wang, H. Y. Guo,
Eur. J. Med. Chem. 2011, 46, 4267–4273.
[5] 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. 1997, 40, 3584–3593.
[6] G. Emmer, Tetrahedron 1992, 48, 7165–7172.
[7] Y. Dejaegher, N. M. Kuz’menok, A. M. Zvonok, N. De Kimpe, Chem. Rev.
2002, 102, 29–60.
[8] S. J. Harnett, A. P. Fraise, J. M. Andrews, G. Jevons, N. P. Brenwald, R.
Wise, J. Antimicrob. Chemother. 2004, 53, 783–792.
[9] J. A. Wiles, B. J. Bradbury, M. J. Pucci, Expert Opin. Ther. Pat. 2010, 20,
1295–1319.
General procedure for the synthesis of target compounds 16s–
z: To a solution of (S)-14e,g,h/(R)-14h (1 mmol) and Et3N (3 mmol)
in MeCN (10 mL) was added 15c–f (0.8 mmol) at room tempera-
ture. The reaction mixture was stirred overnight at 508C, and con-
centrated under reduced pressure. The residue was dissolved in
a solution of 5% NaOH (8 mL) and stirred for 1 h at 508C. After
cooling to room temperature, the mixture was adjusted to pH 7.0–
7.5 with 5% AcOH, and extracted with CH2Cl2. The combined ex-
tracts were concentrated under reduced pressure. The residue was
dissolved in 20% AcOH (10 mL), stirred for 0.5 h at 508C, and fil-
tered. The filtrate was adjusted to pH 6.5–7.5 by 15% NaOH and
extracted by CH2Cl2. The combined extracts were dried over anhy-
drous MgSO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography (silica gel), elut-
ing with CH2Cl2 and MeOH (10:1, v/v) to afford the title compounds
16s–z.
[10] M. Fujita, K. Chiba, Y. Tominaga, K. Hino, Chem. Pharm. Bull. 1998, 46,
787–796.
[11] P. Brown, S. H. Calvert, P. C. A. Chapman, S. C. Cosham, A. J. Eglington,
R. L. Elliot, M. A. Harris, J. D. Hinks, J. Lowther, D. J. Merrikin, M. J. Pear-
son, R. J. Ponsford, J. V. Syms, J. Chem. Soc. Perkin Trans. 1 1991, 881–
891.
[12] P. Garner, J. M. Park, J. Org. Chem. 1987, 52, 2361–2364.
[13] A. C. B. Burtoloso, C. R. D. Correia, J. Organomet. Chem. 2005, 690,
5636–5646.
[14] S. Hanessian, J. M. Fu, Can. J. Chem. 2001, 79, 1812–1826.
[15] S. Hanessian, J.-M. Fu, J.-L. Chiara, R. D. Fabio, Tetrahedron Lett. 1993,
34, 4157–4160.
[16] K. Lv, M. L. Liu, L. S. Feng, L. Y. Sun, Y. X. Sun, Z. Q. Wei, H. Y. Guo, Eur. J.
Med. Chem. 2012, 47, 619–625.
[17] J. F. Miller, E. M. Turner, K. S. Gudmundsson, S. Jenkinson, A. Spalten-
stein, M. Thomson, P. Wheelan, Bioorg. Med. Chem. Lett. 2010, 20, 2125–
2128.
[18] S. G. Davies, K. B. Ling, P. M. Roberts, A. J. Russell, J. E. Thomson, P. A.
Woods, Tetrahedron 2010, 66, 6806–6813.
[19] K. Csatayovꢁ, S. G. Davies, J. A. Lee, K. B. Ling, P. M. Roberts, A. J. Russell,
J. E. Thomson, Tetrahedron 2010, 66, 8420–8440.
[20] Y. Chai, M. L. Liu, B. Wang, X. F. You, L. S. Feng, Y. B. Zhang, J. Cao, H. Y.
Guo, Bioorg. Med. Chem. Lett. 2010, 20, 5195–5198.
[21] MIC values 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, NCCLS, Wayne, PA (USA), 2001, M100–S11). MIC was defined as
the lowest concentration of each compound that inhibits visible
growth of bacteria after incubation at 358C for 18–24 h.
MIC determination
All the target compounds 16a–z were screened for their in vitro
antibacterial activity against representative Gram-positive and
Gram-negative strains by using a standard twofold serial dilution
method with agar media.[21] Minimum inhibitory concentration
(MIC) is defined as the minimum concentration of the compound
required to give complete inhibition of bacterial growth after incu-
bation at 358C for 18–24 h.
Received: April 20, 2012
Revised: May 10, 2012
Published online on May 25, 2012
1236
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ChemMedChem 2012, 7, 1230 – 1236