Discovery of Novel 7-[(1R,5S)-1-Amino-5-fluoro-3-Azabicyclo[3.3.0]octan-3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-8-(methoxy or methyl)-quinolones

Article abstract of DOI:10.1248/cpb.c18-00671

A series of 8-methoxy or 8-methylquinolones bearing novel 3-Aminooctahydrocyclopenta[c]pyrrole derivatives at the C-7 position was synthesized, and the pharmacological, physicochemical, and toxicological properties of the individual compounds were evaluat

Full text of DOI:10.1248/cpb.c18-00671

Vol. 67, No. 1
Chem. Pharm. Bull. 67, 47–58 (2019)
47
Regular Article
Highlighted Paper selected by Editor-in-Chief
Discovery of Novel 7-[(1R,5S)-1-Amino-5-fluoro-3-azabicyclo[3.3.0]octan-
3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-8-(methoxy or methyl)-
quinolones
,a
Satoshi Komoriya,* Takashi Odagiri,a Hiroaki Inagaki,a Masatoshi Nagamochi,a Rie Miyauchi,b
Ken-ichi Yoshida,a Takahiro Kitamura,c and Hisashi Takahashia
b
a R&D Division, Daiichi Sankyo Co., Ltd.; 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan: Quality and
Safety Management Division, Daiichi Sankyo Co., Ltd.; 3–5–1 Nihonbashi Honcho, Chuo-ku, Tokyo 103–8426,
c
Japan: and R&D Division, Daiichi Sankyo RD Novare Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo 134–8630,
Japan.
Received August 30, 2018; accepted October 16, 2018
A series of 8-methoxy or 8-methylquinolones bearing novel 3-aminooctahydrocyclopenta[c]pyrrole deriv-
atives at the C-7 position was synthesized, and the pharmacological, physicochemical, and toxicological prop-
erties of the individual compounds were evaluated. Novel 8-methylquinolone 7, which includes a 3-amino-
7-fluorooctahydrocyclopenta[c]pyrrole moiety at the C-7 position, showed potent antibacterial activity against
both Gram-positive and negative pathogens. Compound 7 also demonstrated favorable pharmacokinetic and
pharmacodynamic properties and an acceptably safe toxicological profile. Consequently, compound 7 was
selected as a clinical candidate.
Key words 8-methylquinolone; antibacterial activity; multidrug-resistant microorganism; multidrug-resistant
Gram-negative pathogen; multidrug-resistant Acinetobacter baumannii
vofloxacin (LVFX) and ciprofloxacin (CPFX) for comparison.
Introduction
Increasing antibiotic-resistant infections caused by drug- Compound 2 exhibited a broad-spectrum antibacterial effect
resistant microorganisms and the shortage of clinically avail- against Gram-positive organisms, and its potency against
able antibacterial agents present a major global public health Gram-positive pathogens was higher than that of compound 1
threat. Pathogenic bacteria include not only multidrug-resis- or 3 (Table 1). Unfortunately, the results of our PK and safety
tant Gram-positive pathogens like multidrug-resistant Strep- studies with compound 2 indicated low bioavailability8) and
tococcus pneumoniae, vancomycin-resistant Enterococcus, relatively high single-dose toxicity. In previous structure–PK
and community-acquired methicillin-resistant Staphylococcus relationship studies of quinolones, it is already reported that
aureus, but also multidrug-resistant Gram-negative pathogens the structure of the substituent at the C-5, C-6, C-7, or C-8
like multidrug-resistant Acinetobacter baumannii.1–4)
position has a great influence.9–11) In order to improve the PK
In February 2017, the WHO published its first ever list of profile of compound 2, we substituted unique pyrrolidine de-
antibiotic-resistant “priority pathogens”—a catalog of 12 fami- rivatives at the C-7 position and identified the substituent that
lies of bacteria that pose the greatest threat to human health. allowed excellent activity, safety, and PK.
In this news release, carbapenem-resistant Acinetobacter bau-
mannii was identified as one of the pathogens for which new
antibiotics are urgently needed.5)
Comparing the p values12) of compounds 1, 2, and 3, com-
Table 1. Antibacterial Activities (MIC, µg/mL) of Compounds 1, 2, and
3 and Reference Quinolones against Gram-Positive and Negative Bacteria
Novel antibacterial compound 1 (Table 1), as previously
reported,6) exhibits potent in vitro and in vivo activity against
several quinolone-resistant pathogens. Furthermore, compound
1 shows excellent pharmacokinetic (PK) and safety profiles.
Compound 1 possesses a unique tertiary alkyl amine structure
that reduces the risk of mechanism-based inactivation.6)
We recently reported novel quinolone compound 2 bearing
3-aminooctahydrocyclopenta[c]pyrrole at the C-7 position7)
(Fig. 1). The 3-aminooctahydrocyclopenta[c]pyrrole unit fea-
tures a tertiary alkyl amine structure. Similar to compound 1,
compound 2 exhibits a low mechanism-based inactivation risk
(data not shown). Moxifloxacin (3: MFLX) is a commercially
available respiratory quinolone (Fig. 1) that exhibits potent
antibacterial activity and good PK and safety profiles.
3:
MFLX
Bacteria/Compound
1
2
LVFX CPFX
E. coli NIHJ
0.012 0.025 0.012 0.012 ≤0.003
K. pneumoniae TYPE 1
P. aeruginosa PAO1
H. influenzae ATCC49247
M(B). caterrhalis ATCC25238
S. aureus FDA 209-P
S. epidermidis 56500
S. pneumoniae J24
0.05
0.78
0.1
0.1
0.05
0.39
0.025
0.05
0.78
0.78
0.012 0.006 0.012 0.012 0.006
0.05 0.025 0.05 0.025 0.025
0.025 0.012 0.05
0.1
0.1
0.1
0.05
0.05
0.1
0.1
0.1
0.2
0.2
0.78
0.39
0.78
0.78
0.78
0.2
0.05
0.2
0.78
1.56
0.78
S. pyogenes G-36
E. faecalis 19433
0.2
0.1
S. aureus 870307 (MRSA)
S. pneumoniae 104835
0.39
0.39
0.78
0.39
6.25 >6.25
The minimum inhibitory concentrations (MICs) of 1, 2, and
3 against several representative Gram-positive and negative
bacteria are summarized in Table 1, along with the data for le-
3.13 >6.25 >6.25
MFLX, moxifloxacin; LVFX, levofloxacin; CPFX, ciprofloxacin; MRSA, methicil-
lin-resistant Staphylococcus aureus.
*To whom correspondence should be addressed. e-mail: komoriya.satoshi.cn@daiichisankyo.co.jp
© 2019 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
Fig. 1. Structures of Compounds 1, 2, and Moxifloxacin (MFLX) (3)
Fig. 2. Design of 8-Methyl Quinolone Derivative 7
Table 2. Antibacterial Activities (MIC, µg/mL) of Compounds 2, 4, 5, 6, and 7 and Reference Quinolones against Gram-Positive and Negative Bacteria
Bacteria/Compound (Ex. No.)
E. coli NIHJ
2
4
5
6
7
LVFX
CPFX
0.025
0.1
0.006
0.05
0.1
0.05
0.2
≤0.003
0.012
0.2
0.012
0.05
≤0.003
0.025
0.05
K. pneumoniae TYPE 1
P. aeruginosa PAO1
H. influenzae ATCC49247
M(B). caterrhalis ATCC25238
S. aureus FDA 209-P
S. epidermidis 56500
S. pneumoniae J24
0.39
3.13
0.025
0.05
0.025
0.1
0.78
0.006
0.025
0.012
0.05
0.05
0.1
0.78
1.56
0.05
0.1
0.39
≤0.003
0.025
0.012
0.05
≤0.003
0.006
0.006
0.025
0.025
0.025
0.05
0.012
0.025
0.1
0.006
0.025
0.1
0.05
0.1
0.39
0.2
0.05
0.2
0.05
0.2
0.78
0.78
S. pyogenes G-36
0.1
0.2
0.78
1.56
E. faecalis 19433
0.1
0.2
0.2
0.2
0.78
0.78
S. aureus 870307 (MRSA)
S. pneumoniae 104835
0.78
0.39
0.39
0.78
—
0.78
—
0.2
6.25
>6.25
>6.25
0.39
0.1
>6.25
LVFX, levofloxacin; CPFX, ciprofloxacin; MRSA, methicillin-resistant Staphylococcus aureus.
pound 2 (p=5.62) had much lower p value than compound 1 properties of compounds 2, 4, 5, 6, and 7 are summarized in
(p=19.2) or MFLX (3) (p=53.8), and such low lipophilicity Table 3, along with the data for LVFX and MFLX.
is thought to lead to poor permeability causing low bioavail-
ability. Therefore, we introduced a halogen or a methyl group bacterial effect against Gram-positive and negative bacteria.
at the C-7 position to increase the lipophilicity (Fig. 2). Moreover, the p value and mortality associated with com-
The novel quinolone 7 exhibited a broad-spectrum anti-
Synthesis and biological evaluation of a series of 8-me- pound 7 were significantly lower than those associated with
thoxyquinoline-3-carboxylic acid derivatives with modified compound 2. In the micronucleus test, compound 7 exhibited
C-7 substituents showed that fluoro compound 4 exhibited negative responses at a dose of 150mg/kg (IV) in mice (Table
good antibacterial activity, reduced toxicity (mortality), and 3).
improved oral bioavailability, but weak activity against P.
The MICs of compound 7 against several anaerobic species
aeruginosa. In addition, compound 4 exhibited low urinary are summarized in Table 4,14) along with the corresponding
recovery13) (Fig. 2). We also designed its 8-methylquinoline- data for LVFX, CPFX, MFLX, and sitafloxacin (STFX). Com-
3-carboxylic acid analog. Replacement of the 8-methoxy pound 7 exhibited equivalent or higher activities than STFX
group with an 8-methyl group on this scaffold afforded higher against Gram-positive and negative species. The MIC90 values
antibacterial activity against Gram-negative pathogens and of compound 7 against major pathogens that cause hospital-
higher urinary recovery probably owing to its higher hydro- and community-acquired infections are summarized in Table
philicity (Fig. 2).
5,14) along with the corresponding data for LVFX, CPFX, and
STFX. Compound 7 also exhibited potent activities against
these pathogens, including quinolone-resistant organisms.
The IC50 values of compound 7 and LVFX against Esch-
Results and Discussion
Biology The MICs of compounds 2, 4, 5, 6, and 7 against
several representative Gram-positive and negative bacteria are erichia coli and human topoisomerases are summarized in
summarized in Table 2, along with the corresponding data Table 6.14) Compound 7 inhibited E. coli DNA gyrase and
for LVFX and CPFX. The physicochemical and toxicological topoisomerase IV with significantly lower IC50 values, which

Vol. 67, No. 1 (2019)
Chem. Pharm. Bull.
49
Table 3. Physicochemical and Toxicological Properties of Quinolones
a) Apparent partition coefficient, CHCl3–0.1M phosphate buffer (pH 7.4)8); b) aqueous solubility; c) mortality=(the number of dead mice)/(the
number of tested mice); d) using the bone marrow of surviving animals in the IV single-dose toxicity test. MLD, minimum lethal dose.
Table 4. Antibacterial Activity against Anaerobic Speciesa)
MIC (µg/mL)
Strain
7
LVFX
CPFX
MFLX
STFX
Gram-negative
Gram-positive
B. fragilis PA-2-II
≦0.05
≦0.05
0.78
3.13
0.78
12.5
100
1
6.25
3.13
25
0.78
0.2
12.5
25
0.1
≦0.05
0.78
B. fragilis NCTC9343
F. varium ATCC 8501
F. nucleatum IPP 143
P. intermedia ATCC25611
P. melaninogenica JCM6325
0.78
50
0.78
0.03
2
1
0.06
0.03
1
2
1
0.06
C. perfringens 22
≦0.05
0.1
0.78
6.25
12.5
12.5
0.78
0.78
12.5
25
0.78
1.56
3.13
3.13
0.78
0.1
0.2
C. difficile ATCC9689
C. difficile GAI-0547
C. difficile GAI-0796
P. acnes X-18
0.2
0.39
0.2
0.2
50
≦0.05
0.78
≦0.05
a) Microbroth dilution method. LVFX, levofloxacin; CPFX, ciprofloxacin; MFLX, moxifloxacin; STFX, sitafloxacin.
Table 5. Antibacterial Activity against Key Pathogenic Species of Clini-
cal Isolatesa)
Table 6. IC50 Values of Compound 7 and Levofloxacin (LVFX) against
E. coli and Human Topoisomerases
MIC90 (µg/mL)
E. coli
Topoisomerase IV
Human
Species (#)
Compound
7
LVFX
CPFX
STFX
DNA gyrase
Topoisomerase II
(µg/mL)
(µg/mL)
(µg/mL)
P. aeruginosab) (18)
A. baumannii (20)
E. coli (50)
8
1
1
64
8
32
64
16
0.25
64
4
4
7
0.499
1.88
0.0428
0.735
>1024
>1024
2
LVFX
8
1
K. pneumoniae (26)
S. pneumoniae (24)
S. pyogenes (26)
VGSc) (12)
0.25
0.25
0.03
0.5
4
0.5
32
2
0.12
1
LVFX, levofloxacin.
0.06
2
hibited a higher tissue to plasma drug concentration ratio (Kp
value) after oral administration in rats, as well as good oral
bioavailability in monkeys.
64
>64
>64
32
MRSA (49)
>64
32
8
E. faecalis (26)
1
2
In the clinical study of fluoroquinolones, cardiotoxicity, skin
toxicity, and central nervous system (CNS) toxicity have often
become safety concerns.15–18) Therefore, we evaluated the long
QT, photosensitivity, and convulsion risks associated with
compound 7.
a) Clinical isolates from Eurofins’ surveillance (worldwide) and LVFX-surveil-
lance in Japan; b) not included highly resistant P. aeruginosa; c) viridans group
streptococci; LVFX, levofloxacin; CPFX, ciprofloxacin; MFLX, moxifloxacin; STFX,
sitafloxacin.
were approximately 4- and 17-fold, respectively, lower than
Table 814) shows the effect of the drugs on the human ether-
those for LVFX. However, compound 7 had no effect on the à-go-go-related gene (hERG) channel current and the prolon-
activity of human topoisomerase II, similar to LVFX. gation of action potential duration at 90% (APD90), both of
The PK profiles of compound 7 after oral administration which are indicators of cardiotoxicity. Compound 7 produced
(5mg/kg) to rats and oral (5mg/kg) or IV (5mg/kg) adminis- a lower hERG current than MFLX, while its grade in the
tration to monkeys are shown in Table 7.14) Compound 7 ex- APD90 prolongation test was almost equal to that of LVFX.

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Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
Table 7. Pharmacokinetic (PK) Parameters of Compound 7 and Levo-
floxacin (LVFX) in Ratsa) and Monkeysb) (5mg/kg, n=3)
Table 10. Convulsive Activity of Compound 7 and Ciprofloxacin (CPFX)
in Micea)
Rats
Serum
PK parameters
Cmax (µg/mL)
7
LVFX
Incidence
Dose
Compounds (µg/5µL/mouse,
p.o.
1.01
2.00
1.47
3.41
without BPAAb)
with BPAA
i.cist.)
AUC0–8h (µg·h/mL)
Convulsion Mortality Convulsion Mortality
Lung
AUC0–8h (µg·h/mL)
4.56
2.3
5.57
1.6
7
5
15
50
5
2/6
6/6
6/6
1/6
6/6
6/6
0/6
2/6
0/6
0/6
3/6
4/6
0/6
6/6
6/6
6/6
6/6
6/6
0/6
0/6
1/6
6/6
6/6
6/6
c)
Kp
Liver
AUC0–8h (µg·h/mL)
28.5
16.4
4.8
c)
Kp
14.3
CPFX
15
50
Kidney
AUC0–8h (µg·h/mL)
14.9
7.5
19.3
5.6
c)
Kp
a) Determined by intracisternal (i.cist.) administration in 3-week-old male Slc:ddY
mice (5µg/mouse). b) 4-Biphenylacetic acid (BPAA). CPFX (5µg/mouse) was ad-
ministered 30min after the oral administration of BPAA (400mg/kg).
Urinary recovery0–24h (%)
PK parameters
19.2
7
80.0
Monkeys
LVFX
1.68d)
p.o.
Serum
Cmax (µg/mL)
1.31
5.11 15.3d)
AUC0–8h (µg·h/mL)
Urinary recovery0–24h (%)
15.1
49.1e)
IV
Serum
Cmax (µg/mL)
3.55
7.12
AUC0–8h (µg·h/mL)
Urinary recovery0–24h (%)
Bioavailability (%)
38.1
71.8
a) Male Crj:CD rats (seven weeks); b) female cynomolgus monkeys; c) Kp val-
ues indicate tissue–serum concentration ratio; d) value obtained after 20mg/kg oral
administration, which are then divided by 4; e) value obtained after 20mg/kg oral
administration.
Table 8. Effect on Human Ether-à-go-go-Related Gene (hERG) Channel
Current in hERG-Transfected CHO-K1 Cells and the Prolongation of APD90
Fig. 3. Therapeutic Efficacy of Compound 7 and Levofloxacin (LVFX)
in Murine Respiratory Tract Infection Due to S. pneumoniae GE01085
Inhibition in channel current
(%)a)
Prolongation in APD90
(%)a)
Circles represent the pulmonary bacterial counts in each mouse (n=5). Bars
represent the mean S.E.M. of the bacterial counts. Dashed lines represent the
regression line calculated by a parallel line analysis.
Compound
30µM
100µM
300µM
100µM
300µM
7
4.5
0.5
8.2
1.5
26.9
7.3
1.5
1.3
−2.1
7.3
LVFX
MFLX
17.7
34.6
65.6
14.9
44.2
a) All values represent the mean of four replicates. LVFX, levofloxacin; MFLX,
moxifloxacin.
Table 9. Phototoxicity of Compound 7 and Ciprofloxacin (CPFX) in
Micea)
Auricular
Histopathology
Dose
No. of
Compounds
(mg/kg, IV) animals
Erythema Thickening Auriculae Eye
7
100
5
6
6
−
+
−
+
−
+
−
−
CPFX
a) Female BALB/cAnNCrlCrlj mice (initial age: 6–7 weeks) were exposed to UV
A at 1.5mW/cm2 for 4h (21.6J/cm2) immediately after single IV dosing.
Fig. 4. Therapeutic Efficacy of Compound 7 and Levofloxacin (LVFX)
in Rodent Urinary Tract Infection Due to E. coli GK00432
These results indicated that compound 7 possesses a low risk
of QTc prolongation.
Phototoxicity data and the convulsant activity of compound
7 are summarized in Table 914) and Table 10,14) respectively,
along with the corresponding data for CPFX. Compound 7
Circles represent the difference between each renal bacterial count and the mean
renal bacterial counts at the onset of treatment (6.08logCFU/kidney). The mean
renal bacterial count at the onset of treatment is represented as a broken horizontal
line. The black short bars represent the mean of the renal bacterial counts (n=5).
possessed lower phototoxicity and convulsive potential than for LVFX. The antibacterial activity and therapeutic efficacy
CPFX. of compound 7 against S. pneumoniae were 32- and 30-fold,
The in vivo antibacterial activities of compound 7 are respectively, better than those of LVFX. Compound 7 also
shown in Figs. 314) and 4,14) along with the comparative data showed more potent in vivo bactericidal activity against

Vol. 67, No. 1 (2019)
Chem. Pharm. Bull.
51
Reagents and conditions: (a) TBS-Cl, imidazole–DMF, 49%; (b) RuCl3·H2O, NaIO–CCl4, CH3CN, H2O; (c) MeI, NaHCO3–DMF, 85% from 8.
Chart 1. Synthesis of pPyrrolidine Intermediates 9 and 10
Reagents and conditions: (a) LHMDS–THF, then N-fluorobenzenesulfonimide (for 11a) or N-chlorosuccinimide (for 11b), 26% (11a); (b) TBAF, AcOH–THF, 93% (12a)
or 64% (12b from 9); (c) phenylsulfonyl chloride, Et3N, DMAP–DCM, 69% (13a); (d) KHMDS–THF, 58% (14a) or 28% (14b from 12b); (e) trifluoroacetic acid–DCM;
(f) DPPA, Et3N, toluene, reflux; (g) 6N HClaq–1,4-dioxane, heat; (h) (Boc)2O, heat, 81% (15a from 14a) or 64% (15b from 14b); (i) BH3–THF–THF; (j) Et3N, 90% EtOHaq
reflux, 94% (16a from 15a) or 32% (16b from 15b); (k) 10% Pd-C (wt), H2, EtOH, heat, 91% (17a) or crude (17b).
,
Chart 2. Synthesis of Amines 17a and 17b
Reagents and conditions: (a) LHMDS–THF, 76%; (b) NaH–DMF, then MeI, 76%; (c) ethanedithiol, p-toluenesulfonic acid-H2O–toluene, reflux; (d) trimethylsilyldiazo-
methane–MeOH, THF, 87% from 19; (e) Raney-Ni–EtOH, reflux, 65%; (f) NaOHaq–MeOH; (g) DPPA, Et3N, toluene, reflux; (h) 6N HClaq–1,4-dioxane, heat; (i) Red-Al,
heat; (j) (Boc)2O–DCM, MeOH, 52% from 21; (k) 10% Pd-C (wt), H2, EtOH, heat, 95%.
Chart 3. Synthesis of Amine 23
LVFX-resistant E. coli in the kidneys of a rodent urinary tract derivative 15a or 15b. Finally, BH3 reduction of the N-Boc
infection (UTI) model than LVFX. Compound 7 exhibited derivative 15a or 15b, followed by hydrogenation, gave amine
about 30-fold better antibacterial activity in vitro than LVFX 17a or 17b, respectively.
in both a murine respiratory tract infection (RTI) and a rodent
The preparation of amine 23 is depicted in Chart 3. Cycliza-
UTI model. These results reveal that compound 7 has the tion of ester 10 with lithium hexamethyldisilazide (LHMDS),
same target tissue penetration as LVFX. Since it has already followed by methylation and a 3-step decarbonylation, gave
been known that LVFX shows good PK in humans, it can be methyl ester 21. Hydrolysis of methyl ester 21 followed by the
expected that compound 7 will also exhibit favorable efficacy same amine formation, reduction with Red-Al, tert-butoxycar-
in humans.
bonylation, and hydrogenation afforded amine 23.
Chemistry The preparation of key chiral intermediates 9
The secondary amine (17a, 17b, or 23) and quinolonecar-
and 10 is shown in Chart 1. Silylation of the chiral alcohol 87) boxylic acid BF2 chelate 2419) were heated with triethylamine
with tert-butyldimethylsilyl chloride (TBS-Cl) afforded key in dimethyl sulfoxide (DMSO) followed by dechelation and
chiral intermediate 9. Oxidation of 8 followed by esterification deprotection to afford 8-methoxyquinolone 4, 5, or 6 (Chart 4).
gave another key intermediate 10.
The preparation of 8-methylquinolone 7 is shown in Chart
Preparation of amines 17a and 17b is shown in Chart 2. 5. Buchwald–Hartwig coupling of the secondary amine 17a
Fluorination or chlorination of 9, followed by desilylation, and 7-bromo-8-methylquinolone 25, followed by hydrolysis
phenylsulfonylation, and cyclization with potassium hexa- and deprotection, gave 7. 7-Bromo-8-methylquinolone 25 was
methyldisilazide (KHMDS), gave tert-butyl ester 14a or 14b. prepared from commercially available benzoic acid 26 by
Deprotection of the tert-butyl ester 14a or 14b, followed introduction of aminoacrylate, then cyclopropyl amine, and
by diphenylphosphoryl azide (DPPA)-mediated sequential cyclization (Chart 6).
amine formation reactions (acid azide formation, the Curtius
rearrangement, and hydrolysis of the resulting isocyanate),
Conclusion
and tert-butoxycarbonylation gave the corresponding N-Boc
8-Methoxy- or 8-methylquinolone derivatives bearing bicy-

52
Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
Reagents and conditions: (a) Et3N, DMSO, heat; (b) conc. HCl; (c) pH 7.4, 32% (4) or 41% (5 from 16b) or 46% (6).
Chart 4. Synthesis of 8-Methoxyquinolones 4–6
Reagents and conditions: (a) Pd2(dba)3, Xantphos, Cs2CO3–1,4-dioxane, heat; (b) 1N NaOHaq–EtOH; (c) conc. HCl; (d) pH 7.4; (e) crystallization; (f) 1N HCl (1eq); (g)
recrystallization, 38% from 17a.
Chart 5. Synthesis of 8-Methylquinolone 7
Reagents and conditions: (a) Thionyl chloride–DMF, reflux; (b) ethyl 3-dimethylaminoacrylate, Et3N–THF, reflux, 71% from 26; (c) (1R,2S)-2-fluorocyclopropylamine,
Et3N–DCM; (d) Cs2CO3–DMF, 26% from 27.
Chart 6. Synthesis of 7-Bromo-8-methylquinolone 25
clic amines at the C-7 position were synthesized, evaluated for constant(s) in hertz. High-resolution (HR)-MS were obtained
antibacterial activities, and assessed for physicochemical and on a JEOL JMS-700 mass spectrometer under electron impact
pharmacokinetic properties and preliminary safety. These ana- ionization, electrospray ionization, or FAB ionization condi-
logs exhibited broad-spectrum activity against pathogens of tions. The high-resolution mass spectra were recorded on a
major hospital- and community-acquired infections including JEOL JMS-100LP spectrometer. Elemental analyses are indi-
infections by drug-resistant strains.
cated by the symbols of the elements; analytical results were
Specifically, compound 7 exhibited better activity than within 0.4% of the theoretical values. Purities of ≥95% were
LVFX and MFLX against streptococci, staphylococci, en- determined by elemental analysis for all tested compounds.
terococci, E. coli, A. baumannii, and anaerobes. Compound 7 Column chromatography refers to flash column chromatogra-
also showed potent in vivo antibacterial activity in a murine phy conducted on Merck silica gel 60, 230–400 mesh ASTM.
RTI model and a rodent UTI model. Moreover, compound 7 TLC was performed with Merck silica gel 60F254 TLC plates,
exhibited excellent PK and toxicological profiles. As already and compound visualization was accomplished with a 5%
reported by Higuchi et al.,20) compound 7 showed excellent solution of molybdophosphoric acid in ethanol, a UV lamp,
in vitro and in vivo antibacterial activity against multidrug- iodine, or Wako ninhydrin spray.
resistant A. baumannii. Thus, compound 7 was selected as a
(3S)-3-[3-(tert-Butyldimethylsilyloxy)-1-propyl]-5-oxo-1-
candidate for further evaluation as a new-generation, broad- [(1R)-1-phenylethyl]pyrrolidine-3-carboxylic Acid tert-
spectrum quinolone antibiotic. We also expect compound 7 to Butyl Ester (9) (3S)-3-(3-Hydroxy-1-propyl)-5-oxo-1-[(1R)-1-
potentially become an option for antibacterial therapy against phenylethyl]pyrrolidine-3-carboxylic acid tert-butyl ester (8,
multidrug-resistant A. baumannii.
46g, 0.13mol) and imidazole (11.9g, 0.13mol) were dissolved
in N,N-dimethylformamide (DMF, 600mL). After addition
of TBS-Cl (23.2g, 0.15mol) under ice-cooling, the mixture
Experimental
General Melting points were recorded on a Yanaco MP- was stirred at room temperature for 59.5h. The reaction solu-
500D melting point apparatus and were uncorrected. Optical tion was extracted with a 10% citric acid solution and ethyl
rotations were measured in a 0.5-dm cell at 25°C at 589nm acetate. The organic layer was then sequentially washed
1
with a HORIBA SEPA-300 polarimeter. H-NMR spectra were with saturated sodium bicarbonate, water, and brine; dried
determined on a JEOL JNMEX400 spectrometer. Chemi- over anhydrous sodium sulfate; and filtered. The solvent was
cal shifts are reported in ppm relative to tetramethylsilane evaporated under reduced pressure, and the residue was sub-
or sodium 2,2-dimethyl-2-silapentane-5-sulfonate as internal jected to silica gel column chromatography (hexane–ethyl ace-
standards. Significant 1H-NMR peaks are tabulated in the tate=9:1–2:1) to give 29.7g (49%) of the title compound (9)
1
following order: number of protons, multiplicity (s, singlet; as a pale yellow oil. H-NMR (400MHz, CDCl3) δ: 7.37–7.22
d, doublet; t, triplet; q, quartet; m, multiplet), and coupling (5H, m), 5.48 (1H, q, J=7.11Hz), 3.58 (2H, t, J=6.13Hz),

Vol. 67, No. 1 (2019)
Chem. Pharm. Bull.
53
3.34 (1H, d, J=10.05Hz), 3.12 (1H, d, J=10.05Hz), 2.94 (1H, ice-cooling, and the mixture was stirred at room temperature
d, J=16.91Hz), 2.31 (1H, d, J=17.16Hz), 1.86–1.74 (1H, m), for 12.5h. Saturated aqueous sodium bicarbonate was added
1.72–1.62 (1H, m), 1.51 (3H, d, J=7.11Hz), 1.49–1.24 (2H, m), to the reaction solution, and the mixture was stirred for
1.33 (9H, s), 0.88 (9H, s), 0.03 (6H, s). MS (electrospray ion- 30min, followed by extraction with DCM. The organic layer
ization (ESI)) m/z: 462 (M+H)+. HR-MS (ESI) m/z: 462.3078 was sequentially washed with a 10% citric acid solution and
(M+H)+ (Calcd for C26H44NO4Si: 462.3039). IR (attenuated brine, dried over anhydrous sodium sulfate, and filtered. The
total reflectance (ATR)) cm−1: 2948, 2928, 2857, 1721, 1681, solvent was then evaporated under reduced pressure. The
1426, 1299, 1250, 1219, 1156, 1116, 1089, 1080, 1034.
residue was subjected to silica gel column chromatography
(3S)-3-[3-(tert-Butyldimethylsilyloxy)-1-propyl]-4- (hexane–ethyl acetate=8:2–1:1) to give 11.7g (69%) of the
1
fluoro-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylic title compound (13a) as a pale yellow oil. H-NMR (400MHz,
Acid tert-Butyl Ester (11a) (3S)-3-[3-(tert-Butyldimethyl CDCl3) δ: 7.94–7.87 (2H, m), 7.71–7.63 (1H, m), 7.60–7.53 (2H,
silyloxy)-1-propyl]-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3- m), 7.37–7.23 (5H, m), 5.46 (1H, q, J=7.11Hz), 5.15 (1H, d,
carboxylic acid tert-butyl ester (9, 30g, 65mmol) was dis- J=51.48Hz), 4.10–3.98 (2H, m), 3.26–3.15 (2H, m), 1.88–1.50
solved in tetrahydrofuran (THF, 280mL), and the atmosphere (4H, m), 1.55 (3H, s), 1.30 (9H, s).
was replaced with argon. Then, lithium hexamethyldisilazide
(1S,5R)-5-Fluoro-4-oxo-3-[(1R)-1-phenylethyl]-3-azabicy-
(1.0M solution in THF) (78.0mL) was added dropwise at clo[3.3.0]octan-1-ylcarboxylic Acid tert-Butyl Ester (14a)
−15°C, and the mixture was stirred at −5°C for 30min. After (3S)-3-(3-Phenylsulfonyloxy-1-propyl)-4-fluoro-5-oxo-1-
cooling again to −15°C, a solution of N-fluorobenzenesulfon- [(1R)-1-phenylethyl]pyrrolidine-3-carboxylic acid tert-butyl
imide (26.6g, 84mmol) in THF (220mL) was added dropwise, ester (13a, 10.9g, 21.6mmol) was dissolved in THF (350mL),
and the mixture was stirred at room temperature for 17h. The and the atmosphere was replaced with argon. Then, potas-
reaction solution was extracted with a 10% citric acid solution sium hexamethyldisilazide (0.5M solution in toluene, 86.5mL)
and ethyl acetate. Then, the organic layer was washed with was added dropwise at −15°C, and the mixture was stirred
brine, dried over anhydrous sodium sulfate, and filtered. The at 0°C for 1.5h. After cooling to −10°C, saturated aqueous
solvent was then evaporated under reduced pressure. The resi- ammonium chloride (100mL) was added dropwise, and the
due was subjected to silica gel column chromatography (hex- mixture was stirred at room temperature for 30min. The reac-
ane–ethyl acetate=9:1–8:2) to give 8.15g (26%) of the title tion solution was extracted with a 10% citric acid solution and
1
compound (11a) as a pale yellow solid. H-NMR (400MHz, ethyl acetate. The organic layer was then washed with brine,
CDCl3) δ: 7.37–7.23 (5H, m), 5.53–5.44 (1H, m), 5.18 (1H, d, dried over anhydrous sodium sulfate, and filtered. The solvent
J=51.72Hz), 3.64–3.52 (2H, m), 3.32–3.19 (2H, m), 1.92–1.65 was evaporated under reduced pressure, and the residue was
(2H, m), 1.55 (3H, d, J=4.66Hz), 1.33 (9H, s), 0.88 (9H, s), subjected to silica gel column chromatography (hexane–ethyl
0.03 (6H, s). MS (FAB) m/z: 480 (M+H)+. IR (ATR) cm−1: acetate=9:1–7:1) to give 4.36g (56%) of the title compound
1
3421, 2977, 2935, 2877, 1698, 1454, 1369, 1309, 1249, 1153, (14a) as a pale yellow solid. H-NMR (400MHz, CDCl3) δ:
1058, 1035, 1006, 842.
7.38–7.25 (5H, m), 5.58–5.49 (1H, m), 3.63 (1H, d, J=10.3 Hz),
(3S)-4-Fluoro-3-(3-hydroxy-1-propyl)-5-oxo-1-[(1R)-1- 2.91 (1H, dd, J=10.3, 3.2Hz), 2.67–2.56 (1H, m), 2.50–2.38
phenylethyl]pyrrolidine-3-carboxylic Acid tert-Butyl Ester (1H, m), 2.26–2.09 (1H, m), 2.06–1.94 (1H, m), 1.74–1.66 (1H,
(12a) (3S)-3-[3-(tert-Butyldimethylsilyloxy)-1-propyl]-4- m), 1.54 (3H, d, J=7.1Hz), 1.50–1.40 (1H, m), 1.34 (9H, s).
fluoro-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylic MS (ESI) m/z: 348 (M+H)+. HR-MS (ESI) m/z: 348.1983
acid tert-butyl ester (11a, 8.15g, 17mmol) was dissolved in (M+H)+ (Calcd for C20H27FNO3: 348.1975). IR (ATR) cm−1:
THF (25.0mL). Acetic acid (22.0mL) and tetrabutylammo- 2974, 1729, 1686, 1278, 1242, 1156, 1122, 850.
nium fluoride (TBAF, 1.0M solution in THF, 25.0mL) were
(1R,5R)-1-(tert-Butoxycarbonylamino)-5-fluoro-4-oxo-3-
added under ice-cooling, and the mixture was stirred at room [(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octane (15a) (1S,5R)-
temperature for 21.5h. The reaction solution was extracted 5-Fluoro-4-oxo-3-[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]-
with a 10% citric acid solution and ethyl acetate. The organic octan-1-ylcarboxylic acid tert-butyl ester (14a, 4.36g,
layer was then sequentially washed with saturated sodium bi- 12.5mmol) was dissolved in DCM (70mL). Trifluoroacetic
carbonate, water, and brine; dried over anhydrous sodium sul- acid (70mL) was added dropwise, and the mixture was stirred
fate; and filtered. The solvent was evaporated under reduced at room temperature for 6h. The solvent was evaporated under
pressure. The residue was subjected to silica gel column chro- reduced pressure, and then the residue was azeotropically dis-
matography (hexane–ethyl acetate=9:1–8:2–1:1) to give tilled with toluene to give carboxylic acid (3.70g). The result-
5.77g (93%) of the title compound (12a) as a pale yellow oil. ing carboxylic acid was dissolved in toluene. Triethylamine
1H-NMR (400MHz, CDCl3) δ: 7.37–7.22 (5H, m), 5.48 (1H, (3.51mL, 25.2mmol) and diphenylphosphoryl azide (2.98mL,
q, J=7.03Hz), 5.20 (1H, d, J=51.48Hz), 3.69–3.59 (2H, m), 13.8mmol) were added, and the mixture was heated to reflux
3.31–3.21 (2H, m), 1.95–1.72 (2H, m), 1.68–1.43 (2H, m), 1.56 for 5h. The solvent was evaporated under reduced pressure.
(3H, d, J=7.11Hz), 1.33 (9H, s).
Then, 1,4-dioxane (110mL) and 6N hydrochloric acid (110mL)
(3S)-3-(3-Phenylsulfonyloxy-1-propyl)-4-fluoro-5-oxo- were added to the residue, and the mixture was stirred at
1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylic Acid tert- 60°C for 2.5h. After extraction with water and ethyl acetate,
Butyl Ester (13a) (3S)-4-Fluoro-3-(3-hydroxy-1-propyl)- the aqueous layer was made alkaline with a saturated sodium
5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylic acid tert- hydroxide solution and extracted twice with chloroform. The
butyl ester (12a, 12.20g, 33.4mmol) was dissolved in dichlo- organic layers were combined, dried over anhydrous sodium
romethane (DCM, 400mL). Phenylsulfonyl chloride (9.06mL, sulfate, filtered, and then the solvent was evaporated under re-
79.6mmol), triethylamine (10.7mL, 76.8mmol), and 4-dimeth- duced pressure. Di-tert-butyl dicarbonate (11.05g, 50.6mmol)
ylaminopyridine (DMAP, 2.04g, 16.7mmol) were added under was added to the residue, and the mixture was stirred at 75°C

54
Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
for 6h. The reaction solution was concentrated under reduced 2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-
pressure, and then the residue was subjected to silica gel 3-carboxylic acid-BF2 chelate (24, 2.93g, 8.12mmol), and
column chromatography (hexane–ethyl acetate=9:1–1:1) to triethylamine (3.30mL, 23.7mmol) were dissolved in DMSO
give 3.69g (81%) of the title compound (15a) as a pale yel- (30mL), and the solution was heated with stirring in an oil
1
low oil. H-NMR (400MHz, CDCl3) δ: 7.37–7.23 (5H, m), bath at 40°C. After 19h of stirring, 6,7-difluoro-1-[(1R,2S)-
5.50 (1H, q, J=7.1Hz), 5.22 (1H, brs), 3.34 (2H, s), 2.49–2.37 2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-
(1H, m), 2.32–2.03 (3H, m), 2.02–1.90 (1H, m), 1.51 (3H, d, 3-carboxylic acid-BF2 chelate (24, 1.00g, 2.77mmol) was
J=7.1Hz), 1.55–1.48 (1H, m), 1.35 (9H, s). MS (ESI) m/z: 363 added, then this reaction mixture was heated with stirring
(M+H)+. HR-MS (ESI) m/z: 363.2110 (M+H)+ (Calcd for in an oil bath at 40°C for 16h. A mixed solution of ethanol–
C20H28FN2O3: 363.2084).
water=4:1 (50mL) and triethylamine (5mL) was added to
(1R,5S)-1-(tert-Butoxycarbonylamino)-5-fluoro-3-[(1R)- the reaction solution, and the mixture was heated to reflux in
1-phenylethyl]-3-azabicyclo[3.3.0]octane (16a) (1R,5R)-1- an oil bath at 90°C for 1.5h. The reaction solution was con-
(tert-Butoxycarbonylamino)-5-f luoro-4-oxo-3-[(1R)-1- centrated under reduced pressure. The residue was dissolved
phenylethyl]-3-azabicyclo[3.3.0]octane (15a, 3.69g, 10.2mmol) in ethyl acetate and washed with a 10% citric acid solution,
was dissolved in THF (200mL). A 1.20M solution of a bo- water, and brine. The organic layer was dried over anhydrous
rane–THF complex in THF (42.4mL, 50.9mmol) was added sodium sulfate and filtered, and then the solvent was evaporat-
dropwise under ice-cooling, and the mixture was stirred for ed under reduced pressure. The residue was purified by silica
2h while gradually warming to room temperature. The solvent gel flash column chromatography (DCM–methanol=99:1) to
was evaporated under reduced pressure. Under ice-cooling, afford 3.30g of the amine coupling product as a yellow oil.
90% aqueous ethanol (100mL) and triethylamine (100mL) The amine coupling compound was dissolved in concentrated
were added to the residue, and the mixture was heated to hydrochloric acid (20mL) under ice-cooling. After stirring at
reflux for 2h. The solvent was evaporated under reduced pres- room temperature for 1h, the reaction solution was washed
sure, and the residue was extracted with saturated aqueous with chloroform. The aqueous layer was adjusted to pH 12.0
sodium bicarbonate and DCM. The target substance was ex- with a 10M sodium hydroxide solution under ice-cooling and
tracted from the aqueous layer with DCM. The organic layers then adjusted to pH 7.4 with hydrochloric acid. The target sub-
were combined, washed with brine, and dried over anhydrous stance was then extracted from the aqueous layer with chloro-
sodium sulfate. After filtration, the solvent was evaporated form. The organic layers were dried over anhydrous sodium
under reduced pressure, and the resulting residue was sub- sulfate. After filtration, the solvent was evaporated under
jected to silica gel column chromatography (hexane–ethyl reduced pressure, and the resulting residue was purified by
acetate=95:5–90:10) to give 3.33g (94%) of the title com- recrystallization from ethanol to give 769mg (32%) of the title
1
pound (16a) as a pale yellow oil. H-NMR (400MHz, CDCl3) compound (4) as a pale yellow powder. mp: 191–195°C (dec.).
1
δ: 7.32–7.18 (5H, m), 5.38 (1H, brs), 3.22 (1H, q, J=6.37Hz), [α]D24 +97° (c=0.100, 0.1 N NaOH). H-NMR (400MHz, 0.1N
2.92–2.57 (4H, m), 2.12–1.86 (4H, m), 1.80–1.67 (1H, m), NaOD) δ: 8.48 (1H, d, J=1.23Hz), 7.70 (1H, d, J=13.73Hz),
1.63–1.52 (3H, m), 1.42 (9H, s), 1.32 (3H, d, J=6.37Hz). 5.07–4.85 (1H, m), 4.10–4.02 (1H, m), 3.93–3.78 (2H, m),
MS (ESI) m/z: 349 (M+H)+. HR-MS (ESI) m/z: 349.2315 3.70–3.53 (2H, m), 3.66 (3H, s), 2.21–2.04 (2H, m), 2.01–1.83
(M+H)+ (Calcd for C20H30FN2O2: 349.2291). IR (ATR) cm−1: (2H, m), 1.83–1.46 (4H, m). MS (FAB) m/z: 438 (M+H)+.
3458, 2972, 1711, 1487, 1365, 1249, 1161.
(1R,5S)-1-(tert-Butoxycarbonylamino)-5-fluoro-3- F, 12.72%; N, 9.37%. Found: C, 56.03%; H, 5.51%; F, 12.79%;
azabicyclo[3.3.0]octane
(17a) (1R,5S)-1-(tert-Butoxycar- N, 9.66%. IR (ATR) cm−1: 2958, 2871, 1724, 1621, 1513, 1452,
bonylamino)-5-fluoro-3-[(1R)-1-phenylethyl]-3-azabicyclo- 1319, 1056, 929, 804.
Anal. Calcd for C21H22F3N3O4·0.6H2O: C, 56.27%; H, 5.22%;
[3.3.0]octane (16a, 700mg, 2.0mmol) was dissolved in ethanol
tert-Butyl (3S)-4-Chloro-3-(3-hydroxy-1-propyl)-5-oxo-1-
(30mL). Ten percent palladium–carbon (50% wet, 1.01g) [(1R)-1-phenylethyl]pyrrolidine-3-carboxylate (12b) Start-
was added, and the mixture was stirred under a hydrogen ing with 9 (960mg, 2.08mmol) and NCS (333mg, 2.50mmol)
atmosphere at 50°C for 15h. The catalyst was removed by and following the procedure for the preparation of 11a gave
filtration, and then the filtrate was concentrated under reduced 11b as a pale yellow solid. No further purification was at-
pressure. The resulting residue was subjected to silica gel tempted on this compound, which was used directly in the
column chromatography (DCM–methanol=98:2–>95:5) to next step. Starting with 11b and following the procedure
give 446mg (91%) of the title compound (17a) as a pale yel- for the preparation of 12a afforded 12b (506mg, 64% from
1
low oil.
9) as a pale yellow oil. H-NMR (CDCl3) δ: 7.34–7.26 (5H,
[α]D23 −15° (c=0.100, MeOH). 1H-NMR (400MHz, m), 5.49–5.40 (1H, m), 4.75 (1H, s), 3.68–3.61 (2H, m),
CDCl3) δ: 5.29 (1H, brs), 3.47–3.18 (2H, m), 2.93–2.79 (2H, 3.39–3.34 (2H, m), 3.24 (0.75H, d, J=10.0Hz), 3.14 (0.25H, d,
m), 2.15–1.71 (6H, m), 1.45 (9H, s). MS (FAB) m/z: 245 J=10.9Hz), 1.95–1.80 (2H, m), 1.68–1.40 (2H, m), 1.52 (3H,
(M+H)+. HR-MS (ESI) m/z: 245.16453 (M+H)+ (Calcd for d, J=7.1Hz), 1.28 (9H, s). MS (ESI) m/z: 382 (M+H)+.
tert-Butyl (1S,5R)-5-chloro-4-oxo-3-[(1R)-1-phenylethyl]-
1702, 1560, 1284, 1245, 1176, 1101, 1035, 1016, 966, 917, 889, 3-azabicyclo[3.3.0]octan-1-ylcarboxylate (14b) Starting
867, 701. with 12b (270mg, 0.71mmol) and following the procedure
C12H22FN2O2: 245.16653). IR (ATR) cm−1: 3280, 3212, 2952,
7-[(1R,5S)-1-Amino-5-f luoro-3-azabicyclo[3. 3.0]- for the preparation of 13a gave 13b as a pale yellow solid. No
octan-3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4- further purification was attempted on this compound, which
dihydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid (4) was used directly in the next step. Starting with 13b and
(1R,5S)-1-(tert-Butoxycarbonylamino)-5-fluoro-3-azabicyclo[3.3.0]- following the procedure for the preparation of 14a afforded
1
octane (17a, 1.32g, 5.40mmol), 6,7-difluoro-1-[(1R,2S)- 14b (73mg, 28% from 12b) as a pale yellow solid. H-NMR

Vol. 67, No. 1 (2019)
Chem. Pharm. Bull.
55
(CDCl3) δ: 7.37–7.26 (5H, m), 5.54 (1H, q, J=7.1Hz), 3.51 an ice bath and a 1N hydrochloric acid solution (2.2L) was
(1H, d, J=10.7Hz), 3.02 (1H, d, J=10.7Hz), 2.76–2.71 (1H, added at an internal temperature of 10°C or lower, followed
m), 2.52–2.45 (1H, m), 2.38–2.30 (1H, m), 2.02–1.96 (1H, by extraction with 2L of chloroform. The aqueous layer was
m), 1.74–1.60 (2H, m), 1.53 (3H, d, J=7.1Hz), 1.45 (9H, s). extracted with chloroform (1L×3). Then, the organic layers
MS (ESI) m/z: 364 (M+H)+. HR-MS (ESI) m/z: 364.1710 were combined, washed with brine (2L×2), and dried over
(M+H)+ (Calcd for C20H27ClNO3: 364.1680). IR (ATR) cm−1: anhydrous sodium sulfate. After filtration, the solvent was
2968, 1730, 1691, 1249, 1239, 1157, 1122.
concentrated under reduced pressure. The resulting crude
(1R,5R)-1-(tert-Butoxycarbonylamino)-5-chloro-4-oxo-3- product was dissolved in DMF (370mL). Sodium bicarbonate
[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octane (15b) Start- (37.9g, 0.451mol) and methyl iodide (70.7g, 0.498mol) were
ing with 14b (332mg, 0.912mmol) and following the proce- added with stirring at room temperature, and the mixture was
dure for the preparation of 15a gave 15b (222mg, 64%) as stirred for 3d. The reaction solution was poured into ice water
1
a colorless oil. H-NMR (CDCl3) δ: 7.37–7.26 (5H, m), 5.50 (1.8L), followed by extraction with ethyl acetate (1.8, 0.5L).
(1H, q, J=6.9Hz), 5.25 (1H, brs), 3.66 (1H, brd, J=10.0 Hz), The organic layers were combined, washed with brine, and
2.96 (1H, d, J=6.8Hz), 2.76–2.69 (1H, m), 2.55–2.51 (1H, m), then dried over anhydrous sodium sulfate. After filtration, the
2.18–2.08 (1H, m), 1.98–1.84 (2H, m), 1.51 (3H, d, J=7.1Hz), filtrate was concentrated under reduced pressure; the residue
1.65–1.50 (1H, m), 1.40 (9H, s). MS (ESI) m/z: 379 (M+H)+.
was subjected to silica gel column chromatography (hexane–
(1R,5R)-1-(tert-Butoxycarbonylamino)-5-chloro- ethyl acetate=1:1), and the fraction containing the target
3-[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octane (16b) substance was concentrated under reduced pressure. The
Starting with 15b (217mg, 0.573mmol) and following the resulting solid was dissolved in ethyl acetate, washed with a
procedure for the preparation of 16a gave 16b (67.3mg, 32%) 10% sodium thiosulfate solution, and dried over anhydrous
as a colorless oil. [α]D25.1 +103.5° (c=0.228, 0.1N NaOH). sodium sulfate. After filtration, the filtrate was concentrated
1H-NMR (CDCl3) δ: 7.29–7.19 (5H, m), 5.53 (1H, brs), 3.22 under reduced pressure, and the resulting solid was dried to
(1H, q, J=6.8Hz), 3.07 (1H, brd, J=8.8Hz), 2.93 (1H, brd, give 35.0g (85%) of the title compound 10 as a pale yellow
1
J=8.7Hz), 2.82–2.71 (1H, m), 2.64–2.57 (1H, m), 2.27–2.15 brown solid. H-NMR (400MHz, CDCl3) δ: 7.35–7.25 (5H, m),
(2H, m), 2.09–2.06 (2H, m), 1.74–1.67 (2H, m), 1.55 (9H, s), 5.48 (1H, q, J=7.1Hz), 3.68 (3H, s), 3.33 (1H, d, J=10.3 Hz),
1.31 (3H, d, J=6.6Hz). MS (ESI) m/z: 365 (M+H)+. HR-MS 3.13 (1H, d, J=10.3Hz), 2.93 (1H, d, J=16.8Hz), 2.34–2.20
(ESI) m/z: 365.2000 (M+H)+ (Calcd for C20H30ClN2O2: (3H, m), 2.13–1.94 (2H, m), 1.51 (3H, d, J=7.1Hz), 1.32 (9H,
365.1996). IR (ATR) cm−1: 3428, 2971, 2927, 1716, 1481, 1453, s). MS (ESI) m/z: 376 (M+H)+. HR-MS (ESI) m/z: 376.2140
1365, 1160.
7-[(1R,5R)-1-Amino-5-chloro-3-azabicyclo[3.3.0]octan- 2977, 1740, 1712, 1670, 1304, 1170, 1151.
(M+H)+ (Calcd for C21H30NO5: 376.2124). IR (ATR) cm−1:
3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropan-1-yl]-1,4-
(1S,5R)-4,6-Dioxo-3-[(1R)-1-phenylethyl]-3-aza-
dihydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid (5) bicyclo[3.3.0]octan-1-ylcarboxylic Acid tert-Butyl Ester
Starting with 16b (63.0mg, 0.17mmol) and following the (18) (3S)-3-(2-Methoxycarbonyl-1-ethyl)-5-oxo-1-[(1R)-1-
procedure for the preparation of 17a gave 17b as a color- phenylethyl]pyrrolidine-3-carboxylic acid tert-butyl ester
less oil. No further purification was attempted on this com- (10, 35.0g, 93.2mmol) was dissolved in THF (1L). A 2M
pound, which was used directly in the next step. Starting lithium diisopropylamide–heptane–THF–ethylbenzene solu-
with 17b and following the procedure for the preparation of tion (100mL, 200mmol) was added dropwise in a nitrogen
4 afforded 5 (33.9mg, 41% from 16b) as a colorless pow- atmosphere at an internal temperature of −69°C over 30min.
der. mp: 161–163°C. [α]D24 +100.8° (c=0.104, 0.1 N NaOH). After stirring at the same temperature for 1h, the reaction
1H-NMR (400MHz, 0.1N NaOD) δ: 8.48 (1H, s), 7.70 (1H, solution was poured into a 1N hydrochloric acid solution (2L)
d, J=14.2Hz), 5.07–4.85 (1H, m), 4.19–4.08 (2H, m), 3.92 in an ice bath, followed by extraction with ethyl acetate (2L,
(1H, d, J=11.8Hz), 3.76–3.63 (5H, m), 2.48–2.41 (1H, m), then 1L). The organic layers were combined, washed with
2.30–2.25 (1H, m), 2.08–1.87 (4H, m), 1.70–1.51 (2H, m), 1.19 brine, and dried over anhydrous sodium sulfate. After filtra-
(1.8H, t, J=7.2Hz). Anal. Calcd for C21H22ClF4N3O3·0.6EtOH: tion, the filtrate was concentrated under reduced pressure,
C, 55.38%; H, 5.36%; N, 8.73%; F, 7.89%; Cl, 7.36%. Found: and the precipitated solid was collected by filtration to give
C, 55.24%, H, 4.91%; N, 8.85%; F, 8.27%; Cl, 6.92%. MS 22.2g (69%) of the title compound 18 as pale red crystals.
(ESI) m/z: 454 (M+H)+. HR-MS (ESI) m/z: 454.1359 The filtrate was further concentrated to give 2.88g of the title
(M+H)+ (Calcd for C21H23ClF2N3O4: 454.1345). IR (ATR) compound as pale red crystals. The filtrate was concentrated
cm−1: 3384, 3075, 2880, 1728, 1621, 1513, 1453, 1360, 1318, under reduced pressure, and the residue was subjected to silica
1188, 1136, 1120, 1103, 1056.
(3S)-3-(2-Methoxycarbonyl-1-ethyl)-5-oxo-1-[(1R)-1- give 2.09g (6.5%) of the title compound 18 as colorless crys-
phenylethyl]pyrrolidine-3-carboxylic Acid
tert-Butyl tals. [α]D25 −46.5° (c=1.009, MeOH). 1H-NMR (400MHz,
gel column chromatography (hexane–ethyl acetate=1:1) to
Ester (10) Carbon tetrachloride (550mL), acetonitrile CDCl3) δ: 7.37–7.26 (5H, m), 5.50 (1H, q, J=7.1Hz), 3.38
(550mL), and water (550mL) were dissolved in (3S)-3-(3- (1H, d, J=10.5Hz), 3.23 (1H, d, J=10.5Hz), 2.55–2.35 (3H,
hydroxy-1-propyl)-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3- m), 2.05–1.94 (1H, m), 1.53 (3H, d, J=7.1Hz), 1.38 (9H, s).
carboxylic acid tert-butyl ester (8, 38.2g, 0.110mol), and MS (ESI) m/z: 344 (M+H)+. HR-MS (ESI) m/z: 344.1870
ruthenium (III) chloride hydrate (456mg, 2.20mmol) and (M+H)+ (Calcd for C20H26NO4: 344.1862). IR (ATR) cm−1:
sodium periodide (94.1g, 0.440mol) were sequentially added. 3442, 2975, 1742, 1732, 1671, 1424, 1366, 1261, 1244, 1221,
The mixture was stirred in a water bath with a thermostatic 1170, 1155, 1144.
circulator at 15°C for 2.5h while maintaining the internal
(1S,5R)-5-Methyl-4,6-dioxo-3-[(1R)-1-phenylethyl]-3-
temperature at 20–25°C. The reaction solution was cooled in azabicyclo[3.3.0]octan-1-ylcarboxylic Acid tert-Butyl Ester

56
Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
(19) DMF (2.0mL) was added to sodium hydride 1H-NMR (400MHz, CDCl3) δ: 7.39–7.24 (5H, m), 5.56–5.44
(152mg, 3.48mmol) in an argon atmosphere. A solution of (1H, m), 3.64 (3H, s), 3.51 (1H, d, J=10.05Hz), 3.02–2.96
(1S,5R)-4,6-dioxo-3-[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]- (1H, m), 2.49–2.26 (2H, m), 1.91–1.44 (4H, m), 1.52 (3H, d,
octan-1-ylcarboxylic acid tert-butyl ester (18, 1.00g, J=7.35Hz), 1.12 (3H, s).
2.91mmol) in DMF (8.0mL) was added dropwise to this sus-
(1S,5S)-1-(tert-Butoxycarbonylamino)-5-methyl-4-
pension under ice-cooling, and the mixture was stirred at 0°C oxo-3-[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octane
for 30min. Subsequently, methyl iodide (0.217mL, 3.49mmol) (22) (1S,5R)-5-Methyl-4-oxo-3-[(1R)-1-phenylethyl]-3-
was added dropwise under ice-cooling, and the mixture was azabicyclo[3.3.0]octan-1-ylcarboxylic acid methyl ester (21,
stirred at room temperature for 2.5h. The reaction solu- 130mg, 0.43mmol) was dissolved in methanol (5.0mL). A
tion was ice-cooled, and then the reaction was quenched 1N NaOH solution (1.5mL) was added dropwise under ice-
with water, followed by extraction with ethyl acetate. The cooling, and the mixture was stirred at room temperature for
organic layer was then washed with water and brine, dried 4h. Then, a 1N NaOH solution (1.5mL) was added dropwise,
over anhydrous sodium sulfate, and filtered. The solvent was and the mixture was stirred at room temperature for 15h.
evaporated under reduced pressure, and the residue was sub- NaOH (93mg) was again added, and the mixture was stirred
jected to silica gel column chromatography (hexane–ethyl at room temperature for 6h. NaOH (90mg) was added again,
acetate=2:1–1:1) to give 0.79g (76%) of the title compound and the mixture was stirred at room temperature for 4h, and
19 as a pale yellow solid. No further purification was at- then at 50°C for 1h. The reaction solution was made weakly
tempted on this compound, which was used directly in the acidic with hydrochloric acid, and the solvent was evaporated
1
next step. H-NMR (400MHz, CDCl3) δ: 7.41–7.23 (5H, m), under reduced pressure. The resulting residue was extracted
5.48 (1H, q, J=6.62Hz), 3.40 (1H, d, J=10.54Hz), 3.13 (1H, with DCM and dilute hydrochloric acid. The organic layer
d, J=10.54Hz), 2.63–2.40 (3H, m), 1.96–1.83 (1H, m), 1.54 was dried over anhydrous sodium sulfate and filtered, and
(3H, d, J=7.11Hz), 1.39 (9H, s), 1.22 (3H, s).
then the solvent was evaporated under reduced pressure.
(1S,5R)-6,6-Ethanediyldimercapto-5-methyl-4-oxo- The resulting residue was dissolved in toluene (5.0mL). Tri-
3-[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octan-1-ylcarbox- ethylamine (0.132mL, 0.95mmol) and diphenylphosphoryl
ylic Acid Methyl Ester (20) (1S,5R)-5-Methyl-4,6-dioxo-3- azide (0.111mL, 0.52mmol) were added, and the mixture was
[(1R)-1-phenylethyl]-3-azabicyclo[3.3.0]octan-1-ylcarboxylic heated to reflux for 3h. The reaction solution was extracted
acid tert-butyl ester (19, 0.28g, 0.78mmol) was dissolved in with ethyl acetate and saturated aqueous sodium bicarbon-
toluene (14mL). Toluenesulfonic acid monohydrate (155mg, ate. Then, the organic layer was washed with brine, dried
0.81mmol) and ethanedithiol (0.14mL, 1.7mmol) were added, over anhydrous sodium sulfate, and filtered. The solvent was
and the mixture was heated to reflux for 9h. The solvent evaporated under reduced pressure. 1,4-Dioxane (2.0mL) and
was evaporated under reduced pressure, and the resulting 6N hydrochloric acid (2.0mL) were added to the resulting
residue was subjected to silica gel column chromatography residue, and the mixture was stirred at 50°C for 15h. After
(DCM–methanol=98:2) to give the target 1-position carbox- extraction with water and ethyl acetate, the aqueous layer was
ylic acid (289mg) as a pale yellow solid. The carboxylic acid made alkaline with a saturated NaOH solution and extracted
(289mg) was dissolved in THF (10mL) and methanol (3.0mL). with chloroform twice. The organic layers were combined,
Trimethylsilyldiazomethane (1.7mL) was added under ice- dried over anhydrous sodium sulfate, and filtered, and then
cooling, and the mixture was stirred at room temperature the solvent was evaporated under reduced pressure. Toluene
for 2h. The solvent was evaporated under reduced pressure, (3.0mL) and Red-Al™ (65% solution in toluene, 0.50mL) were
and the resulting residue was subjected to silica gel column sequentially added to the resulting residue, and the mixture
chromatography (hexane–ethyl acetate=3:2) to give 267mg was stirred at 80°C for 2.5h. A 3N NaOH solution was added
(87%) of the title compound 20 as a pale yellow solid. No fur- to the reaction solution under ice-cooling, and the layers were
ther purification was attempted on this compound, which was separated with toluene. The organic layer was dried over an-
1
used directly in the next step. H-NMR (400MHz, CDCl3) δ: hydrous sodium sulfate and filtered, and then the solvent was
7.39–7.23 (5H, m), 5.52 (1H, q, J=6.86Hz), 3.60 (3H, s), 3.55 evaporated under reduced pressure. The resulting residue was
(1H, d, J=10.05Hz), 3.44–3.31 (1H, m), 3.28–3.19 (1H, m), dissolved in DCM (10mL) and methanol (5.0mL). Di-tert-
3.16 (1H, d, J=10.05Hz), 2.79–2.70 (1H, m), 2.54–2.44 (1H, butyl dicarbonate (560mg, 2.57mmol) was added, and the
m), 2.26–2.15 (1H, m), 1.81–1.70 (1H, m), 1.59–1.52 (2H, m), mixture was stirred at room temperature for 16h. The reaction
1.56 (3H, d, J=7.11Hz), 1.33 (3H, s).
solution was subjected to silica gel column chromatography
(1S,5R)-5-Methyl-4-oxo-3-[(1R)-1-phenylethyl]-3-azabi- (DCM–methanol=98:2) to give 77mg (52%) of the title com-
cyclo[3.3.0]octan-1-ylcarboxylic Acid Methyl Ester (21) pound 22 as a pale yellow solid. No further purification was
(1S,5R)-6,6-Ethanediyldimercapto-5-methyl-4-oxo-3-[(1R)-1- attempted on this compound, which was used directly in the
1
phenylethyl]-3-azabicyclo[3.3.0]octan-1-ylcarboxylic
acid next step. H-NMR (400MHz, CDCl3) δ: 7.33–7.16 (5H, m),
methyl ester (20, 266mg, 0.68mmol) was dissolved in etha- 4.79 (1H, brs), 3.17–3.00 (1H, m), 2.74–2.58 (2H, m), 2.53–2.44
nol (10mL). Raney nickel (2.0mL) was added dropwise, and (1H, m), 2.27–2.13 (1H, m), 2.08–1.89 (2H, m), 1.74–1.62 (2H,
the mixture was heated to reflux for 5.5h. The catalyst was m), 1.60–1.24 (2H, m), 1.41 (9H, s), 1.28 (3H, d, J=6.59Hz),
removed by filtration, and then the filtrate was concentrated 1.07 (3H, s).
under reduced pressure. The resulting residue was subjected
to silica gel column chromatography (hexane–ethyl ace- 3-azabicyclo[3.3.0]octane
(1S,5S)-1-(tert-Butoxycarbonylamino)-5-methyl-4-oxo-
(23) (1S,5S)-1-(tert-Butoxyc
tate=7:3–1:1) to give 133mg (65%) of the title compound 21 arbonylamino)-5-methyl-4-oxo-3-[(1R)-1-phenylethyl]-3-
as a pale yellow solid. No further purification was attempted azabicyclo[3.3.0]octane (22, 77mg, 0.22mmol) was dissolved
on this compound, which was used directly in the next step. in ethanol (6.0mL). 10% palladium–carbon (50% wet, 69mg)

Vol. 67, No. 1 (2019)
Chem. Pharm. Bull.
57
was added, and the mixture was stirred in a hydrogen at- reduced pressure, and ethyl acetate and water were added
mosphere at 45°C for 19.5h. After removing the catalyst by to the residue to separate the layers. The organic layer was
filtration, the filtrate was concentrated under reduced pressure washed with brine and dried over anhydrous sodium sulfate,
to give 50mg (95%) of the title compound 23 as a pale yellow and the solvent was evaporated under reduced pressure. The
oil. No further purification was attempted on this compound, residue was subjected to flash column chromatography (hex-
1
which was used directly in the next step. H-NMR (400MHz, ane–ethyl acetate=9:1–1:1–1:2) to give the title compound
CDCl3) δ: 4.68 (1H, brs), 3.36–3.19 (1H, m), 3.00–2.58 (4H, (25, 2.98g, 26%) as a colorless powder. mp: 191–195°C (dec.).
1
m), 2.40–1.89 (4H, m), 1.81–1.26 (2H, m), 1.44 (9H, s), 1.07 [α]D25 −166.5° (c=0.159, CHCl3). H-NMR (CDCl3) δ: 8.56
(3H, s).
(1H, d, J=3.2Hz), 8.06 (1H, d, J=8.1Hz), 4.98–4.73 (1H,
7-[(1R,5R)-1-Amino-3-aza-5-methylbicyclo[3.3.0]octan- m), 4.40 (2H, q, J=7.1Hz), 3.91–3.82 (1H, m), 2.85 (3H, s),
3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-1,4-di- 1.61–1.22 (2H, m), 1.41 (3H, t, J=7.1Hz). MS (ESI) m/z: 386
hydro-8-methoxy-4-oxoquinoline-3-carboxylic Acid (6) (M+H)+. HR-MS (ESI) m/z: 386.0229 (M+H)+ (Calcd for
Starting with 23 (79.1mg, 0.22mmol) and following the proce- C16H15BrF2NO3: 386.0203). IR (ATR) cm−1: 3083, 2976, 1724,
dure for the preparation of 4 afforded 6 (47mg, 46%) as a pale 1624, 1610, 1456, 1404, 1387, 1312, 1239, 1173, 1126, 1047,
yellow solid. mp: 109–113°C (dec.). [α]D24 +78.2° (c=0.135, 1031, 1022, 1006.
1
0.1 N NaOH). H-NMR (400MHz, 0.1N NaOD) δ: 8.45 (1H,
7-[(1R,5S)-1-Amino-5-fluoro-3-azabicyclo[3.3.0]octan-
s), 7.66 (1H, d, J=14.5Hz), 4.79–4.85 (1H, m), 4.00–4.10 3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-1,4-dihydro-
(1H, m), 3.61 (3H, s), 3.51–3.75 (3H, m), 3.34–3.44 (1H, m), 8-methyl-4-oxoquinoline-3-carboxylic Acid Hydrochloride
1.94–2.07 (1H, m), 1.43–1.93 (7H, m), 1.10 (3H, s). MS (FAB) Dihydrate (7) To a solution of tris(dibenzylideneacetone)-
m/z: 434 (M+H)+. Anal. Calcd for C22H25F2N3O4·2H2O: C, dipalladium (0) (7.75g, 8.46mmol), 4,5-bis(diphenyl)phosphino-
56.28%; H, 6.23%; F, 8.09%; N, 8.95/%. Found: C, 56.57%; H, 9,9-dimethylxanthene (14.7g, 25.4mmol), ethyl 7-bromo-
6.24%; F, 8.19%; N, 9.01%. IR (ATR) cm−1: 2942, 2877, 1612, 6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-
1573, 1448, 1434, 1392, 1349, 1342, 1311, 1301, 1290, 1272, 4-oxoquinoline-3-carboxylate (25, 14.2g, 36.8mmol) and
821, 804.
(1R,5S)-1-(tert-butoxycarbonyl amino)-5-fluoro-3-azabicyclo-
Ethyl
2-(4-Bromo-2,5-difluoro-3-methylbenzoyl)-3-di- [3.3.0]octane (17a, 6.90g, 28.2mmol) in 1,4-dioxane (345mL)
methylaminoacrylate (27) 2,5-Difluoro-4-bromo-3-methyl- was added cesium carbonate (18.4g, 56.5mmol), and the
benzoic acid (26, 10.7g, 42.4mmol) was dissolved in toluene mixture was stirred at 110°C for 23h under a nitrogen atmo-
(160mL). Thionyl chloride (5.00mL, 63.9mmol) and DMF sphere. The reaction mixture was diluted with water (400mL)
(5.0mL) were added, and the mixture was heated to reflux and extracted with ethyl acetate (600mL×1, 250mL×1).
for 2h. The solvent was evaporated under reduced pressure, The organic layer was dried with anhydrous sodium sulfate
and the residue was dissolved in THF (300mL). Ethyl 3-di- and concentrated under reduced pressure. The residue was
methylaminoacrylate (7.30mL, 50.9mmol) and triethylamine purified by silica gel column chromatography (chloroform–
(7.60mL, 54.5mmol) were added, and the mixture was heated methanol=100:0–99:1) to afford a solid. To a solution of
to reflux for 3h. The solvent was evaporated under reduced this solid in ethanol (273mL) was added a 1N aqueous solu-
pressure, and DCM and water were added to the residue to tion of NaOH (68.2mL) in an ice bath, and the mixture was
separate the layers. Then, the organic layer was dried over an- stirred at room temperature for 1h. To this reaction solution
hydrous sodium sulfate, and the solvent was evaporated under was added ethanol (327mL) and a 1N aqueous solution of
reduced pressure. The residue was subjected to flash column NaOH (27.3mL), and the mixture was stirred at room tem-
chromatography (hexane–ethyl acetate=2:1–1:1–1:2) to give perature for 13h. To this reaction solution was added a 1N
the title compound (27, 11.4g, 71%) as a yellow oil. No further aqueous solution of hydrochloric acid (95.5mL) in an ice bath,
purification was attempted on this compound, which was used and the organic layer was concentrated under reduced pres-
1
directly in the next step. H-NMR (CDCl3) δ: 7.81–7.74 (1H, sure. The aqueous solution was diluted with water (150mL)
m), 7.27–7.16 (1H, m), 4.00 (2H, q, J=7.1Hz), 3.31 (3H, brs), and extracted with chloroform (250mL×1, 150 mL×1). The
2.89 (3H, brs), 2.35 (3H, d, J=2.9Hz), 0.97 (3H, t, J=7.1 Hz). organic layer was dried with anhydrous sodium sulfate and
Ethyl 7-Bromo-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]- concentrated under reduced pressure. The residue was puri-
1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylate
(25) fied by silica gel column chromatography (n-hexane–ethyl
Ethyl 2-(4-bromo-2,5-difluoro-3-methylbenzoyl)-3-dimethyl- acetate=100:0–2:1–1:5–0:100) to afford a pale yellow
aminoacrylate (27, 11.4g, 30.2mmol) was dissolved in DCM solid. The solid was dissolved in concentrated hydrochloric
(200mL). (1R,2S)-2-Fluorocyclopropylamine tosylate (8.24g, acid (30mL) in an ice bath, and the aqueous solution was
33.3mmol) was added, and the mixture was cooled to −25°C. washed with chloroform (100mL×5). To the aqueous layer
Triethylamine (6.60mL, 47.4mmol) was added dropwise to was added a saturated solution of NaOH to adjust the pH to
the reaction solution at −25°C, and the mixture was stirred 12.0. To the solution was added water (1.8L), and the pH of
at −15°C for 1h and at 0°C for 2.5h. The solvent was evapo- the basic aqueous solution was adjusted with hydrochloric
rated under reduced pressure, and ethyl acetate and water acid to pH 7.4. The solution was extracted with chloroform
were added to the residue to separate the layers. The organic (1.5 L×1, 800mL×1). The organic layer was dried with
layer was washed with brine and dried over anhydrous sodium anhydrous sodium sulfate and concentrated under reduced
sulfate. The solvent was evaporated under reduced pressure to pressure. The residue was purified by recrystallization from
give an aminoacrylate as a yellow oil. The resulting amino- 2-propanol–methanol=10:1 to afford the crude (6.14g,
acrylate was dissolved in DMF (350mL). Cesium carbonate 52%)
of
7-[(1R,5S)-1-amino-5-fluoro-3-azabicyclo[3.3.0]-
(19.8g, 60.9mmol) was added, and the mixture was stirred at octan-3-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropane]-1,4-
room temperature for 12h. The solvent was evaporated under dihydro-8-methyl-4-oxoquinoline-3-carboxylic acid.

58
Chem. Pharm. Bull.
Vol. 67, No. 1 (2019)
Subsequently, the crude product (3.05g, 7.24mmol) was murine RTI model, bacteria were intranasally inoculated in
dissolved in 1N hydrochloric acid (7.19mL). The reaction mix- six-week-old male CBA/JNCrlj mice. The mice (n=5/group)
ture was stirred at 40°C for 30min. The mixture was diluted were subcutaneously injected with the dissolved compounds at
with water (7mL) and distilled under reduced pressure to af- 2 and 8h after inoculation. For the rodent UTI model, bacteria
ford 3.48g of crude product. This (1.16g) was recrystallized were transurethrally inoculated into the bladders of seven-
from 10% H2O–isopropanol to afford 0.87g (73%) of the title week-old female Crl:CD(SD)(IGS) rats and then, the urethrae
compound 7 as colorless powder. mp: 227–230°C (dec.). [α]D24 were clamped for 2h to prevent urine flow. The rats (n=5/
−153.9° (c=0.104, 0.1 N NaOH). 1H-NMR (400MHz, 0.1N group) were infused with the dissolved compounds through
NaOD) δ: 8.49 (1H, s), 7.72 (1H, d, J=13.8Hz), 5.10–4.90 the tail vein for 2h at 4h post-inoculation. The bacterial num-
(1H, m), 4.15–4.05 (1H, m), 3.95–3.80 (1H, m), 3.60–3.40 bers in the lungs (mice) or kidneys (rats) were examined on
(2H, m), 3.35 (1H, d, J=10.5Hz), 2.63 (3H, s), 2.00–2.15 the day following the inoculations.
(3H, m), 1.90–1.80 (1H, m), 1.80–1.55 (3H, m), 1.35–1.20
(1H, m). MS (FAB) m/z: 422 (M+H)+. Anal. Calcd for
Acknowledgments We thank Dr. Kiyoshi Takasuna and
C21H22F3N3O3·HCl·2H2O: C, 51.07%; H, 5.51%; F, 11.54%; Dr. Koichi Goto for assistance with the safety assessment, Ms.
N, 8.51%; Cl, 7.18%. Found: C, 50.85%; H, 5.39%; F, 11.51%; Megumi Chiba, Dr. Ryo Okumura, Dr. Saito Higuchi, and Dr.
N, 8.48%; Cl, 7.31%. IR (ATR) cm−1: 3477, 3333, 2861, 1696, Yuichi Kurosaka for assistance with biological testing. We are
1616, 1519, 1450, 1373, 1361, 1317, 1138, 1124, 1026, 981, 969, grateful to Dr. Makoto Takemura for helpful discussion.
805.
In Vitro Antibacterial Activity The MICs of the com-
Conflict of Interest The authors declare no conflict of
pounds tested in this study were determined by the 2-fold interest.
microdilution method using Mueller–Hinton broth (Difco
Laboratories, Detroit, MI, U.S.A.) with an inoculum size of
approximately 105 colony forming unit (CFU) per well. The
MIC was defined as the lowest concentration that prevented
visible bacterial growth after incubation at 35°C for 18h.
Animal Experiments The care and use of animals and
the experimental protocols were approved by the Experimen-
tal Animal Care and Use Committee of Daiichi Sankyo Co.,
Ltd.
Single IV Dose Toxicity The test compounds were dis-
solved in 0.1N NaOH in saline at various concentrations.
Solutions were administered IV to male Slc:ddY mice (six
weeks old) at dose levels of 50, 100, and 150mg/kg (10mL/kg,
0.2mL/min). The number of dead mice was counted on day 7.
Bone Marrow Micronucleus Test The test compounds
were dissolved in 0.1N NaOH in saline at different concentra-
tions. The solution was administered intravenously to male
Slc:ddY mice (six weeks old) at dose levels of 50, 100, and
150mg/kg (10mL/kg, 0.2mL/min). At 24 and 48h after dosing
of the compounds, ca. 5µL of peripheral blood was collected
from the tail vein of each mouse. The blood was dropped onto
an acridine orange-coated glass slide and covered immediately
References and Notes
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2) Rice L. B., J. Infect. Dis., 197, 1079–1081 (2008).
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D., Rice L. B., Scheld M., Spellberg B., Bartlett J., Clin. Infect. Dis.,
48, 1–12 (2009).
4) Larkin M., Lancet Infect. Dis., 3, 322 (2003).
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used. The animals were administered drug samples at a single
IV dose (5mg/kg) as an aqueous solution. The concentrations
of the compounds were determined by a microbiological assay
(agar well dilution method) using B. subtilis ATCC6051. The
mean values of four rats are reported.
In Vivo Antibacterial Activity S. pneumoniae GE01085
and E. coli GK00432 were used as causative pathogens for the
models of murine RTI and rodent UTI, respectively. For the