Design, synthesis and biological evaluations of novel 7-[3-(1-aminocycloalkyl)pyrrolidin-1-yl]-6-desfluoro-8-methoxyquinolones with potent antibacterial activity against multi-drug resistant Gram-positive bacteria

Article abstract of DOI:10.1016/j.bmc.2009.08.026

A series of novel 6-desfluoro [des-F(6)] and 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyquinolones bearing 3-(1-aminocycloalkyl)pyrrolidin-1-yl substituents at the C-7 position (1-6) was synthesized to obtain potent drugs for nosocomial infect

Full text of DOI:10.1016/j.bmc.2009.08.026

Bioorganic & Medicinal Chemistry 17 (2009) 6879–6889
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluations of novel
7-[3-(1-aminocycloalkyl)pyrrolidin-1-yl]-6-desfluoro-8-methoxyquinolones
with potent antibacterial activity against multi-drug resistant
Gram-positive bacteria q
a
a
b
a
a
Rie Miyauchi a, , Katsuhiro Kawakami , Masao Ito , Norikazu Matsuhashi , Hitoshi Ohki , Hiroaki Inagaki ,
*
Hisashi Takahashi a, Makoto Takemura a
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
b Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2009
Revised 11 August 2009
Accepted 13 August 2009
Available online 20 August 2009
A series of novel 6-desfluoro [des-F(6)] and 6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyqu-
inolones bearing 3-(1-aminocycloalkyl)pyrrolidin-1-yl substituents at the C-7 position (1–6) was synthe-
sized to obtain potent drugs for nosocomial infections caused by Gram-positive pathogens. The des-F(6)
compounds 4–6 exhibited at least four times more potent activity against representative Gram-positive
bacteria than ciprofloxacin or moxifloxacin. Among the derivatives, 7-[(3R)-3-(1-aminocyclopropan-1-
yl)pyrrolidin-1-yl] derivative 4, which showed favorable profiles in preliminary toxicological and non-
clinical pharmacokinetic studies, exhibited potent antibacterial activity against clinically isolated
Gram-positive pathogens that had become resistant to one or more antibiotics.
Keywords:
6-Desfluoro-quinolones
Type II topoisomerases
Gram-positive bacteria
Antibacterial agents
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
available in clinical use, but they show some problems, for example,
resistance mutations and/or side effects.5,18 These problems have
Multi-drug-resistant Gram-positive pathogens, such as methicil-
lin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Strep-
tococcus pneumoniae (PRSP), and vancomycin-resistant enterococci
(VRE), have become a serious problem in the medical community.2–
5 One particularlyalarming sign istheacquisitionofresistance tovan-
comycin (VRE and vancomycin-resistant staphylococcal strains), an
antibioticgenerallyregardedastheagentoflastresortforhospital-ac-
quired infections.2,4–6 In the field of quinolone antibacterial agents,
various attempts have been made to obtain potent drugs for resistant
Gram-positivebacteria, andtrovafloxacin,7 moxifloxacin,8 gemifloxa-
cin,9 and gatifloxacin,10 etc., have beendeveloped and introducedinto
clinical use over the last few years. Considering that the incidence of
Gram-positive bacterial resistance to antibacterial agents has been
growing, however, the antibacterial activities of these newer quino-
lones are not potent enough and bacteria resistance to these agents
will be problematic in the near future.11,12 There are a few agents
other than quinolones, such as teicoplanin,13 quinupristin/dalfopri-
stin,14 and linezolid,15 tigecycline,16 daptomycin,17 which are now
been the driving force for the development of new antibacterial
agents that would be applicable to infections caused by multi-drug-
resistant Gram-positive pathogens.
In our previous paper, we reported that several quinolone deriv-
atives bearing 3-(1-amino-1-substituted-methyl)pyrrolidin-1-yl
groups, including 3-(1-aminocycloalkyl)pyrrolidin-1-yl substitu-
ent, at the C-7 position showed potent antibacterial activity against
Gram-positive bacteria.19 Although 7-[3-(1-aminocycloalkyl)pyrr-
olidin-1-yl]quinolone derivatives exhibit the most potent activity
among them, they possess higher genotoxicity than 7-(piperazin-
1-yl) or 7-(3-aminopyrrolidin-1-yl) quinolone derivatives. The
high genotoxicity of 7-(3-aminomethylpyrrolidin-1-yl)quinolone
derivatives has also been reported by other groups.20–23 As a meth-
od to reduce this genotoxicity, we reported the usefulness of intro-
ducing a (1R,2S)-2-fluorocyclopropan-1-yl substituent into the N-1
position instead of a cyclopropyl substituent.24–26 Furthermore, 8-
methoxyquinolone derivatives were reported to exhibit potent
antibacterial activity against Gram-positive bacteria and show
reduced phototoxicity in comparison with several quinolone
derivatives.27,28 Therefore, we fully used the scientific knowledge
of the structure–activity relationships of quinolone antibacterials
and a new 1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyquino-
q
See Ref. 1.
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8344.
E-mail address: miyauchi.rie.hr@daiichisankyo.co.jp (R. Miyauchi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.026

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R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
O
N
O
the in vitro antibacterial activity, and the toxicity profiles of the
8-methoxyquinolones 1–6.
R6
N
COOH
R6
N
COOH
H
N
OCH3
*
2. Chemistry
R10
OCH3
N
H
H2N
F
F
We planned to synthesize compounds 1–6 by an aromatic
nucleophilic substitution reaction from 8-methoxyquinolonecar-
boxlic derivatives 12, 13 and appropriate 3-(1-aminoalkyl)pyrrol-
idines 9–11, of which the peripheral amino groups were
protected by tert-butoxycarbonyl groups (Schemes 1 and 2).
The synthesis of the difluoroboron chelate derivative of the 6,7-di-
fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-8-methoxyquinolone-
3-carboxilic acid 12 is illustrated in Scheme 3. Thus, the reaction of
the acid chloride of benzoic acid 14 with ethyl 3-(dimethyl-
amino)acrylate, reported by Ataka et al.,34 followed by substitution
of the dimethylamino group with (1R,2S)-2-fluoro-1-cyclopropyl-
amine, gave crude enaminoketoester. Cyclization of the resultant
product with potassium carbonate in N,N-dimethylformamide
yielded ethyl 6,7-difluoro-8-methoxyquinolone-3-carboxylate 15.
The acidic hydrolysis of 15, followed by treatment of the resultant
6,7-difluoro-8-methoxyquinolone-3-carboxylate with boron tri-
fluoride diethyl etherate in diethyl ether provided difluoroboron
chelate 12.
The synthesis of the 7-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-
yl]-8-methoxyquinolone-3-carboxilic acid 13 is illustrated in
Scheme 4. Treatment of ketoester 17, reported by Ledoussal
et al.35 with ethyl orthoformate in acetic anhydride followed by
the reaction with (1R,2S)-2-fluoro-1-cyclopropylamine p-toluene-
sulfonate in the presence of triethylamine provided the crude ena-
minoketoester. Cyclization of the resultant product with sodium
hydride in 1,4-dioxane yielded ethyl 6-desfluoro-8-methoxyquino-
lone-3-carboxylate 18. Acidic hydrolysis of 18 provided quinolon-
ecarboxylic acid 13.
1: R6 = F, R10 = H
3: R6 =F
6: R6 =H
2: R6 =F, R10 = CH3
O
N
4 (DX-619): R6 =H, R10 = H
5: R6 =H, R10 = CH3
R6
N
COOH
H
H
OCH3
H2N
7
:
(PEG 9215875) R6=F
8 (PEG 9262932): R6=H
Figure 1. Chemical structure of 8-methoxyquinolones. (* The compound is
enantiomeric, although the absolute configuration has not been determined.)
lone having the (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl
substituent at the C-7 position (1, Fig. 1) was designed and synthe-
sized to obtain a highly potent compound against Gram-positive
bacteria with reduced genotoxicity. As we expected, 1 showed
highly potent antibacterial activity, but showed a positive response
in a micronucleus test and was toxic in a chromosome injuring
test.
Recently, favorable safety profiles of 6-desfluoro [des-F(6)]-
quinolones have been elucidated by a number of groups.29–31 In par-
ticular, it was reported by Ledoussal et al. that the genotoxicity of
des-F(6)-quinolone 8 (PEG 9262932) was less than that of 6-fluoro
(6-F)-quinolone 7 (PEG 9215875).32 As another approach to reduce
the toxicity of 7-[(3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-
yl]quinolone derivatives, we planned to utilize this strategy for
compound 1, and designed and synthesized several des-F(6)-quino-
lone compounds having the (3R)-3-(1-aminocyclopropan-1-yl)
pyrrolidin-1-yl substituent 4 (DX-619),1 (3R)-3-(1-methylaminocy-
clopropan-1-yl)pyrrolidin-1-yl substituent (5), or 3-(1-aminocy-
clobutan-1-yl)pyrrolidin-1-yl substituent (6)33 at the C-7 position.
In this paper, we draw parallels between des-F(6)-quinolones
4–6 and 6-F-quinolones 1–3. In addition, we report the synthesis,
The synthesis of (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-
yl substituents 936 is illustrated in Scheme 5. We designed a facile
synthetic route for 9 by using the intramolecular Horner–Wads-
worth–Emmons reaction of ketophosphonate 20. Treatment of the
amino keto ester 19, reported in our previous paper,37 with diethyl-
phosphonoacetic acid in the presence of 1,10-Carbonyldiimidazole
provided the condensation product 20. The subsequent intramolec-
O
O
F
COOH
R8
F
F
COOBF2
R9
R10
NH
*
R8
R9
R10
N
N
N
OCH3
N
OCH3
*
Boc
N
F
F
H
9:
1-3
R8,R9 = -(CH2)2-, R10 = H
12
10: R8,R9 = -(CH2)2-, R10 = CH3
11: R8,R9 = -(CH2)3-, R10 = H
Scheme 1. * The compound is enantiomeric, although the absolute configuration has not been determined (see Ref. 33).
O
O
H
N
COOH
R8
H
F
COOH
R9
R10
NH
*
R8
R9
R10
N
N
N
*
Boc
OCH3
4-6
OCH3
13
N
F
F
H
9: R8,R9 = -(CH2)2-, R10 = H
10: R8,R9 = -(CH2)2-, R10 = CH3
11: R8,R9 = -(CH2)3-, R10 = H
Scheme 2. * The compound is enantiomeric, although the absolute configuration has not been determined (see Ref. 33).

R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
6881
O
N
CH3
CH3
Boc
F
F
COOH
F
N
N
F
F
COOEt
N
H
a
b
a, b, c, d
Boc
Boc
H
H
H
O
O
N
N
N
OCH3
OCH3
F
Ph
Ph
Ph
25
27
28
14
15
O
N
O
CH3
F
F
COOH
F
F
COOBF2
N
e
c
f
Boc
H
N
N
H
10
OCH3
OCH3
F
F
16
12
Scheme 6. Reagents and conditions: (a) NaH, CH3I/DMF; (b) BH3–THF/THF, then
K2CO3/H2O, reflux; (c) H2, Pd–C/EtOH.
Scheme 3. Reagents and conditions: (a) SOCl2/toluene, reflux; (b) ethyl 3-(dimeth-
ylamino)acrylate, Et2N/THF; (c) (1R,2S)-2-fluoro-1-cyclopropylamine p-toluenesul-
fonate, Et3N/CH2Cl2; (d) K2CO3/DMF; (e) concentrated aqueous HCl, AcOH, reflux; (f)
BF3–OEt2/Et2O.
and a subsequent Curtius rearrangement using diphenylphosphoryl
azide (DPPA) and tert-butyl alcohol. Reduction of the amide moiety
of 25 with the borane–tetrahydrofuran complex provided N-(1-
phenylethyl)pyrrolidine derivative 26. Deprotection of the 1-phen-
ylethyl group by catalytic hydrogenation in ethanol yielded the
C-7 substituent 9. C-7 substituent 9 was used for the next reaction
without purification.
The synthesis of (3R)-3-(1-methylaminocyclopropan-1-yl)pyrr-
olidin-1-yl substituent 10 is illustrated in Scheme 6. Treatment of
the amide 25 with methyl iodide in the presence of sodium hydride
provided N-methylation product 27. Reduction of the amide moi-
ety of 27 with the borane–tetrahydrofuran complex provided N-
(1-phenylethyl)pyrrolidine derivative 28. Deprotection of the 1-
phenylethyl group by catalytic hydrogenation in ethanol yielded
the C-7 substituent 10. C-7 substituent 10 was used for the next
reaction without purification.
The reaction of the resultant crude products 9,10 with difluo-
roboron chelate 12, followed by removal of the chelates under ba-
sic conditions and deprotection of the tert-butoxycarbonyl groups
under acidic conditions, gave the 6-fluoro-1-[(1R,2S)-2-fluorocy-
clopropan-1-yl]-8-methoxyquinolone-3-carboxilic acid having
the 3-(1-aminocyclopropyl)pyrrolidine substituents 1–2, as shown
in Scheme 7. The 7-[3-(1-aminocyclobutan-1-yl)pyrrolidine] com-
pound 3 was synthesized by the same procedure as described in
Scheme 7, from C-7 substituent 11, reported in our previous
paper.33
O
O
N
O
F
COOEt
d
COOH
COOEt
a, b, c
F
N
OCH3
F
F
OCH3
OCH3
F
F
17
18
13
Scheme 4. Reagents and conditions: (a) Ac2O, CH(OEt)3, reflux; (b) (1R,2S)-2-
fluoro-1-cyclopropylamine p-toluenesulfonate, Et3N/CH2Cl2; (c) NaH/1,4-dioxane;
(d) concentrated aqueous HCl, AcOH, reflux.
ular Horner–Wadsworth–Emmons reaction was proceeded by using
potassium tert-butoxide in toluene below 0 °C to yield cyclic ena-
mide 21.38 Hydrogenation of 21 catalyzed by platinum carbon gave
a mixture of two diastereomers 22 (3.5:1), which were separated by
silica gel column chromatography. The major isomer 23 was con-
verted to the tert-butoxycarbonylamino compound 25 by hydrolysis
O
P
O
a
EtO
EtO
HN
COOEt
N
COOEt
O
O
Ph
Ph
19
20
Finally, as illustrated in Scheme 8, the pyrrolidines of 9–11 and
quinolonecarboxylic acid 13 were heated with triethylamine in
dimethylsulfoxide followed by deprotection of tert-butoxycarbonyl
groups under acidic condition to give des-F(6)-1-[(1R,2S)-2-fluoro-
cyclopropan-1-yl]-quinolone compounds having 3-(1-amin-
ocycloalkyl)pyrrolidin-1-yl substituent, 4–6, respectively. The
hydrochloride of compound 4 was obtained as prisms, and the
absolute configuration was determined by X-ray crystallographic
analysis.
The 1-cyclopropanyl-quinolone compounds 7, 8 were synthe-
sized by the same procedure as described in Scheme 7, reported
by Domagala et al.,39,40 or in Scheme 8, reported by Ledoussal
et al., respectively.35
COOEt
COOEt
c
b
O
O
N
N
Ph
Ph
21
22
COOEt
COOH
e
d
H
H
O
O
N
N
Ph
Ph
23
24
Boc
Boc
3. Results and discussion
Boc
N
H
N
f
g
h
N
H
H
H
H
H
The minimum inhibitory concentrations (MICs) of 1–8 against
several representative Gram-positive and Gram-negative bacteria
are summarized in Table 1, along with the data for garenoxacin,30
trovafloxacin, moxifloxacin, gatifloxacin, and ciprofloxacin41 for
comparison. The synthesized compounds 1–8 exhibited 4–520
times more potent activity against Gram-positive bacteria than
the reference quinolones, trovafloxacin, moxifloxacin, gatifloxacin,
or ciprofloxacin. In particular, the des-F(6)-1-[(1R,2S)-2-fluorocy-
O
N
N
N
H
Ph
Ph
25
26
9
Scheme 5. Reagents and conditions: (a) (EtO)2P(O)CH2COOH, CDI/THF; (b) tert-
BuOK/toluene; (c) H2, 5% Pt–C/EtOAc; (d) silica gel column chromatography; (e)
aqueous NaOH/EtOH; (f) diphenylphosphoryl azide, Et3N/toluene, then tert-BuOH;
(g) BH3–THF/THF, then K2CO3/H2O, reflux; (h) H2, Pd–C/EtOH.

6882
R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
O
O
N
R8
F
F
COOBF2
F
COOH
R9
R10
a, b, c
NH
*
R8
R9
N
N
N
*
Boc
R10
OCH3
OCH3
N
F
F
H
12
9:
1:
R8,R9 = -(CH2)2-, R10 = H
R8,R9 = -(CH2)2-, R10 = H
2: R8,R9 = -(CH2)2-, R10 = CH3
3: R8,R9 = -(CH2)3-, R10 = H
10: R8,R9 = -(CH2)2-, R10 = CH3
11: R8,R9 = -(CH2)3-, R10 = H
Scheme 7. Reagents and conditions: (a) Et3N/DMSO; (b) Et3N/80% aqueous EtOH, reflux; (c) concentrated aqueous HCl.
O
N
O
N
R8
H
F
COOH
H
N
COOH
R9
R10
NH
a, b
*
R8
R9
N
*
Boc
R10
OCH3
OCH3
N
F
F
H
9:
10:
11:
4:
R8,R9 = -(CH2)2-, R10 = H
R8,R9 = -(CH2)2-, R10 = CH3
R8,R9 = -(CH2)3-, R10 = H
13
R8,R9 = -(CH2)2-, R10 = H
5: R8,R9 = -(CH2)2-, R10 = CH3
6: R8,R9 = -(CH2)3-, R10 = H
Scheme 8. Reagents and conditions: (a) Et3N/DMSO, heat; (b) concentrated aqueous HCl.
clopropan-1-yl]-quinolone compounds 4–6 were at least four
times more potent than the newer quinolones, garenoxacin, trova-
floxacin, moxifloxacin, or gatifloxacin, which were designed for
Gram-positive infections. Against Gram-negative bacteria except
for Pseudomonas aeruginosa, 1–8 exhibited potency comparable
with that of trovafloxacin and ciprofloxacin. The compounds pos-
sessing a fluorine atom at the C-6 position 1–3 exhibited 1–4 times
more potent activity against both Gram-positive and Gram-nega-
tive bacteria than the des-F(6) compounds 4–6. Thus, concerning
substitution at the C-6 position about our compounds 1–6, the
rank of inhibitory activity was fluorine (F) > hydrogen (H). How-
ever, compound 7 and des-F(6) compound 8 showed almost iden-
tical antibacterial activities, and the introduction of a fluorine atom
to the C-6 position of 8 did not affect antibacterial activity
substantially.
substituent at the C-7 position, were less toxic than compound 6,
which has the 3-(amincyclobutan-1-yl)pyrrolidin-1-yl substituent,
or 8, which has the (3R,10S)-3-(1-aminoethyl) pyrrolidin-1-yl sub-
stituent. Concerning micronuclei-forming toxicity, compounds 4
and 5 showed different profiles according to their N-substituent.
Compound 5, which has N-methyl substituent, was more toxic
than compound 4, which has (3R)-3-(1-aminocyclopropan-1-
yl)pyrrolidin-1-yl substituent. Thus, compound 5 showed a posi-
tive response, even though it has a combination of des-F(6) and
the (1R,2S)-2-fluorocyclopropan-1-yl group at the N-1 position,
which are reported to reduce genotoxicity.23–28 The des-F(6) com-
pounds 4, 6 and 8 showed negative responses as we expected. The
results indicate the advantage of removing the fluorine atom at the
C-6 position for reducing intravenous single-dose toxicity and
micronuclei-forming toxicity.
The results of the intravenous single-dose toxicity study and the
micronucleus test are summarized in Tables 2 and 3. Concerning
intravenous single-dose toxicity, des-F(6) compounds 4–6 or 8
were less toxic than compounds 1–3 or 7, which possesses a fluo-
rine atom at the C-6 position. In particular, compounds 4 and 5,
which have the (3R)-3-(1-aminocyclopropan-1-yl)pyrrolidin-1-yl
Table 4 shows the solubilities to water and apparent partition
coefficients of these compounds. The des-F(6) compounds 4, 6 or
8 were more soluble than the fluorinated compounds 1, 3 or 7.
However, des-F(6) compounds(4, 5 or 8) and the corresponding
fluorinated compounds(1, 2 or 7) showed the almost same appar-
ent partition coefficients. These data indicate that the removing
Table 1
Antibacterial activity MIC (
lg/ml) of the compounds 1–8 and reference compounds
Compound
Organism
S. aureus FDA
S. epidermidis
S. pneumoniae
S. pyogenes
S. mitis
E. faecalis
E. coli
K. pneumoniae
P. aeruginosa
209-P
56,500
J-24
G-36
IID685
ATCC19433
NIHJ
type II
PAO1
1
2
3
4
5
6
7
8
60.003
60.003
60.003
60.003
60.003
60.003
0.0006
60.003
0.013
0.025
0.013
0.05
0.05
60.003
0.006
60.003
0.006
0.012
0.012
0.012
0.006
0.05
0.05
0.1
0.2
0.2
60.003
60.003
60.003
0.006
0.006
0.006
N.T.
60.003
0.05
0.025
0.025
0.2
60.003
60.003
60.003
0.006
0.012
0.006
0.012
0.012
0.2
0.1
0.2
0.39
1.56
0.003
0.003
0.003
0.003
0.003
0.003
0.025
0.006
0.1
0.05
0.1
0.2
0.78
0.012
0.025
0.025
0.025
0.05
0.05
0.05
0.05
0.2
0.2
0.2
0.39
0.78
60.003
60.003
60.003
60.003
0.006
0.012
0.012
0.012
60.003
0.012
0.012
0.012
0.025
0.05
0.05
0.05
0.05
0.1
0.025
0.05
0.05
0.05
0.025
0.2
0.2
0.39
0.39
0.39
0.39
N.T.
0.2
TVFXa
GRNXa
MFLXa
GFLXa
CPFXa
0.2
0.78
0.78
0.78
0.05
0.006
0.006
60.003
0.39
a
Abbreviations are as follows: TVFX = trovafloxacin; GRNX = garenoxacin; MFLX = moxifloxacin; GFLX = gatifloxacin; CPFX = ciprofloxacin.

R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
6883
Table 2
against both strains of VRE (Enterococcus faecalis and Enterococcus
faecium) among the listed compounds.
Intravenous single-dose toxicity of 1–8 in mice
The bacterial strains were collected by the Levofloxacin Surveil-
lance Group from patients in Japan in 2007. The MICs against lev-
ofloxacin-resistant MRSA of compound 4 were determined and the
results are shown in Table 6, along with the data for garenoxacin,
compound 8, moxifloxacin, ciprofloxacin, levofloxacin and vanco-
mycin for comparison. The MIC at which 90% of isolates are inhib-
ited (MIC90) of compound 4 for levofloxacin-resistant MRSA was
Dose (mg/kg)
Mortality (dead/tested)
1
2
3
4
5
6
7
8
150
100
50
2/2
2/2
5/5
2/2
2/2
5/5
2/2
1/3
0/5
N.T.
1/4
0/2
N.T.
2/2
0/5
0/5
1/1
4/4
4/4
2/2
2/2
0/5
2/3
N.T.a
a
Not tested.
1 lg/ml, which was superior to those of the reference quinolones
garenoxacin, compound 8, moxifloxacin, ciprofloxacin, and levo-
floxacin. In particular, compound 4 was 64 times and eight times
more potent than the des-F(6) quinolones garenoxacin and com-
pound 8, which were designed for Gram-positive infections. Com-
Table 3
Micronuclei-forming toxicity of 1–8 in mice
Result (dose, mg/kg)
1
2
3
4
5
6
7
8
b
c
N.T.a
N.T.
N.T.
ꢀ
(150)
+
(100)
ꢀ (100)
N.T.
ꢀ (50)
a
b
c
Not tested.
Negative.
Positive.
Table 6
Antibacterial activities of
4 and reference compounds against clinically isolated
levofloxacin-resistant MRSA (LVFX-r MRSA: The bacterial strains were collected by
the Levofloxacin Surveillance Group from patients in Japan in 2007) (MIC:
l
g/ml)
LVFX-r MRSAa (24b)
fluorine atom at the C-6 position contributes to improving the sol-
ubility in water with similar lipophilicity.
Compound
c
c
c
Range
MIC50
MIC80
MIC90
The MICs against clinically isolated resistant Gram-positive bac-
teria of compound 4, which exhibited the lowest single-dose toxic-
ity and a negative response in the micronucleus test, were
determined and the results are shown in Table 5. Compound 4
exhibited more potent activity than the other quinolone antibacte-
rial agents (gatifloxacin, moxifloxacin, or garenoxacin), or the other
categories of drugs (vancomycin, teicoplanin, or linezolid) and the
activity was that of with quinupristin/dalfopristin against cipro-
floxacin-resistant MRSA (Bacterial strains were collected by the
Levofloxacin Surveillance Group from patients in Japan in
200042). Against penicillin-intermediate resistant S. pneumoniae
(PISP)+PRSP, 4 exhibited the most potent activity among the listed
compounds. Compound 4 also exhibited the most potent activity
4
60.03–1
0.25–64
0.12–8
1–32
8–>64
4–>64
0.5–2
0.12
2
0.5
4
>64
16
1
1
64
8
32
>64
>64
1
1
64
8
32
>64
>64
1
GRNXc
8
MFLXc
CPFXc
LVFXc
VCMc
a
The bacterial strains were collected by the Levofloxacin Surveillance Group
from patients in Japan in 2007.
b
No. of strains.
c
Abbreviations are as follows: GRNX = garenoxacin; MFLX = moxifloxacin;
CPFX = ciprofloxacin; LVFX = levofloxacin; VCM = vancomycin MIC50 = MIC at which
50% of isolates are inhibited; MIC80 = MIC at which 80% of isolates are inhibited;
MIC90 = MIC at which 90% of isolates are inhibited.
Table 4
Physicochemical profiles of 1–8
Compound
1
2
3
4
5
6
7
8
Aqueous solubility (
lg/ml)
221
74.1
173
93.9
180
26.9
480
75.9
161
>85.9
604
9.9
89
9.9
>10,000
9.5
a
P0
a
Apparent partition coefficient, CHCl3—0.1 M phosphate buffer (pH 7.4).
Table 5
Antibacterial activities of 4 and reference compounds against clinically isolated ciprofloxacin-resistant MRSA (CPFX-r MRSA), PRSP, and VRE (MIC:
l
g/ml)
Compound
Organisms
CPFX-r MRSAa (99b)
PISP+PRSPc (50b)
MIC50
VRE (E. faecalis) (18b)
VRE (E. faecium) (19b)
d
d
Range
MIC50
MIC90
Range
MIC90
Range
MIC50
MIC90
Range
MIC50
MIC90
4
60.03–1
1–128
1–64
0.25–64
4–>128
0.5–2
0.25–4
0.12–4
0.5–2
0.06
4
2
1
8
1
1
0.5
1
0.5
64
32
32
32
2
1
0.5
1
0.008–0.06
0.25–1
0.12–0.5
0.03–0.25
16–64
0.25–1
N.T.
0.03
0.5
0.25
0.12
32
0.03
0.5
0.5
0.12
64
0.5
0.015–0.5
0.25–32
0.12–32
0.06–8
16–>128
8–>128
0.25–128
0.5–16
2
0.25
16
16
4
0.5
32
32
8
0.06–4
0.5–32
0.5–32
0.25–32
2–64
32–>128
0.5–64
0.5–4
0.25
4
4
2
GFLXd
MFLXd
GRNXd
ABKd
32
32
32
32
>128
64
2
8
2
2
16
>128
32
0.5
2
VCMd
TEICd
QPR/DPRd
LNZd
0.5
>128
64
8
>128
128
16
2
0.5–4
1–2
1
1
1
2
2
2
2
a
b
c
MIC range of ciprofloxacin is 8–>64
No. of strains.
MIC range of benzylpenicillin is 1–4
l
g/ml. (Bacterial strains were collected by the Levofloxacin Surveillance Group from patients in Japan in 2000.42
)
lg/ml.
d
Abbreviations are as follows: GFLX = gatifloxacin; MFLX = moxifloxacin; GRNX = garenoxacin; ABK = arbekacin; VCM = vancomycin; TEIC = teicoplanin; QPR/DPR = quinu-
pristin/dalfopristin; LNZ = linezolid; MIC50 = MIC at which 50% of isolates are inhibited; MIC90 = MIC at which 90% of isolates are inhibited.

6884
R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
pound 4 showed excellent antibacterial activity, even against the
latest (2007) clinically isolated levofloxacin-resistant MRSA.
The potent antibacterial activity of compound 4 described in
this paper is due, in part, to its potent inhibitory activity against
bacterial type II topoisomerases. The inhibitory effects of com-
pound 4 against type II topoisomerases of S. aureus were deter-
mined as 50% inhibitory concentrations (IC50s) and the results
are shown in Tables 7 and 8. Against wild-type topoisomerase IV
and altered topoisomerase IV with quinolone-resistant mutation
of S. aureus, IC50s of compound 4 were 0.835 lg/ml and 7.81 lg/ml,
respectively (Table 8). Compound 4 showed the most potent inhib-
itory activities against wild type and altered quinolone-resistant
target enzymes. Inhibitory activities of 4 against altered enzymes
were the same level as those of comparator quinolones against
wild type enzyme. The ratio of the IC50s of 4 against wild-type en-
zyme to altered one was also the lowest among the quinolone
tested. From these results the potent antibacterial activity of com-
pound 4 against various types of quinolone-resistant MRSA may
due to the potent inhibitory activity against both target enzymes.
The pharmacokinetic profiles of compound 4 (20 mg/kg) to rats
and monkeys are shown in Table 9. Compound 4 exhibited high
plasma concentration and area under the time–concentration
curve (AUC) and showed a good distribution pattern in the liver,
the kidney, and especially the lung. Compound 4 was expected to
exhibit a high plasma concentration and a long half-life time in hu-
mans. The excellent in vitro activities (Tables 1, 5 and 6) and phar-
macokinetic profiles (Table 7) of compound 4 showed a promising
result compound 4 exhibited potent in vivo efficacies on gram-po-
sitive bacteria such as PRSP in animal models of pneumonia. In-
deed, in the recent study of Kohno and co-workers,43 compound
4 had high degrees of efficacy against penicillin-susceptible S.
pneumoniae (PSSP) and PRSP in a mouse lung infection model.
(GrlA; S80F) of S. aureus, IC50s of compound 4 were 0.309
lg/ml
and 1.76 g/ml, respectively (Table 7). Against DNA gyrase and al-
l
tered DNA gyrase with quinolone-resistant mutation (GyrA; S84L)
Table 7
Inhibitory activities of 4 and reference compounds against wild-type and altered
topoisomerase IV of Staphylococcus aureus (S. aureus)
a
Compound
IC50
(
lg/ml)
Wild-type (A)
Ser80Pheb (B)
B/A ratio
4
0.309
1.14
1.36
1.85
2.67
1.24
1.76
20.7
24.2
22.5
57.1
22.4
5.70
18.16
17.79
12.16
21.38
18.06
GRNXc
MFLXc
GFLXc
LVFXc
CPFXc
4. Conclusion
a
50% Inhibitory concentration.
Altered topoisomerase IV with quinolone-resistant mutation (GrlA; S80F) of S.
b
We synthesized a series of novel des-F(6) and 6-fluoro-1-
[(1R,2S)-2-fluorocyclopropan-1-yl]-8-metoxylquinolones bearing
3-(1-aminocycloalkyl)pyrrolidin-1-yl substituents at the C-7 posi-
tion. The des-F(6) compound 4–6 exhibited slightly inferior anti-
bacterial activity comparable with that of the 6-fluorinated
compounds 1–3. The des-F(6) compounds 4,6 had at least four
times more potent activities against Gram-positive bacteria than
the reference quinolones. In addition, they showed no micronu-
clei-forming toxicity. Among the des-F(6) compounds, (3R)-3-(1-
aureus.
c
Abbreviations are as follows: GRNX = garenoxacin; MFLX = moxifloxacin;
GFLX = gatifloxacin; CPFX = ciprofloxacin; LVFX = levofloxacin.
Table 8
Inhibitory activities of 4 and reference compounds against wild-type and altered DNA
gyrase of Staphylococcus aureus (S. aureus)
a
Compound
IC50
(
lg/ml)
aminocyclopropan-1-yl)pyrrolidin-1-yl derivative
4
showed
Wild-type (A)
Ser84Leub (B)
B/A ratio
reduced intravenous single-dose toxicity in comparison with 6
and good pharmacokinetic profiles in rats and monkeys. As well,
compound 4 exhibited comparable or greater antibacterial activity
against clinically isolated Gram-positive bacteria that had become
resistant to one or more antibacterial agents in comparison with
the other quinolones tested, vancomycin, teicoplanin, quinupri-
stin/dalfopristin, or linezolid. Compound 4 has been found to exhi-
bit excellent antibacterial activity against other clinically isolated
bacteria44 in addition to those we describe in this paper, and was
selected for further preclinical and clinical evaluation. Thus, com-
pound 4 should be a promising candidate for the treatment of seri-
4
0.835
3.59
4.38
3.22
9.41
13.4
7.81
>512
>512
287
>512
>256
9.35
GRNXc
MFLXc
GFLXc
LVFXc
CPFXc
>143
>117
89.1
>54.4
>19.1
a
50% Inhibitory concentration.
Altered DNA gyrase with quinolone-resistant mutation (GyrA; S84L) of S.
aureus.
Abbreviations are as follows: GRNX = garenoxacin; MFLX = moxifloxacin;
GFLX = gatifloxacin; CPFX = ciprofloxacin; LVFX = levofloxacin.
b
c
Table 9
Pharmacokinetic parameters of 4 in rats and monkeys at a dose of 20 mg/kg
Animal
Route
Tissue
Parameters (units)a
C5min
(l
g/mL or
lg/g)
t1/2 (h)
AUC0–6h
(lg h/mL or
lg h/g)
AUC ratio (tissue/serum)
BAb
Rats
po
iv
Serum
Serum
Liver
Kidney
Lung
6.7c
11.0
48.5
47.2
32.4
1.3
1.2
1.1
1.4
1.2
9.8d
14.5d
66.1d
64.4d
45.5d
67.4%
1.0
4.6
4.4
3.1
Monkeys
po
iv
Serum
Serum
3.7c
12.3
6.0
4.4
37.5e
64.4e
58.2%
a
b
c
Mean values of four animals.
Bioavailability, calculated from AUC ratio.
Cmax
0–6 h.
0–24 h.
.
d
e

R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
6885
ous human infections due to multi-drug resistant gram-positive
bacteria. The clinical trials of compound 4 are in progress.
m), 3.83–3.88 (1H, m), 4.10 (3H, d, J = 2.2 Hz), 4.39 (2H, q,
J = 7.1 Hz), 4.85 (1H, br d, J = 63.0 Hz), 8.05 (1H, dd, J = 8.6,
10.0 Hz), 8.57 (1H, d, J = 1.2 Hz). [
Calcd for C16H14F3NO4: C, 56.31; H, 4.13; F, 16.70; N, 4.10. Found:
a
]D: ꢀ24.3 (c 0.989, CHCl3). Anal.
5. Experimental
5.1. Chemistry
C, 56.09; H, 4.11; F, 16.90; N, 4.22.
5.1.2. 6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-
dihydro-8-methoxy-4-oxoquinoline-3-carboxylate (16)
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. Melting points
were taken on a Yanako MP-500D melting point apparatus and are
uncorrected. Optical rotations were measured in a 0.5-dm cell at
25 °C at 589 nm with a HORIBASEPA-300 polarimeter. 1H NMR spec-
tra were determined on a JEOL JNM-EX400 spectrometer. Chemical
shifts are reported in parts per million relative to tetramethylsilane
or sodium 2,2-dimethyl-2-silapentane-5-sulphonate as internal
standards. Significant 1H NMR data are tabulated in the order: num-
ber of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quar-
tet; m, multiplet; br, broad), coupling constant(s) in hertz. Infrared
(IR) spectra were obtained on a HORIBA FT-720 spectrometer or a
JASCO FT/IR-6100 typeA. High-resolution mass spectra were ob-
tained on a JEOL JMS-700 mass spectrometer under electron impact
ionization conditions (EI), electron spray ionization conditions (ESI)
or fast atom bombardment ionization conditions (FAB). Elemental
analyses are indicated only by the symbols of the elements; analyt-
ical results were within 0.4% of the theoretical values unless other-
wise noted, which indicates P95% purity of the tested compounds.
Column chromatography refers to flash column chromatography
conducted on Merck Silica Gel 60, 230–400 mesh ASTM. Thin-layer
chromatography (TLC) was performed with Merck Silica Gel 60
F254 TLC plates, and compound visualization was effected with a
5% solution of molybdophosphoric acid in ethanol, UV-lamp, iodine,
or Wako Ninhydrin Spray. Reagents and solvents were purchased
from Sigma Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Indus-
tries, and Kanto Kagaku and used without purification.
A mixture of 15 (201 g, 590 mmol), 700 mL of AcOH, and 700 mL
of concd HCl aq was heated for reflux for 12 h. After cooling the mix-
ture, 2000 mL of cold water was added, and the precipitate was col-
lected by filtration. The resultant solid was washed with water (2ꢁ),
EtOH and Et2O, to give crude product. Recrystallization from ace-
tone/EtOH gave 16 (91.3 g, 49%) as a colorless powder. mp 184–
186 °C. IR (KBr pellet) cmꢀ1: 3458, 3105, 3088, 3025, 2958, 2923,
2851, 2733, 1733, 1621, 1569, 1508, 1487, 1460, 1401, 1380, 1365,
1346, 1320, 1284, 1246, 1229, 1185, 1135, 1115, 1104, 1063, 1057,
1015. 1H NMR (CDCl3) d: 1.64–1.75 (2H, m), 3.97–4.00 (1H, m),
4.17 (3H, d, J = 2.2 Hz), 4.91 (1H, br d, J = 63.2 Hz), 8.05 (1H, dd,
J = 8.6, 10.0 Hz), 8.84 (1H, s), 14.31 (1H, s). [
Anal. Calcd for C14H10F3NO4: C, 53.68; H, 3.22; F, 18.20; N, 4.47.
a
]D: ꢀ9.4 (c 0.953, CHCl3).
Found: C, 53.69; H, 3.18; F, 18.01; N, 4.54.
5.1.3. {6,7-Difluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-
dihydro-8-methoxy-4-oxoquinoline-3-yl}carboxyl
difluoroborate (12)
Compound 16 (90.3 g, 288 mmol) was suspended in 1000 mL of
Et2O and 653 mL of boron trifluoride ethyl etherate was added to
the suspension and the suspension was stirred at ambient temper-
ature for 24 h. The precipitate was collected by filtration. The resul-
tant solid was washed with Et2O to give 12 (96.5 g, 93%) as a
colorless powder. mp 206–208 °C. IR (KBr pellet) cmꢀ1: 3433,
3065, 2969, 2857, 1724, 1628, 1579, 1555, 1498, 1480, 1412,
1363, 1329, 1267, 1241, 1223, 1192, 1139, 1113, 1068, 1053. 1H
NMR (CDCl3) d: 1.77–1.98 (2H, m), 4.30 (3H, d, J = 2.9 Hz), 4.38–
4.44 (1H, m), 5.03 (1H, br d, J = 62.5 Hz), 8.17 (1H, dd, J = 8.1,
5.1.1. Ethyl 6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-
1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate (15)
8.8 Hz), 9.14 (1H, s). [a]D: +166.2 (c 0.157, CHCl3). Anal. Calcd for
C14H9BF5NO4: C, 46.58; H, 2.51; F, 26.31; N, 3.88. Found: C,
46.43; H, 2.54; F, 26.22; N, 3.90.
To
a solution of 2,4,5-trifluoro-3-methoxybenzoic acid 14
(206 g, 1.00 mol) in 2000 mL of toluene at ambient temperature,
DMF (2 mL) and thionyl chloride (109 ml, 1.50 mol) were added
dropwise. The mixture was stirred at 80 °C for 16 h. The mixture
was concentrated in vacuo to give a brown oil. To a stirred solution
of ethyl 3-(dimethylamino)acrylate34 (172 g, 1.20 mol) and trieth-
ylamine (184 mL, 1.32 mol) in 1500 mL of THF in an ice bath was
added dropwise a solution of the brown oil in 500 mL of THF.
The mixture was refluxed for 5 h, and concentrated in vacuo. The
residue was diluted with CH2Cl2, and the solution was washed
with water, brine, dried over Na2SO4, and concentrated in vacuo
to give a brown oil. To a stirred solution of the brown oil in
2200 mL of CH2Cl2 in an ice bath was added (1R,2S)-2-fluorocyclo-
propylamine p-toluenesulfonic acid salt (224 g, 904 mmol), and
then a solution of triethylamine (139 mL, 995 mmol) in 300 mL
of CH2Cl2 was added dropwise. Stirring was continued for 2 h in
the ice bath. The mixture was diluted with CH2Cl2, and the solution
was washed with water, brine, dried over Na2SO4, and concen-
trated. To a stirred solution of the residue in 2000 mL of DMF on
an ice bath was added K2CO3 (276 g, 764 mmol) portionwise. The
mixture was stirred at ambient temperature for 72 h. After the
mixture was poured into ice-cooled 2 N HCl aq, the precipitate
was collected by filtration and washed with water (3ꢁ), EtOH,
and Et2O to give 15 (213 g, 82%) as a colorless powder. mp 161–
163 °C. IR (KBr pellet) cmꢀ1: 3431, 3092, 3002, 2984, 2964, 2936,
2910, 2872, 1724, 1618, 1604, 1570, 1495, 1471, 1405, 1346,
1330, 1288, 1265, 1224, 1175, 1143, 1111, 1095, 1068, 1054,
1019. 1H NMR (CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 1.56–1.68 (2H,
5.1.4. Ethyl 7-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-
dihydro-8-methoxy-4-oxoquinoline-3-carboxylate (18)
A
mixture of ethyl 2,4-difluoro-3-methoxybenzoylacetate
(1735) (141 g, 546 mmol), ethyl orthoformate (227 mL, 1.37 mol)
and 500 mL of Ac2O was heated at 120 °C for 6 h. The mixture
was concentrated in vacuo to give a brown oil. To a stirred solution
of the brown oil in 1000 mL of CH2Cl2 in an ice bath was added
(1R,2S)-2-fluorocyclopropylamine p-toluenesulfonic acid salt
(165 g, 668 mmol), and then a solution of triethylamine (118 mL,
846 mmol) in 382 mL of CH2Cl2 was added dropwise. Stirring
was continued at ambient temperature for 6 h. The mixture was di-
luted with CH2Cl2, and the solution was washed with water, brine
(2ꢁ), dried over Na2SO4, and concentrated. To a stirred solution of
the residue in 1200 mL of 1,4-dioxane on an ice bath was added
NaH (60% mineral oil dispersion, 31.2 g, 780 mmol) portionwise.
The mixture was stirred at ambient temperature for 1 h. After
the mixture was poured into ice-cooled 1 N HCl aq, the precipitate
was collected by filtration and washed with water (3ꢁ), EtOH and
Et2O (3ꢁ) to gave 18 (131 g, 75%) as a yellow powder. mp 153–
155 °C dec.. IR (KCl pellet) cmꢀ1: 3429, 3094, 3071, 2981, 2950,
2906, 2839, 1727, 1631, 1611, 1595, 1564, 1497, 1446, 1401,
1373, 1350, 1329, 1313, 1271, 1252, 1192, 1172, 1128, 1076,
1043, 1017. 1H NMR (CDCl3) d: 1.41 (3H, t, J = 7.1 Hz), 1.55–1.65
(2H, m), 3.85–3.90 (1H, m), 4.03 (3H, d, J = 2.0 Hz), 4.40 (2H, q,
J = 7.1 Hz), 4.87 (1H, br d, J = 62.3 Hz), 7.20 (1H, dd, J = 9.0,
10.5 Hz), 8.24 (1H, dd, J = 6.1, 9.0 Hz), 8.57 (1H, s). [
a]D: ꢀ27.2 (c

6886
R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
0.464, CHCl3). Anal. Calcd for C16H15F2NO4: C, 59.44; H, 4.68; F,
11.75; N, 4.33. Found: C, 59.40; H, 4.67; F, 11.58; N, 4.42.
stirred at ambient temperature for 17 h under a hydrogen atmo-
sphere. The mixture was filtrated and concentrated in vacuo to
give a colorless oil. The oil was separated to two diastereomers
by column chromatography eluting with AcOEt/hexane = 2:3. 23
(9.0 g, 74%) as a pale yellow oil. The spectral data of 23 obtained
by this procedure were identical to those reported.38 1H NMR
(CDCl3) d: 0.63–0.65 (2H, m), 1.13 (3H, t, J = 7.1 Hz), 1.12–1.19
(2H, m), 1.52 (3H, d, J = 7.3 Hz), 2.17 (1H, dd, J = 9.0, 16.8 Hz),
2.46 (1H, dd, J = 9.3, 16.3 Hz), 2.67–2.76 (2H, m), 3.47 (1H, t,
J = 8.3 Hz), 3.96–4.11 (2H, m), 5.51 (1H, q, J = 7.3 Hz), 7.26–7.35
(5H, m). MS (ESI) m/z 302 (M+H+). High-resolution MS (ESI) Calcd
5.1.5. 7-Fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-
dihydro-8-methoxy-4-oxoquinoline-3-carboxylate (13)
A mixture of 18 (131 g, 406 mmol), 1000 mL of AcOH, and
1000 mL of concd HCl aq was heated for reflux for 6 h. After cooling
the mixture, 3000 mL of cold water was added, and the precipitate
was collected by filtration. The resultant solid was washed with
water (2ꢁ), EtOH and Et2O, to give crude product. Recrystallization
from MeCN gave 13 (92.8 g, 77%) as a colorless powder. mp 232–
234 °C. IR (KCl pellet) cmꢀ1: 3427, 3090, 3060, 3019, 2949, 2858,
2589, 1726, 1616, 1560, 1515, 1476, 1443, 1400, 1375, 1320,
1269, 1230, 1206, 1186, 1171, 1133, 1123, 1105, 1069, 1044,
1015. 1H NMR (CDCl3) d: 1.55–1.73 (2H, m), 4.01 (1H, m), 4.10
(3H, d, J = 2.2 Hz), 4.90 (1H, br d, J = 62.7 Hz), 7.35 (1H, dd, J = 9.0,
for C18H23NO3+H: 302.1756. Found: 302.1745. [
1.136, CHCl3).
a]D: ꢀ93.4 (c
5.1.9. 1-{(3S)-5-Oxo-1-[(S)-1-phenylethyl]pyrrolidin-3-
yl}cyclopropanecarboxylic acid (24)
10.2 Hz), 8.27 (1H, dd, J = 5.9, 9.0 Hz), 8.76 (1H, s). [
0.165, 0.1 N NaOH aq). Anal. Calcd for C14H11F2NO4: C, 56.95; H,
3.76; F, 12.87; N, 4.74. Found: C, 56.87; H, 3.76; F, 12.63; N, 4.82.
a
]D: 40.0 (c
To a solution of 23 (10.5 g, 34.9 mmol) in 70 mL of EtOH was
added 1 N NaOH aq (70 mL, 70.0 mmol) at ambient temperature,
and the mixture was stirred at this temperature for 15.5 h, and
then at 40 °C for 3 h. The reaction mixture was concentrated in va-
cuo, and the residual aqueous solution was washed with AcOEt.
Then the aqueous layer was acidified with concd HCl aq under
ice cooling, and the mixture was extracted with CHCl3 (3ꢁ). The
combined organic layer was dried over Na2SO4, and concentrated
in vacuo to give 18 (9.4 g, 99%) as a white solid. The spectral data
of 24 obtained by this procedure were identical to those re-
ported.38 mp 184–186 °C. IR (KBr pellet) cmꢀ1: 3418, 3091, 3008,
2991, 2945, 2921, 2887, 2711, 2579, 2538, 2511, 2094, 1972,
1852, 1704, 1627, 1581, 1498, 1455, 1422, 1385, 1376, 1358,
1331, 1309, 1292, 1258, 1240, 1219, 1191, 1159, 1093, 1067,
1035, 1006. 1H NMR (CDCl3) d: 0.72–0.74 (2H, m), 1.21–1.23 (2H,
m), 1.52 (3H, d, J = 7.3 Hz), 2.17 (1H, dd, J = 8.8, 16.8 Hz), 2.48
(1H, dd, J = 9.5, 16.8 Hz), 2.66–2.78 (2H, m), 3.50 (1H, t,
5.1.6. N-[(1-Ethoxycarbonylcycloprop-1-yl)carbonylmethyl]-N-
[(S)-1-phenylethyl]-2-(diethoxy- phosphoryl)acetamide (20)
To
a solution of diethoxyphosphorylacetic acid (15.1 g,
76.8 mmol) in 120 mL of THF on an ice bath was added 1,10-car-
bonyldiimidazole (13.7 g, 84.5 mmol). The mixture was stirred at
ambient temperature for 1 h, and then a solution of ethyl 1-{N-
[(S)-1-phenylethyl]aminoacetyl}cyclopropanecarboxylate (19)37
(17.6 g, 64.0 mmol) in 30 mL of THF was added dropwise over
20 min below 0 °C. After stirring the solution for 1 h at ambient
temperature, the solution was diluted with AcOEt, and washed
with aqueous 1 N HCl solution, aqueous saturated NaHCO3 solu-
tion, and brine. The resultant solution was dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude product was purified
by silica gel column chromatography, eluting with AcOEt/hex-
ane = 2:1 to yield crude 20 (28.7 g) as a yellow oil. 1H NMR (CDCl3)
d: 1.14–1.68 (16H, m), 2.85–4.69 (2H, m), 3.18–4.55 (2H, m), 4.06–
4.22 (6H, m), 5.42–6.05 (1H, m), 7.26–7.37 (5H, m). MS (ESI) m/z
454 (M+H+). High-resolution MS (ESI) Calcd for C22H32NO7P+H:
J = 9.3 Hz), 5.51 (1H, q, J = 7.3 Hz), 7.25–7.34 (5H, m).
[a]D:
ꢀ116.8 (c 0.787, CHCl3). Anal. Calcd for C16H19NO3: C, 70.31; H,
7.01; N, 5.12. Found: C, 69.91; H, 6.75; N, 5.06.
5.1.10. (3R)-3-[1-(tert-Butoxycarbonylamino)cyclopropan-1-
yl]-5-oxo-1-[(S)-1-phenylethyl]pyrrolidine (25)
454.1995. Found: 454.1993. [
a]D: ꢀ30.3 (c 0.945, CHCl3).
To a solution of 24 (9.40 g, 34.4 mmol) in 95 mL of toluene was
added triethylamine (9.60 mL, 69.0 mmol) followed by diphenyl-
phosphoryl azide (10.4 g, 37.9 mmol) dropwise at ambient temper-
ature. The mixture was stirred at ambient temperature for 1 h and
then heated to reflux for 1.5 h. To the mixture was added 95 mL of
tert-butylalcohol, and the mixture was heated for reflux for another
15 h. Thesolventwasremovedinvacuo, andtheresiduewaspurified
by silica gel column chromatography eluting with CHCl3/metha-
nol = 50:1 to yield 25 (10.7 g, 90%) as a colorless oil. Recrystallization
from n-hexane–CH2Cl2 gave a colorless powder. mp 130–132 °C. IR
(KBr pellet) cmꢀ1: 3379, 3080, 3064, 3025, 3007, 2978, 2940, 2913,
2147, 1694, 1680, 1584, 1501, 1453, 1428, 1390, 1365, 1303, 1277,
1248, 1226, 1166, 1155, 1094, 1072, 1055, 1027. 1H NMR (CDCl3)
d: 0.56–0.84 (4H, m), 1.37 (9H, s), 1.51 (3H, d, J = 7.3 Hz), 2.32–2.44
(3H, m), 2.79 (1H, dd, J = 7.3, 10.0 Hz), 3.36 (1H, m), 4.66 (1H, br s),
5.1.7. Ethyl 1-{5-oxo-1-[(S)-1-phenylethyl]-2,5-dihydro-1H-
pyrrol-3-yl}cyclopropanecarboxylate (21)
To a solution of 20 (25.0 g, 55.2 mmol) in 250 mL of toluene was
added potassium tert-butoxide (7.40 g, 66.2 mmol) portionwise
over 1 h below 0 °C. After the solution was stirred for 15 min at
ambient temperature, an aqueous 10% citric acid solution was
added dropwise to the solution at 0 °C. The resultant suspension
was diluted with AcOEt and filtered through Celite, and then the
organic layer of the filtrate was separated and washed with aque-
ous saturated NaHCO3 solution, and brine. The resultant solution
was dried over anhydrous Na2SO4 and concentrated in vacuo.
The crude product was purified by silica gel column chromatogra-
phy, eluting with AcOEt/hexane = 1:2 to yield 21 (12.1 g, 73%) as a
red-yellow oil. The spectral data of 21 obtained by this procedure
were identical to those reported.38 1H NMR (CDCl3) d: 1.13–1.15
(2H, m), 1.18 (3H, t, J = 6.8 Hz), 1.60 (3H, d, J = 7.3 Hz), 1.61–1.64
(2H, m), 3.80 (1H, d, J = 19.5 Hz), 4.09 (2H, q, J = 6.8 Hz), 4.13 (1H,
q, J = 19.5 Hz), 5.56 (1H, q, J = 7.3 Hz), 5.85 (1H, t, J = 1.5 Hz),
7.25–7.37 (5H, m). MS (ESI) m/z 300 (M+H+). High-resolution MS
5.50 (1H, q, J = 7.3 Hz), 7.26–7.34 (5H, m). [
a
]D: ꢀ114.7 (c 0.716,
CHCl3). Anal. Calcd for C20H28N2O3ꢂ0.1H2O: C, 69.38; H, 8.21; N,
8.09. Found: C, 69.06; H, 7.81; N, 8.38.
5.1.11. (3R)-3-[1-(tert-Butoxycarbonylamino)cyclopropan-1-
yl]-1-[(S)-1-phenylethyl]pyrrolidine (26)
(ESI) Calcd for C18H21NO3+H: 300.1600. Found: 300.1587. [a]D:
ꢀ45.7 (c 1.104, CHCl3).
To a solution of 25 (10.4 g, 30.2 mmol) in 95 mL of THF was
added 1 N BH3–THF (90.7 mL, 90.7 mmol) dropwise at 0 °C. After
the solution was stirred for 16 h at ambient temperature, K2CO3
(25.0 g, 181 mmol) solution in 72 mL of water was added dropwise
to the solution. The mixture was heated for reflux for another 1.5 h.
The solvent was removed in vacuo, and the residue was diluted
5.1.8. Ethyl 1-{(3S)-5-oxo-1-[(S)-1-phenylethyl]pyrrolidin-3-
yl}cyclopropanecarboxylate (23)
To a solution of 21 (12.1 g, 40.5 mmol) in 300 mL of EtOAc was
added 5% Pt/C (2.40 g, containing 50% water), and the mixture was

R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
6887
with AcOEt, and washed with brine. The resultant solution was
5.1.15. (3R)-3-[1-(tert-Butoxycarbonyl-N-
dried over anhydrous Na2SO4 and concentrated in vacuo. The crude
product was purified by silica gel column chromatography eluting
with CHCl3/methanol = 30:1 to yield 26 (8.20 g, 82%) as a colorless
oil. 1H NMR (CDCl3) d: 0.62 (2H, br s), 0.75–0.88 (2H, m), 1.35 (3H,
d, J = 6.6 Hz), 1.41 (9H, s), 1.63 (2H, br s), 1.88–1.92 (1H, m), 2.14–
2.17 (1H, m), 2.27–2.34 (2H, m), 2.63 (2H, br s), 3.15 (1H, t,
J = 6.6 Hz), 5.10 (1H, br s), 7.23–7.33 (5H, m). MS (ESI) m/z 331
(M+H+). High-resolution MS (ESI) Calcd for C20H30N2O2+H:
methylamino)cyclopropan-1-yl]pyrrolidine (10)
To a solution of 28 (7.19 g, 20.9 mmol) in 78 mL of EtOH was
added 10% Pd/C (3.9 g, containing 50.0% water), and the mixture
was stirred at 40 °C for 4 h under a hydrogen atmosphere. The cat-
alyst was filtered off, and the filtrate was concentrated in vacuo.
The residue was used for the next reaction without purification.
The crude product 10 (4.38 g) was yielded as a colorless oil. 1H
NMR (CDCl3) d: 0.80 (4H, br s), 1.46 (9H, s), 1.80–1.81 (1H, m),
2.05 (1H, br s), 2.28–2.42 (1H, m), 2.54 (1H, br s), 2.84 (3H, s),
2.88–2.96 (3H, m).
331.2386. Found: 331.2422. [
a]D: ꢀ27.9 (c 0.531, CHCl3).
5.1.12. (3R)-3-[1-(tert-Butoxycarbonylamino)cyclopropan-1-
yl]pyrrolidine (9)
To a solution of 26 (270 mg, 0.817 mmol) in 15 mL of EtOH was
added 10% Pd/C (270 mg, containing 52.0% water), and the mixture
was stirred at 40 °C for 3 h under a hydrogen atmosphere. The cat-
alyst was filtered off, and the filtrate was concentrated in vacuo.
The residue was used for the next reaction without purification.
The crude product 9 (185 mg) was yielded as a colorless oil. 1H
NMR (CDCl3) d: 0.69 (2H, br s), 0.79 (2H, br s), 1.42 (9H, s), 1.43–
1.50 (1H, m), 1.86–1.88 (1H, m), 2.15–2.19 (1H, m), 2.68–2.72
(1H, m), 2.90–3.07 (3H, m), 4.92 (1H, br s). MS (ESI) m/z 227
(M+H+). High-resolution MS (ESI) Calcd for C12H22N2O2+H:
5.1.16. 7-[(3R)-3-(1-Aminocyclopropan-1-yl)pyrrolidin-1-yl]-6-
fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-
methoxy-4-oxoquinolin-3-carboxylic acid (1)
To a solution of crude 9 (1.20 mmol) in 10 mL of DMSO was added
12 (217 mg, 0.6 mmol), triethylamine (0.174 mL, 1.25 mmol), and
the mixture was stirred at ambient temperature for 25 h under a
nitrogen atmosphere. The solvent was removed in vacuo, and the
residue was added to water, and then the mixture was stirred at
ambient temperature for 1 h. The precipitate was collected by filtra-
tion and washed with water, and then was dissolved in 20 mL of
MeOH/water = 4:1. To the solution was added triethylamine
(0.3 mL), and the mixture was heated for reflux for another 4.5 h.
The solvent was removed in vacuo, and the residue was dissolved
CHCl3, and thesolutionwas washedwith 10%citric acidaq andbrine.
The resultant solution was dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. To the residue was added 10 mL of concd
HCl aq at 0 °C, and the mixture was stirred for 1 h at ambient temper-
ature. The solution was washed with CH2Cl2, alkalified with satd
NaOH aq at 0 °C, and then the pH of the solution was adjusted to
7.4 with concd HCl aq and dil HCl aq The resultant solution was ex-
tracted with chloroform (4ꢁ), and the combined organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by semi-separable TLC eluting with an un-
der layer solution of CHCl3/MeOH/water = 7:3:1 to give the crude
product. The crude product was recrystallized from EtOH to yield 1
(181 mg, 72%) as a pale yellow powder. mp 195–197 °C. IR (KBr pel-
let) cmꢀ1: 3369, 3311, 3094, 3052, 3028, 3004, 2977, 2953, 2933,
2893, 2869, 1729, 1623, 1516, 1458, 1402, 1370, 1355, 1338, 1318,
1296, 1231, 1207, 1187, 1161, 1122, 1097, 1050, 1039, 1015. 1H
NMR (0.1 N NaOD/D2O) d: 0.60 (4H, s), 1.34–1.60 (2H, m), 1.71–
1.82 (1H, m), 1.99–2.07 (1H, m), 2.20–2.29 (1H, m), 3.46–3.65 (2H,
m), 3.60 (3H, s), 3.69–3.78 (1H, m), 3.98–4.07 (1H, m), 4.93–4.96,
5.12–5.15 (1H, m), 7.60 (1H, d, J = 13.7 Hz), 8.43 (1H, d, J = 2.9 Hz).
227.1760. Found: 227.1772. [a]D: +6.9 (c 1.055, CHCl3).
5.1.13. (3R)-3-[1-(tert-Butoxycarbonyl-N-methylamino)
cyclopropan-1-yl]-5-oxo-1-[(S)-1-phenylethyl]pyrrolidine
(27)
To a stirred solution of 25 (7.87 g, 22.8 mmol) in 100 mL of DMF
on an ice bath was added NaH (60% mineral oil dispersion, 1.40 g,
30.2 mmol) portionwise. After the solution was stirred for 5 min at
ambient temperature, methyl iodide (8.11 mL, 130 mmol) was
added dropwise to the solution. The mixture was stirred at 40 °C
for 24 h, and then satd NH4Cl aq was added dropwise at 0 °C. The
mixture was extracted with AcOEt (2ꢁ), and the organic layers
were combined and washed with water (2ꢁ), dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude product was purified
by silica gel column chromatography eluting with CHCl3/metha-
nol = 30:1 to yield 27 (7.53 g, 92%) as a colorless oil. Recrystalliza-
tion from n-hexane–CH2Cl2 gave a colorless powder. mp 80–82 °C.
IR (KBr pellet) cmꢀ1: 3443, 3088, 2978, 2927, 1692, 1677, 1494,
1477, 1452, 1423, 1363, 1270, 1252, 1232, 1170, 1152, 1058,
1040, 1031, 1014. 1H NMR (CDCl3) d: 0.82–0.84 (4H, m), 1.32
(9H, s), 1.38 (3H, s), 1.51 (3H, d, J = 7.1 Hz), 1.61 (3H, d,
J = 16.6 Hz), 2.43 (1H, m), 2.68–2.81 (3H, m), 3.21 (1H, m), 5.48–
5.50 (1H, m), 7.26–7.36 (5H, m). [
Anal. Calcd for C21H30N2O3: C, 70.36; H, 8.44; N, 7.81. Found: C,
a
]D: ꢀ102.2 (c 0.968, CHCl3).
[
a
]D: ꢀ123.1 (c 0.515, 0.1 N NaOH aq). Anal. Calcd for C21H23F2N3O4:
70.33; H, 8.30; N, 7.78.
C, 60.14; H, 5.53; N, 10.02. Found: C, 60.02; H, 5.45; N, 9.92.
5.1.14. (3R)-3-[1-(tert-Butoxycarbonyl-N-methylamino)
cyclopropan-1-yl]-1-[(S)-1-phenylethyl]pyrrolidine (28)
5.1.17. 6-Fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-
methoxy-7-[(3R)-3-(1-methylaminocyclopropan-1-yl)pyrrolidin-1-
yl]-4-oxoquinolin-3-carboxylic acid (2)
To a solution of 27 (7.53 g, 21.0 mmol) in 70 mL of THF was
added 1 N BH3–THF (63.0 mL, 63.0 mmol) dropwise at 0 °C. After
the solution was stirred for 20 h at ambient temperature, K2CO3
(7.22 g) solution in 72 mL of water was added dropwise to the
solution. The mixture was heated for reflux for another 1.5 h. The
solvent was removed in vacuo, and the residue was diluted with
AcOEt, and washed with brine. The resultant solution was dried
over anhydrous Na2SO4 and concentrated in vacuo. The crude
product was purified by silica gel column chromatography eluting
with CHCl3/methanol = 50:1 to yield 28 (7.19 g, 99%) as a colorless
oil. 1H NMR (CDCl3) d: 0.72–0.74 (4H, m), 1.35 (9H, s), 1.36 (3H, s),
1.61–1.62 (1H, m), 1.84–1.85 (1H, m), 1.96–1.98 (1H, m), 2.26–2.27
(1H, m), 2.50–2.58 (2H, m), 2.79 (3H, s), 2.98–2.99 (1H, m), 3.14–
3.19 (1H, m), 7.27–7.30 (5H, m).
Following the procedures described for 1, the title compound
was prepared in 83% yield from 10 and 12 as a pale yellow powder.
mp 208–209 °C. IR (KBr pellet) cmꢀ1: 3348, 3092, 3040, 3004,
2970, 2937, 2873, 2790, 2671, 1730, 1622, 1515, 1456, 1367,
1356, 1317, 1272, 1228, 1188, 1158, 1134, 1122, 1093, 1055,
1019, 1009. 1H NMR (0.1 N NaOD/D2O) d: 0.53–0.69 (4H, m),
1.32–1.59 (3H, m), 1.91–2.02 (1H, m), 2.34 (3H, s), 2.85–2.95
(1H, m), 3.29–3.38 (1H, m), 3.51–3.62 (2H, m), 3.57 (3H, s), 3.70–
3.79 (1H, m), 3.98–4.07 (1H, m), 4.95–4.98, 5.09–5.13 (1H, m),
7.66 (1H, d, J = 14.2 Hz), 8.39 (1H, d, J = 2.9 Hz). [
0.525, 0.1 N NaOH aq). Anal. Calcd for C22H25F2N3O4: C, 60.96; H,
a
]D: ꢀ123.4 (c
5.81 N, 9.69. Found: C, 60.79; H, 5.73; N, 9.55.

6888
R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
5.1.18. 7-[(3R)-3-(1-Aminocyclobutan-1-yl)pyrrolidin-1-yl]-6-
fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-
methoxy-4-oxoquinolin-3-carboxylic acid (3)
1.56–1.58 (2H, m), 1.99–2.01 (1H, m), 2.34 (3H, s), 2.87–2.89
(1H, m), 3.15–3.17 (1H, m), 3.521–3.54 (3H, m), 3.53 (3H, s),
4.00–4.02 (1H, m), 5.02 (1H, br d, J = 64.5 Hz), 7.03 (1H, s), 7.92
Following the procedures described for 1, the title compound
was prepared in 27% yield from 1132 and 12 as a pale yellow pow-
der. mp 123–139 °C. 1H NMR (0.1 N NaOD/D2O) d: 1.33–1.40 (1H,
m), 1.50–1.60 (1H, m), 168–1.79 (2H, m), 1.86–1.88 (3H, m),
2.03–2.07 (1H, m), 2.14 (2H, br s), 2.40–2.49 (1H, m), 3.50–3.52
(3H, m), 3.56 (3H, s), 3.67–3.71 (1H, m), 3.98–4.03 (1H, m), 7.66
(1H, d, J = 14.6 Hz), 8.42 (1H, d, J = 2.9 Hz). Anal. Calcd for
C22H25F2N3O4ꢂ0.75H2O: C, 59.12; H, 5.98; N, 9.40. Found: C,
58.94; H, 5.70; N, 9.13.
(1H, d, J = 7.0 Hz), 8.39 (1H, s). [
aq). Anal. Calcd for C22H26FN3O4: C, 63.60; H, 6.31; N, 10.11. Found:
C, 63.36; H, 6.31; N, 9.97.
a
]D: ꢀ119.7 (c 0.295, 0.1 N NaOH
5.1.22. 7-[(3R)-3-(1-Aminocyclobutan-1-yl)pyrrolidin-1-yl]-1-
[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-methoxy-4-
oxoquinolin-3-carboxylic acid (6)
Following the procedures described for 4, the title compound
was prepared in 93% yield from 1132 and 13 as a pale yellow pow-
der. mp 174–176 °C. 1H NMR (DMSO-d6) d: 1.30–1.45 (1H, m),
1.45–1.60 (1H, m), 1.60–1.70 (1H, m), 1.70–2.50 (8H, m), 3.30–
3.40 (1H, m), 3.40–3.50 (1H, m), 3.50 (3H, s), 3.56–3.60 (2H, m),
4.03–4.09 (1H, m), 5.00–5.22 (1H, m), 7.09 (1H, d, J = 9.1 Hz),
7.92 (1H, d, J = 9.1 Hz), 8.57 (1H, d, J = 3.4 Hz). MS (ESI) m/z 416
5.1.19. 7-[(3R)-3-(1-Aminocyclopropan-1-yl)pyrrolidin-1-yl]-1-
[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-methyl-4-
oxoquinolin-3-carboxylic acid (4 = DX-619)
To a solution of crude 9 (0.731 mmol) in 2 mL of DMSO was
added triethylamine (0.5 mL) and 13 (180 mg, 0.61 mmol), and
the mixture was heated at 100 °C for 13 h under a nitrogen atmo-
sphere. The solvent was removed in vacuo, and the residue was
dissolved with CHCl3, and the solution was washed with 10% citric
acid aq and brine. The resultant solution was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. To the residue was
added 5 mL of concd HCl aq at 0 °C, and the mixture was stirred
for 1 h at ambient temperature. The solution was washed with
CH2Cl2, alkalified with satd NaOH aq at 0 °C, and then the pH of
the solution was adjusted to 7.4 with concd HCl aq and dil HCl
aq The resultant solution was extracted with chloroform (4ꢁ),
and the combined organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
semi-separable TLC eluting with an under layer solution of
CHCl3/MeOH/water = 7:3:1 to give the crude product. The crude
product was recrystallized from 2-propanol to yield 4 (146 mg,
60%) as a pale yellow powder. mp 189–192 °C. IR (KBr disk) cm
ꢀ1: 3373, 3315, 3091, 3003, 2976, 2935, 2836, 1903, 1714, 1618,
1518, 1439, 1371, 1313, 1261, 1219. 1H NMR (0.1 N NaOD/D2O)
d: 0.56 (4H, br s), 1.31–1.37 (1H, m), 1.50–1.56 (1H, m), 1.77–
1.78 (1H, m), 2.02–2.04 (1H, m), 2.19–2.21 (1H, m), 3.31–3.32
(1H, m), 3.49–3.51 (3H, m), 3.50 (3H, s), 4.00–4.02 (1H, m), 4.93–
4.94, 5.09–5.10 (1H, m), 7.01 (1H, s), 7.90 (1H, d, J = 9.03 Hz),
(M+H+). [
a
]D: ꢀ42.5 (c 1.01, 0.1 N NaOH aq). Anal. Calcd for
C22H26FN3O4ꢂ0.75H2O: C, 61.60; H, 6.46; F, 4.43; N, 9.80. Found:
C, 61.39; H, 6.41; F, 4.43; N, 9.73.
5.2. X-ray crystallographic study
All measurements were made on a Rigaku AFC7R diffractometer
(Cu Ka
radiation; k = 1.54178 Å, graphite monochrometer,
x
ꢀ 2h
scans, 2hmax = 120.1°). The crystal data and parameters are summa-
rized below. The structures were solved by direct methods and re-
fined by full-matrix least-square and Fourier techniques. The non-
hydrogen atoms were refined anisotropically. The hydrogen atoms
were refined isotropically (dC–H = 0.95 Å) and hydrogen positions
were calculated assuming ideal geometries. Acidic hydrogen atom
of the hydrochloride of 4 was refined with respect to its coordi-
nates. All calculations were performed by using the CrystalStruc-
ture 3.6.0 crystallographic software package of Rigaku/MSC and
Rigaku Corporation.
5.2.1. Crystal data and structure analysis. The hydrochloride of 4
A
colorless prism-shaped crystal was formed from EtOH:
C21H24FN3O4ꢂHCl; FW = 437.89; sample dimensions, 0.30 ꢁ 0.30 ꢁ
0.18 mm; orthorhombic, space group P212121; a = 12.0699(21) Å,
b = 6.7639(15) Å, c = 16.4648(13) Å, V = 1340.7(4) Å3, Z = 2; dcalcd
8.39 (1H, d, J = 3.2 Hz). [
Calcd for C21H24FN3O4: C, 62.83; H, 6.03; N, 10.47. Found: C, 62.50;
a
]D: ꢀ50.8 (c = 0.240, 0.1 N NaOH aq). Anal.
= 1.20 g/cm3; F000 = 508.00; = 16.86 cmꢀ1
l ; Flack parameter =
H, 6.04; N, 10.26.
ꢀ0.02(3). The final cycle of full-matrix least-squares refinement was
based on 4611 observed reflections and 327 variable parameters
and converged at R = 0.111 (Rw = 0.239). Lists of the atomic coordi-
nates, anisotropic thermal parameters, and bond lengths and angles
are stored at the Cambridge Crystal Crystallographic Data Centre,
UK, CCDC-743566.
5.1.20. 7-[(3R)-3-(1-Aminocyclopropan-1-yl)pyrrolidin-1-yl]-1-
[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-dihydro-8-methyl-4-
oxoquinolin-3-carboxylic acid hydrochloride (hydrochloride of
4)
To a solution of 4 (13.0 g, 32.3 mmol) in 130 mL of EtOH was
added 1 N HCl aq (34.0 mL) dropwise at 0 °C. The solution was stir-
red for 5 min at 0 °C and concentrated in vacuo. The residue was
recrystallized from EtOH to yield the hydrochloride of 4 (13.4 g,
5.3. Biology
5.3.1. In vitro antibacterial activity
83%) as a pale yellow powder. mp 225–227 °C. IR (ATR) cmꢀ1
:
The MICs of the compounds tested in this study were deter-
mined by the twofold micro broth dilution method using Muel-
ler-Hinton broth (Difco Laboratories, Detroit, MI) with an
inoculum size of approximately 105 CFU per well. The MIC was de-
fined as the lowest concentration that prevented visible bacterial
growth after incubation at 37 °C for 18 h.
2887, 2665, 1705, 1610, 1514, 1431, 1363, 1311, 1232, 1200.
[
a
]D: ꢀ69.2 (c 0.404, MeOH). Anal. Calcd for C21H24FN3O4ꢂHClꢂ
EtOHꢂH2O: C, 55.03; H, 6.63; N, 8.37. Found: C, 55.20; H, 6.25; N,
7.99.
5.1.21. 1-[(1R,2S)-2-Fluorocyclopropan-1-yl]-1,4-dihydro-8-
methoxy-7-[(3R)-3-(1-methylaminocyclopropan-1-
5.3.2. Intravenous single-dose toxicity
yl)pyrrolidin-1-yl]-4-oxoquinolin-3-carboxylic acid (5)
Following the procedures described for 4, the title compound
was prepared in 50% yield from 10 and 13 as a pale yellow powder.
mp 213–215 °C. IR (KBr disk) cmꢀ1: 3352, 3095, 3051, 2939, 2837,
2787, 1716, 1699, 1616, 1520, 1439, 1358, 1319, 1259, 1221. 1H
NMR (0.1 N NaOD/D2O) d: 0.57–0.61 (4H, m), 1.33–1.40 (1H, m),
The test compounds were dissolved in 0.1 N NaOH in saline at
different concentrations. The solution was administered intrave-
nously to five week-old male Slc:ddY mice (n = 5 for each dose)
at a speed of 0.2 mL/min. The total volume of administration was
adjusted to 10 mL/kg of body weight. The number of dead mice
was counted on day 7.

R. Miyauchi et al. / Bioorg. Med. Chem. 17 (2009) 6879–6889
6889
Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Anaheim,
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5.3.3. Micronucleus test
Five-week-old male Slc:ddY mice were used in this test. The test
compounds were dissolved in 0.1 N NaOH in saline, and the solu-
tion was administered intravenously in each group of five mice.
At 24 h and 48 h after treatment, approximately 5 lL of peripheral
blood was collected from a tail blood vessel of each mouse. The
blood was dropped onto an acridine orange-coated glass slide
and covered immediately with a coverslip. For each animal, 1000
reticulocytes were examined for micronuclei by fluorescence
microscopy, and the frequency of micronucleated reticulocytes
(MNRET) was expressed as a percentage. Statistical analysis was
performed by the Kastenbaum and Bowman method.45
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5.3.4. Anti-topoisomerase activity
To determine the inhibitory activities of quinolones against tar-
get enzymes, GyrA and GyrB proteins of DNA gyrase, and GrlA and
GrlB proteins of topoisomerase IV were purified separately using a
protein fusion and purification system (New England Biolabs, MA).
DNA gyrase activity was measured by supercoiling assay using re-
laxed pBR322 as a substrate and topoisomerase IV activity was
measured by decatenation assay using kinetoplast DNA as a sub-
strate. The 50% inhibitory concentration (IC50) was determined as
the drug concentration that reduced the supercoiling and the
decatenation activity seen with drug-free control by 50%, respec-
tively. The altered GyrA (a substitution of Leu for Ser-84) and GrlA
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dure after mutation was introduced by site-directed mutagenesis.
5.3.5. Pharmacokinetic studies
Seven-week-old male Crj:CD rats or five/six-year-old female
cynomolgus monkeys (n = 4) were used. The animals were admin-
istered drug samples in a single intravenous or oral dosing (20 mg/
kg) as an aqueous solution. The concentrations of the compounds
were determined by a microbiological assay (agar well dilution
method) using Bacillus subtilis ATCC 6051 or ATCC 6633. The mean
values of the 4 animals are shown.
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Acknowledgements
33. The C-7 substituent moiety of compound 3 or 6 was prepared by our reported
method. (Takemura, M.; Kimura, Y.; Kawakami, K.; Kimura, K.; Ohki, H.;
Matsuhashi, N.; Kawato, H. PCT Int. WO9623782, 1996.) The quinolone
derivatives having this isomer (isomer 11) of the C-7 substituent have been
become clear that the compounds are higher activity the corresponding
quinolones bearing an enantiomer of 11.
34. Ataka, K.; Oku, M.; Omori, K.; Kimura, T.; Iwata, M. Jpn. Kokai Tokkyo Koho
JP63316757, 1988.
35. Ledoussal, B.; Almstead, J. K.; Gray, J. L.; Hu, X. E. PCT Int. WO9914214,
1999.
36. The racemate of compound 9 was reported by Chu, D. T. (Chu, D. T.; Li, Q.;
Cooper, C. S.; Fung, A. K. L.; Lee, C. M.; Plattner, J. J.; Ma, Z.; Wang, W.-B. PCT Int.
WO9639407, 1996.).
37. Inagaki, H.; Takeda, T.; Miyauchi, R.; Kawakami, K.; Takahashi, H.; Takemura,
M. Heterocycles 2004, 63, 699.
We thank the Medicinal Safety Research Laboratories for the
safety assessment, as well as, Biological Research Laboratories IV
for the biological evaluation. In particular, we thank Dr. Katsuko
Fujikawa and Ms. Megumi Chiba for the biological tests against
levofloxacin-resistant MRSA, as well as, Dr. Mayumi Tanaka, Dr.
Takaaki Akasaka and Dr. Hiroko Kanda for the determination of
inhibitory activities against target enzymes. We also thank Mr.
Hidetaka Sakurai for the X-ray crystal analyses.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.026.
38. Compounds 21–24 were reported in our previous paper. (Inagaki, H.; Miyauchi,
S.; Miyauchi, R.; Kawato, H.; Ohki, H.; Matsuhashi, N.; Kawakami, K.;
Takahashi, H.; Takemura, M. J. Med. Chem. 2003, 46, 1005.).
39. Domagala, J. M.; Hagen, S. E.; Joannides, T.; Kiely, J. S.; Laborde, E.; Schroeder,
M. C.; Sesnie, J. A.; Shapiro, M. A.; Suto, M. J.; Vanderroest, S. J. Med. Chem. 1993,
36(7), 871.
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