Discovery of WQ-3810: Design, synthesis, and evaluation of 7-(3-alkylaminoazetidin-1-yl)fluoro-quinolones as orally active antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2015.08.015

Novel 7-(3-alkylaminoazetidin-1-yl)fluoroquinolones were designed, synthesized, and evaluated for their antibacterial activities and oral absorption rates. Against Gram-negative bacteria, 10a-e, which have various alkyl groups containing different numbers

Full text of DOI:10.1016/j.ejmech.2015.08.015

Accepted Manuscript
Discovery of WQ-3810: Design, Synthesis, and Evaluation of 7-(3-
alkylaminoazetidin-1-yl)fluoro-quinolones as Orally Active Antibacterial Agents
Kenji Itoh, Yasuhiro Kuramoto, Hirotaka Amano, Daichi Kazamori, Akira Yazaki
PII:
S0223-5234(15)30199-9
10.1016/j.ejmech.2015.08.015
EJMECH 8051
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 July 2015
Accepted Date: 6 August 2015
Please cite this article as: K. Itoh, Y. Kuramoto, H. Amano, D. Kazamori, A. Yazaki, Discovery of
WQ-3810: Design, Synthesis, and Evaluation of 7-(3-alkylaminoazetidin-1-yl)fluoro-quinolones as
Orally Active Antibacterial Agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.08.015.
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ACCEPTED MANUSCRIPT
O
Novel
7-(3-alkylaminoazetidin-1-yl)fluoroquinolones
were
COOH
N-1 position
F
10a: R = H
10b: R = Me
10c: R = Et
10d: R = n-Pr
10e: R = i-Pr (WQ-3810)
10f: R = t-Bu
designed, synthesized, and evaluated for their antibacterial
activities and oral absorption rates. In this series of compounds,
WQ-3810 was identified as an orally active fluoroquinolone with a
potent in vitro activity.
C-7 position
N
N
F
R
N
H
F
N
H2N

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Discovery of WQ-3810: Design, Synthesis, and Evaluation of
7-(3-alkylaminoazetidin-1-yl)fluoro-quinolones as Orally Active
Antibacterial Agents
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Kenji Itoh a,*, Yasuhiro Kuramoto a, Hirotaka Amano a, Daichi Kazamori a, Akira Yazaki a
a
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Drug Discovery Laboratory, Wakunaga Pharmaceutical Co., Ltd., 1624 Shimokotachi,
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Koda-cho, Akitakata-shi, Hiroshima 739-1195, Japan
* Corresponding author.
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E-mail address: itoh_k@wakunaga.co.jp (Kenji Itoh)
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Kewords
Antibacterial agent; Fluoroquinolone; Gram-negative bacteria; Oral absorption;
Alkylaminoazetidine.
Abstract
Novel 7-(3-alkylaminoazetidin-1-yl)fluoroquinolones were designed, synthesized, and
evaluated for their antibacterial activities and oral absorption rates. Against Gram-negative
bacteria, 10a-e, which have various alkyl groups containing different numbers of carbon
atoms (C0-C3) at the C-7 alkylaminoazetidine position, showed potent and similar
antibacterial activities, whereas the activity of 10f (C4,
t-Bu) was significantly lower than
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those of 10a-e. Conversely, the oral absorption rates of 10a-e in rats increased depending on
the number of carbon atoms in the alkyl groups; 10d (C3, n-Pr) and 10e (C3, i-Pr) had high
oral absorption rates (> 90 % at 10 mg/kg). These results demonstrated that the introduction
of alkyl groups onto C-7 aminoazetidine is useful for the improvement of the oral absorption
rates of these drugs while maintaining their antibacterial activities. As a conclusion, from
this series of fluoroquinolones, WQ-3810 (10e), having 3-isopropylaminoazetidine as the C-7
substituent, was identified as an orally active antibacterial agent with a potent in vitro
activity.
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1. Introduction
Since the discovery of norfloxacin as the first fluoroquinolone (FQ) [1], FQs have become a
significant class of useful antibacterial agents. In an effort to improve the profiles of FQs as
antibacterial drugs, a number of structure-activity relationships (SARs) have been
established and reviewed [2-7]. In those SARs, aminopyrrolidines and piperazines were
identified as the most effective C-7 substituents to enhance the antibacterial activities of FQs
[2, 5]. The introduction of alkyl groups onto those C-7 substituents has also enhanced their
antibacterial activities against Gram-positive bacteria [8], improved their pharmacokinetic
profiles [5], and reduced their cytotoxicities against mammalian cells [7]. Furthermore, a
recent study reported that the introduction of a methyl group at the geminal position of the
amino group of C-7 aminopyrrolidine was effective for avoiding the mechanism-based
inhibition of cytochrome P450 3A4 [9].
In our effort to create a useful FQ with a potent in vitro activity against refractory
drug-resistant pathogens, we discovered the highly active compound (10a), whose structure is
characterized by its unique N-1 and C-7 substituents, 2,4-difluoro-5-aminopyridine and
3-aminoazetidine, respectively. In the course of SAR studies of this series of FQs, we
accumulated several important findings as follows: the introduction of an amino group in the
N-1 substituent resulted in the reduction of the phototoxicity, even when groups (halogen
atoms) that induce phototoxicity were introduced at the C-8 position [10]; a four-membered
ring (aminoazetidine) substituent at the C-7 position gave a higher antibacterial activity than
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a five-membered ring (aminopyrrolidine) substituent in combination with the N-1 substituent
difluoro-aminopyridine; and the C-8 methyl group was effective for reducing the cytotoxicity
without the loss of in vitro activity [11]. However, the proto-type compound 10a has a defect in
its pharmacokinetic properties, its poor oral absorption. Until now, there have been few
studies evaluating the structure-pharmacokinetic relationships of FQs having a C-7
aminoazetidine. Jordi et al. [12-14] demonstrated that introduction of a methyl or ethyl group
to the C-7 aminoazetidine improved the pharmacokinetic properties; the area under the
plasma concentration-time curve (orally in mice) increased, but the antibacterial activity
against Gram-negative bacteria was reduced. It is unclear how longer alkyl groups (> C3)
effect the antibacterial activities and pharmacokinetics of 7-(3-alkylaminoazetidin-1-yl)FQs.
The aim of this study was to improve the oral absorption of 10a by introducing an alkyl
group and to clarify the effect of this introduction. To achieve this goal, we prepared various
compounds with different alkyl groups (C0-C4) on the C-7 alkylaminoazetidine (10a-f) and
evaluated their antibacterial activities, especially against Gram-negative bacteria, as well as
their oral absorption rates in rats. In addition, we investigated the effects of the alkyl groups
on the inhibitory activities of the drugs against DNA gyrase, which is one of the molecules
targeted by FQs, and the physiochemical properties (solubility and membrane permeability)
of the FQs.
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2. Chemistry
WQ-3810 10e and its derivatives 10a-d and f were synthesized according to previously
reported
methods
[15,
16].
Scheme
1
shows
the
synthesis
of
2-amino-6-tert-butylamino-3,5-difluoropyridine (4) as a N-1 substituent of 10a-f. Stepwise
aminations of 2,3,5,6-tetrafluoropyridine (1) with tert-butylamine and benzylamine gave
di-protected diaminopyridine (3). The second amination with benzylamine required a high
reaction temperature (160°C) due to the low activity for the amination at the C-6 position of 2.
The benzyl group of 3 was deprotected selectively by Pd/C-catalyzed hydrogenation to give the
mono-protected
diaminopyridine
4.
Scheme
2
shows
the
synthesis
of
1-(3,5-difluoro-6-aminopyridin-2-yl)-8-methylquinolone carboxylic acid (8). The reaction of
benzoylaceate (5) with triethyl orthoformate under reflux conditions and the removal of
excess reagents gave benzoylacrylate (6). The reaction of 6 with 4 and subsequent cyclization
under basic conditions gave quinolone ester 7. To obtain 8-methylquinolone carboxylic acid (8),
the deprotection of the tert-butyl group and the hydrolysis of the ethyl ester of 7 were
achieved under acidic conditions.
As shown in scheme 3, compounds 10a-f were obtained by aromatic nucleophilic
substitution at the C-7 position of 8-methylquinolone carboxylic acid (8) with various
3-alkylaminoazetidines 9a-f, which were synthesized according to a method similar to
established procedures [17, 18]. The reactivity at the C-7 position of 8 is low due to the
electron donating effect of the C-8 methyl group. We already reported that the C-7
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substitution reaction of an 8-methylquinolone core with a 3-alkylaminoazetidine was
accelerated by the presence of Li+ ions [19]. Accordingly, the syntheses of 10a-f were conducted
in the presence of LiCl and/or LiOH
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3. Results
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3.1 In vitro activity
The half maximal inhibitory concentration (IC50) and the minimum inhibitory
concentration (MIC) values of 10a-f and levofloxacin (LVFX) were determined for Escherichia
coli DNA gyrase and Gram-negative bacteria (three strains of E. coli and 15 strains of 11 other
Gram-negative bacteria), respectively (Table 1). 10a-f have various alkyl groups of different
lengths and sizes on the C-7 alkylaminoazetidine. These compounds similarly inhibited E. coli
DNA gyrase potently with IC50 values ranging from 0.078 to 0.33 mg/L. Conversely, the
antibacterial activities of 10a-e were higher than that of 10f; the MIC values for E coli (3
strains) and other Gram-negative bacteria (11 species, 15 strains) were from 0.004 to 0.012
(10a-e) and 0.038 mg/L (10f) and from 0.061 to 0.21 (10a-e) and 0.57 mg/L (10f), respectively.
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3.2 Oral absorption in rats
The urinary and biliary excretions (% of dose) of 10a-e were measured following their oral
(p.o.) or intravenous (i.v.) administration to rats (Table 2). The excretions of 10a-e into bile
were analyzed after the conversion of glucuronide metabolites of 10a-e into their
unmetabolized forms by alkaline hydrolysis. The total excretions of 10d (C3,
n-Pr) and 10e
(C3, i-Pr) in urine and bile after oral administration were 91 and 92 % of the dose, respectively,
indicating their excellent oral absorption rates (> 90%). The high total recoveries (85-91%) of
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10a-c following intravenous administration indicated that these compounds were primary
excreted in their unmetabolized forms or as glucuronide metabolites. Therefore, the oral
absorption rates (%) of 10a-c were calculated by dividing their total recoveries (p.o.) by the
total recoveries (i.v.). The calculated oral absorption rates of 10a (C0, non-alkylated), 10b (C1,
Me), and 10c (C2, Et) were 9.6, 31, and 76 %, respectively.
3.3 Solubility and membrane permeability
The solubilities and membrane permeabilities of 10a-e were measured (Table 3). The
solubilities in Japanese Pharmacopoeia disintegration test solution 2 (JP2) were not very
high and ranged from 3.1 to 45 mg/L. The permeability coefficients (Peffs) of 10d and 10e
in the parallel artificial membrane permeability assay (PAMPA) were 1.1 × 10-6 and 1.2 ×
10-6 cm/s, respectively, which were four to 10 times higher than those of 10a-c (1.2 × 10-7 to
2.9 × 10-7 cm/s).
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4 Discussion
In this study, we attempted to improve the oral absorption of our proto-type FQ 10a by
performing several SAR studies. We determined the MICs against clinically important
Gram-negative pathogens (18 strains of 12 species) and the oral absorption rates (in rats) of
10a-f, which have various C-7 alkylaminoazetidine substituents (C0-4). Additionally, we
evaluated their inhibitory activities against DNA gyrase, solubilities in JP2, and membrane
permeabilities in a PAMPA study. DNA gyrase is the primary drug target of FQs [20], and the
solubility and membrane permeability in the gastrointestinal tract are two major determining
factors for the oral absorption of a drug.
In the MIC assay, the antibacterial activities of 10a-e, containing a C0-C3 alkyl group, were
found to be similar against Gram-negative bacteria; however, 10f (C4, t-Bu) was significantly
less potent (Table 1). To address the reason for the differences between the antibacterial
activities of 10a-e and 10f, we measured the IC50 values of 10a-f against E. coli DNA gyrase
and examined the correlation between their IC50s and MICs for E. coli (three strains).
Contrary to the higher MIC value of 10f compared to those of 10a-e, 10a-f exhibited similar
inhibition rates of E. coli DNA gyrase, although a good correlation between the IC50 and MIC
values of the FQs was reported [21]. This result suggested that the less potent antibacterial
activity of 10f was not due to its weaker inhibitory activity against the target enzyme.
To exert a potent antibacterial activity, a drug must have enough exposure to its target in
addition to strong inhibition of the target molecules. The bacterial uptake of drugs is greatly
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affected by components of the cell wall structure characteristic of bacteria. Especially,
Gram-negative bacteria have a hydrophilic outer membrane in the cell wall, which serves as
an effective permeability barrier against lipophilic antibiotics including FQs. The influence of
the lipophilicities of FQs on their uptake by Gram-negative bacteria has been studied [22-24],
and an inverse correlation between the lipophilicity of a FQ and its uptake by E. coli was
reported [24]. The cLogD values and the retention times of 10a-f (data not shown) for
reverse-phase HPLC indicated that the lipophilicities of 10a-f increased depending on the
number of carbon atoms in the alkyl groups, and 10f has the highest lipophilicity. Therefore,
the impaired bacterial uptake of 10f due to its higher lipophilicity might be a plausible reason
for its less potent antibacterial activity, although this speculation should be confirmed by the
future evaluation of the bacterial uptakes of 10a-f.
The oral absorption rates of 10a-e were evaluated in rats by measuring the total recoveries
(% of dose) of their unmetabolized forms and glucuronide metabolites. The total recoveries of
10d and 10e (p.o.) were over 90%, indicating that both compounds were almost completely
absorbed from the gastrointestinal tract. The oral absorption rates (%) of 10a-e increased with
an increase in the number of carbon atoms in the alkyl group. These results indicated that
introduction of alkyl groups at the C-7 aminoazetidine of 10a enhanced the oral absorption by
increasing the lipophilicities of the compounds. The oral absorption of drugs depends on their
physicochemical properties. Considering the process of the intestinal absorption of drugs, the
solubility of a drug in gastrointestinal tract fluid and its intestinal membrane permeability
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are two major determinants for its oral absorption. The dissolution of drugs in
gastrointestinal fluid is the first requirement; only drug molecules dissolved in the fluid can
be available for absorption across the intestinal membrane and thereafter be delivered to
their site of action by systemic circulation. Due to the lipid nature of the intestinal membrane,
a drug’s lipophilicity is considered to be an important factor for its penetration. To address the
reason for the improvement of the oral absorption of 10a by the introduction of the alkyl
groups, we evaluated the solubilities and permeabilities of 10a-e (Table 3). The solubility
study used JP2, which is one of the most commonly used test mediums as a simulated
gastrointestinal tract fluid. The solubilities of 10a-e in JP2 were not extremely high, and their
solubilities were all at a similar level. The membrane permeabilities were evaluated using a
PAMPA model, which has been reported to be useful for predicting the oral absorption rates of
drugs [25]. In this report, a sigmoidal relationship has been observed between the Peff value in
the PAMPA study and the oral absorption in humans; most of the drugs with Peff values
greater than 1.0 × 10-6 cm/s had good oral absorption rates. The Peff values of 10a-e increased
depending on the number of carbon atoms in the alkyl groups, and the values of 10d (1.1 × 10-6
cm/s) and 10e (1.2 × 10-6 cm/s) were greater than 1.0 × 10-6 cm/s (Table 3). These results
demonstrated that the oral absorption rates of 10a-e in rats was in good correlation with the
Peff values, suggesting that the introduction of alkyl groups to the C-7 aminoazetidine of 10a
improved the oral absorption through the enhancement of the membrane permeabilities of
the drugs due to their increased lipophilicities.
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5 Conclusion
This study evaluated the effects of various alkyl groups (C0-C4) of
7-(3-alkylaminoazetidin-1-yl)FQs 10a-f on the anti-Gram-negative activities and oral
absorption rates of the drugs. The alkyl group had little influence on the inhibitory
activity against E. coli DNA gyrase, but a t-Bu group (10f, C4) decreased the antibacterial
activity against Gram-negative bacteria, including E. coli, which might be due to its
reduced bacterial uptake due to an excessive increase in its lipophilicity. Conversely, the
increased lipophilicity by the introduction of alkyl groups enhanced oral absorption by
enhancing the membrane permeability. Consequently, WQ-3810 (10e, i-Pr) was identified
as an orally active FQ with a potent in vitro activity. Our subsequent evaluations have
demonstrated that WQ-3810 is highly active against quinolone-resistant pathogens and
has a good pharmacokinetic profile as well as a low potential for side effects related to
this class of antibiotics [26, 27].
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6 Experimental
6.1. Chemistry
6.1.1. General Methods
All chemicals used were of reagent grade. Reactions were monitored by thin-layer
chromatography on silica gel (Merck 60F 254) plates using UV light (254 nm) for detection.
NMR spectra were recorded with a JEOL JNM-ECP 500 at 500 MHz (1H NMR) and Varian
VNMRS 500 at 125 MHz (13C NMR). Chemical shifts are expressed in ppm (
δ) relative to
internal tetramethylsilane, and the coupling constants (
J values) are given in hertz (Hz).
Mass spectra (MS) were obtained with a Finnigan LTQ mass spectrometer with ESI.
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6.1.2. 2-(tert-Butylamino)-3,5,6-trifluoropyridine (2)
A mixture of 2,3,5,6-tetrafluoropyridine (1, 11.0 g, 72.8 mmol), tert-butylamine (18.5 g, 253
mmol) and MeCN (40 mL) was stirred at 60 °C for 3 d. The reaction mixture was evaporated,
and the residue was partitioned between CHCl3 (100 mL) and water (50 mL). The organic
layer was separated, dried over Na2SO4 and evaporated to give 2 as pale yellow oil. (9.7 g, 66%
yield); 1H NMR (CDCl3):
1H).
δ
1.45 (s, 9H), 4.40 (brs, 1H), 7.16 (ddd,
J
= 7.0 Hz, 8.0 Hz, 9.0 Hz,
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6.1.3. 2-Benzylamino-6-(tert-butylamino)-3,5-difluoropyridine (3)
A mixture of 2-(tert-butylamino)-3,5,6-trifluoropyridine (2, 9.7 g, 47.5 mmol), benzylamine
(15.5 g, 145 mmol) and
N-methylpyrrolidone (20 mL) was stirred at 160 °C for 1 d. After
cooling to room temperature, the reaction mixture was diluted with CHCl3. The solution was
washed with water (500 mL × 3), dried over Na2SO4 and evaporated to give crude 3 (16.5 g).
The obtained crude 3 was used for next reaction without further purification.
6.1.4. 2-Amino-6-(tert-butylamino)-3,5-difluoropyridine (4)
A mixture of crude 2-benzylamino-6-(tert-butylamino)-3,5-difluoropyridine (3, 10.7 g, 36.7
mmol), 10% Pd/C (1.1 g), conc. HCl (3.8g) and MeOH (60 mL) was stirred at room temperature
for 1 d under hydrogen atmosphere. After the catalyst was filtered off, the filtrate was
evaporated, and the residue was purified by silica gel column chromatography (2:1
1
CHCl3/hexane → CHCl3) to give 4 as pale brown oil. (3.3 g, 44% yield); H NMR (CDCl3):
δ
1.43 (s, 9H), 4.11 (brs, 2H), 6.94 (t,
J = 10.0 Hz, 1H).
6.1.5. Ethyl 1-[6-(tert-butylamino)-3,5-difluoropyridin-2-yl]-6,7-difluoro-8-methyl-4-oxo-
1,4-dihydroquinoline-3-carboxylate (7)
A mixture of ethyl 2,4,5-trifluoro-3-methylbenzoyl acetate (5, 3.4 g, 13 mmol), acetic
anhydride (3.2 g, 31 mmol) and triethyl orthoformate (2.3 g, 16 mmol) was refluxed for 4 h.
The reaction mixture was evaporated to give a crude 6, which was dissolved in EtOH (5 mL).
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To
the
solution
was
added
dropwise
a
solution
of
2-amino-6-(tert-butylamino)-3,5-difluoropyridine (4, 2.7 g, 13 mmol) in EtOH (20 mL) at 0 °C,
and the mixture was stirred at room temperature for 20 min. The reaction mixture was
evaporated, and the residue was subjected to silica gel column chromatography (1:8 ethyl
acetate/hexane). The fraction was evaporated, and the residue was dissolved in DMF (10 mL).
To the solution was added K2CO3 (1.4 g, 9.8 mmol), and the mixture was stirred at 100 °C for
50 min. The reaction mixture was partitioned between ethyl acetate and water, and the
organic layer was separated, dried over MgSO4 and evaporated. The residue was triturated
with EtOH, and the precipitate was collected by filtration and washed with Et2O to give 7 as a
pale yellow solid. (2.6 g, 44% yield); 1H NMR (CDCl3):
δ 1.34-1.48 (m, 12H), 1.82 (d, J = 3.0 Hz,
3H), 4.40 (q, J = 7.0 Hz, 2H), 4.75 (brs, 1H), 7.23 (t,
8.50 (s, 1H).
J = 9.0 Hz, 1H), 8.22 (t, J = 10.0 Hz, 1H),
6.1.6.
1-(6-Amino-3,5-difluoropyridin-2-yl)-6,7-difluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-c
arboxylic acid (8)
A
mixture
of
ethyl
1-[6-(tert-butylamino)-3,5-difluoropyridin-2-yl]-6,7-difluoro-8-methyl-4-oxo-1,4-dihydroquinol
ine-3-carboxylate (7, 2.5 g, 5.5 mmol) and conc. HCl (10 mL) was refluxed overnight. After
cooling to room temperature, the precipitate was collected by filtration and washed in turn
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with EtOH and Et2O to give 8 as a pale yellow solid. (1.7 g, 84% yield); 1H NMR (DMSO-
d6): δ
1.84 (s, 3H), 6.91 (brs, 2H), 8.03 (t,
J = 9.0 Hz, 1H), 8.25 (t, J = 9.0 Hz, 1H), 8.93 (s, 1H).
6.1.7.
7-(3-Aminoazetidin-1-yl)-1-(6-amino-3,5-difluoropyridin-2-yl)-6-fluoro-8-methyl-4-oxo-1,4
-dihydroquinoline-3-carboxylic acid (10a)
To a suspension of 3-aminoazetidine hydrochloride (9a, 283 mg, 2.0 mmol) in DMSO (2 mL)
were added LiOH monohydrate (165 mg, 3.9 mmol), tetramethylguanidine (300 mg, 2.6 mmol),
LiCl
(165
mg,
3.9
mmol)
and
1-(6-amino-3,5-difluoropyridin-2-yl)-6,7-difluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carbo
xylic acid (8, 477 mg, 1.3 mmol), and the mixture was stirred at 30-35 °C for 40 h. The
reaction mixture was washed with Et2O (5 mL × 5) by decantation to remove DMSO. To the
residue was added water (3 mL), and the pH of the suspension was adjusted to pH 6 with
aqueous 10% citric acid. The precipitate was collected by filtration, which was suspended in
water (10 mL), and the pH of suspension was adjusted to pH 6 with AcOH. The suspension
was stirred at 80 °C for 20 h. The precipitate was collected by filtration, which was suspended
in EtOH (10 mL), and the suspension was refluxed for 2 h. The precipitate was collected by
filtration, and dried under reduced pressure to give 10a as a white solid. (220 mg, 40% yield);
1H NMR (DMSO-
d6): δ 1.63 (s, 3H), 3.68-3.82 (m, 2H), 3.87-3.98 (m, 1H), 4.37-4.52 (m, 2H),
6.84 (brs, 2H), 7.76 (d,
J
= 13.5 Hz, 1H), 7.96 (dd,
J
= 9.5 Hz, 9.5 Hz, 1H), 8.71 (s, 1H); 13C
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NMR (DMSO-
d
6):
δ
17.73 (s), 44.04 (d, J = 5 Hz), 67.05 (s), 67.34 (s), 108.03 (s), 109.12 (d, J =
22 Hz), 113.03 (d,
J
= 4 Hz), 114.54 (dd, J = 24, 21 Hz), 117.45 (d, J = 7 Hz), 134.74 (dd, J = 13,
4 Hz), 138.78 (s), 142.53 (dd, 250, 5 Hz), 145.40 (dd, 260, 5 Hz), 146.60 (d,
J = 14 Hz), 147.32 (d,
J
= 10 Hz), 150.14 (s), 151.35 (d,
J = 247 Hz), 165.86 (s), 177.13 (d, J = 3 Hz); MS-ESI (+): m/z
420 [M+H]+.
6.1.8.
1-(6-Amino-3,5-difluoropyridin-2-yl)-6-fluoro-8-methyl-7-[3-(methylamino)azetidin-1-yl]-4-oxo
-1,4-dihydro-quinoline-3-carboxylic acid (10b)
The title compound 10b was prepared from 3-(methylamino)azetidine hydrochloride (9b,
310 mg, 2.0 mmol) according to the similar procedure for 10a.
1
Pale yellow solid (380 mg, 67% yield); H NMR (DMSO-
d
6):
δ
1.64 (s, 3H), 2.22 (s, 3H),
3.46-3.54 (m, 1H), 3.82-3.92 (m, 1H), 3.95-4.05 (m, 1H), 4.34-4.50 (m, 2H), 6.85 (brs, 2H), 7.75
= 9.5 Hz, 9.5 Hz, 1H), 8.71 (s, 1H); 13C NMR (DMSO-
(d,
J
= 13.5 Hz, 1H), 7.96 (dd,
J
d6): δ
17.71 (s), 33.41 (s), 50.80 (d,
J = 4 Hz), 64.22 (s), 64.42 (s), 108.03 (s), 109.13 (d, J = 23 Hz),
112.81 (d,
J = 4 Hz), 114.57 (dd, J = 24, 21 Hz), 117.41 (d, J = 7 Hz), 134.77 (dd, J = 13, 4 Hz),
138.83 (s), 142.53 (dd,
J = 250, 5 Hz), 145.35 (dd, J = 259, 5 Hz), 146.60 (d, J = 14 Hz), 147.16
(d,
J
= 10 Hz), 150.15 (s), 151.26 (d,
J
= 247 Hz), 165.86 (s), 177.12 (d,
J
= 3 Hz); MS-ESI(+):
m/z 434 [M+H]+.
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6.1.9.
1-(6-Amino-3,5-difluoropyridin-2-yl)-7-[3(ethyl-amino)azetidin-1-yl]-6-fluoro-8-methyl-4-o
xo-1,4-dihydro-quinoline-3-carboxylic acid (10c)
The title compound 10c was prepared from 3-(ethylamino)azetidine hydrochloride (9c, 1.6 g,
9.0 mmol) according to the similar procedure for 10a.
Pale yellow solid (960 mg, 36% yield); 1H NMR (DMSO-
d6): δ 0.98 (t, J = 7.5 Hz, 3H), 1.63 (s,
3H), 3.53-3.63 (m, 1H), 3.82-3.92 (m, 1H), 3.95-4.05 (m, 1H), 4.36-4.51 (m, 2H), 6.83 (brs, 2H),
= 9.5 Hz, 9.5 Hz, 1H), 8.70 (s, 1H); 13C NMR (DMSO-
7.75 (d,
J
= 13.5 Hz, 1H), 7.94 (dd,
J
d6): δ
15.77 (s), 17.69 (s), 45.66 (s), 49.24 (d,
J = 4 Hz), 64.80 (s), 65.05 (s), 109.18 (d, J = 23 Hz),
112.85 (d,
J = 4 Hz), 114.54 (dd, J = 24, 21 Hz), 117.83 (d, J = 6 Hz), 134.94 (dd, J = 13, 4 Hz),
138.75 (s), 142.51 (dd,
J = 249, 5 Hz), 145.32 (dd, J = 260, 5 Hz), 146.56 (d, J = 14 Hz), 146.93
(d,
J = 10 Hz), 149.96 (s), 151.20 (d, J = 247 Hz), 165.87 (s), 176.98 (d, J = 3 Hz); MS-ESI(+):
m/z 448 [M+H]+.
315
316
317
318
319
320
6.1.10.
1-(6-Amino-3,5-difluoropyridin-2-yl)-6-fluoro-8-methyl-4-oxo-7-[3-(
ne-1-yl]-1,4-di-hydroquinoline-3-carboxylic acid (10d)
n-propylamino)azetidi
To a suspension of 3-(n-propylamino)azetidine hydrochloride (9d, 2.7 g, 15 mmol) in DMSO
(6.5 mL) were added LiOH monohydrate (1.2 g, 29 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene
(1.8
mL,
12
mmol)
and
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340
1-(6-amino-3,5-difluoropyridin-2-yl)-6,7-difluoro-8-methyl-4-oxo-1,4-dihydroquinoline-3-carbo
xylic acid (8, 1.1 g, 2.9 mmol), and the mixture was stirred at 60 °C for 3 h. The reaction
mixture was washed with Et2O (5 mL × 5) by decantation to remove DMSO. To the residue
was added water (2 mL), and the pH of the suspension was adjusted to pH 6 with aqueous
10% citric acid. The precipitate was collected by filtration, washed with EtOH and dried
1
under reduced pressure to give 10d as a pale yellow solid. (500 mg, 36% yield); H NMR
(DMSO-d6): δ 0.86 (t, J = 7.5 Hz, 3H), 1.32-1.43 (m, 2H), 1.64 (s, 3H), 2.41 (t, J = 7.5 Hz, 2H),
3.53-3.61 (m, 1H), 3.80-3.91 (m, 1H), 3.95-4.04 (m, 1H), 4.36-4.51 (m, 2H), 6.84 (brs, 2H), 7.76
= 9.5 Hz, 9.5 Hz, 1H), 8.71 (s, 1H); 13C NMR (DMSO-
(d,
J
= 13.5 Hz, 1H), 7.95 (dd,
J
d6): δ
12.20 (s), 17.70 (s), 23.14 (s), 48.95 (s), 49.31 (d,
J = 4 Hz), 64.51 (s), 64.76 (s), 108.02 (s),
109.14 (d,
J = 23 Hz), 112.90 (d, J = 4 Hz), 114.55 (dd, J = 24, 21 Hz), 117.46 (d, J = 7 Hz),
134.75 (dd,
J = 13, 4 Hz), 138.81 (s), 142.51 (dd, J = 250, 5 Hz), 145.44 (dd, J = 259, 5 Hz),
146.60 (d,
177.13 (d,
J
= 14 Hz), 147.16 (d,
J = 10 Hz), 150.17 (s), 151.26 (d, J = 247 Hz), 165.79 (s),
J
= 3 Hz); MS-ESI(+): m/z 462 [M+H]+.
6.1.11.
1-(6-Amino-3,5-difluoropyridin-2-yl)-6-fluoro-7-[3-(isopropylamino)azetidin-1-yl]-8-methy
l-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (10e)
The title compound 10e was prepared from 3-(isopropylamino)azetidine hydrochloride (9e,
14.0 g, 75 mmol) according to the similar procedure for 10a.
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359
Pale yellow solid (12.0 g, 52% yield); 1H NMR (DMSO-d6
) δ 0.93 (d, J = 6.0 Hz, 6H), 1.62 (s,
3H), 2.66-2.75 (m, 1H), 3.60-3.69 (m, 1H), 3.76-3.86 (m, 1H), 3.92-4.02 (m, 1H), 4.39-4.54 (m,
= 9.5 Hz, 9.5 Hz, 1H), 8.69 (s, 1H); 13
C
2H), 6.82 (s, 2H), 7.74 (d,
J
= 14.0 Hz, 1H), 7.94 (dd,
J
NMR (DMSO-
d
J
6): δ 17.73 (s), 23.57 (s), 47.19 (s), 47.51 (d, J = 4 Hz), 65.54 (s), 65.81 (s), 108.03
(s), 109.13 (d,
= 23 Hz), 112.93 (d,
J = 4 Hz), 114.55 (dd, J = 24, 21 Hz), 117.43 (d, J = 7 Hz),
134.76 (dd,
J = 13, 4 Hz), 138.80 (s), 142.49 (dd, J = 250, 5 Hz), 145.35 (dd, J = 259, 5 Hz),
146.59 (d,
177.12 (d,
J
= 14 Hz), 147.23 (d,
J = 10 Hz), 150.15 (s), 151.36 (d, J = 247 Hz), 165.80 (s),
J
= 3 Hz); MS-ESI(+): m/z 462 [M+H]+.
6.1.12.
1-(6-Amino-3,5-difluoropyridin-2-yl)-7-[3-(tert-butyl-amino)azetidin-1-yl]-6-fluoro-8-meth
yl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (10f)
The title compound 10f was prepared from 3-(tert-butylamino)azetidine hydrochloride (9f,
145 mg, 0.7 mmol) according to the similar procedure for 10d.
Pale yellow solid (45 mg, 58% yield); 1H NMR (DMSO-
d6): δ 1.09 (s, 9H), 1.65 (s, 3H), 3.86-4.07
(m, 2H), 4.07-4.18 (m, 1H), 4.47-4.65 (m, 2H), 6.85 (brs, 2H), 7.80 (d,
9.5 Hz, 9.5 Hz, 1H), 8.73 (s, 1H); 13C NMR (DMSO-
J = 13.5 Hz), 7.96 (dd, J =
d
6):
δ
17.75 (s), 29.86 (s), 44.54 (d,
J
= 4 Hz),
= 24, 21
= 250, 5
50.59 (s), 66.84 (s), 67.11 (s), 109.19 (d,
J = 23 Hz), 113.08 (d, J = 4 Hz), 114.50 (dd, J
Hz), 118.10 (d,
J = 7 Hz), 135.00 (dd, J = 13, 4 Hz), 135.59 (s), 138.66 (s), 142.49 (dd, J
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362
Hz), 145.26 (dd,
J
= 259, 5 Hz), 146.56 (d,
J = 14 Hz), 146.92 (d, J = 10 Hz), 149.91 (s), 151.20
(d,
J = 247 Hz), 165.96 (s), 176.91 (d, J
= 3 Hz); MS-ESI(+): m/z 476 [M+H]+.
363
364
365
366
367
368
369
370
371
372
373
374
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379
6.2 in vitro Activity
6.2.1. Antibacterial activity
The in vitro antibacterial activities of 10a-f and LVFX were evaluated against the following
representative organisms: E. coli NIHJ, E. coli KC-14, E. coli 177, Citrobacter freundii
IFO1268, Klebsiella pneumoniae KC-1, K. pneumoniae DT-S, Salmonella typhimurium
IFO13245, Enterobacter cloacae IFO13535, Proteus vulgaris IFO3167, Proteus mirabilis IFO
3849, Serratia marcescens IFO3736, S. marcescens T-55, Morganella morganii W1026,
Providencia rettgeri W1008, Pseudomonas aeruginosa IFO3445, P. aeruginosa E-2, P.
aeruginosa 15846, and Acinetobacter calcoaceticus Ac-54. MICs were determined by an agar
dilution method with Muller Hinton Agar (MHA, Becton Dickinson, NJ, USA) according to
the standard method described by the Japanese Society of Chemotherapy [28]. Approximately
104 cfu of organisms were inoculated onto the MHA plates containing the two-fold dilution
series of 10a-f and LVFX in the concentration range of 128 to 0.002 mg/L, and the plates were
incubated at 37°C for 18 h. The MIC values were determined to be the lowest concentrations
of 10a-f and LVFX that yielded no visible growth of organisms on the MHA plates.
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393
6.2.2. Inhibitory activity against DNA gyrase
The inhibitory activities of 10a-f and LVFX against E. coli DNA gyrase were examined by
the supercoiling assay [29]. After the preincubation of mixtures (20 µL) containing 35 mM
Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% glycerol,
0.1 mg/mL albumin, 0.2 units of E.coli gyrase (New England Biolab, MA, USA), and a series of
diluted solutions of the test compounds at 37°C for 5 min, 250 ng of relaxed pBR322
(Inspiralis, Norwich, UK) was added to initiate the supercoiling reaction. The reaction
mixture was then further incubated at 37°C for 15 min. The reaction was terminated by the
addition of loading buffer, which was followed by electrophoretic analysis in 1% agarose gels.
The gels were viewed and photographed on an ImageQuant4000 instrument (GE Healthcare,
Pollards Wood, UK). The intensities of the bands were calculated using ImageQuant TL (GE
Healthcare). The IC50 values were determined by nonlinear least-squares regression analysis
using Kyplot (version 5.0, Keyence, Osaka, Japan).
394
395
396
397
398
399
6.3. Excretion and oral absorption in rats
Six-week old male Sprague-Dawley rats (Japan SLC and Charles River Japan, Japan) were
used. Before the study, the animals had been acclimated to the laboratory conditions for at
least 1 week. 10a-e were orally or intravenously administered at doses of 10 mg/kg and 5
mg/kg, respectively, to rats fasted overnight. 10a-e were suspended in 0.5% methylcellulose
for oral administration or dissolved in 0.04 N NaOH for intravenous injection. Urine samples
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were collected from intact rats individually housed in a metabolic cage for up to 24 h post-dose.
Bile samples were collected from bile duct-cannulated rats individually kept in a Bollmann
cage for 24 h post-dose. The volumes of the urine and bile samples were recorded, and the
samples were stored at -30°C until analysis. The urine and bile samples were analyzed as
follows: bile samples (100 µL) were alkaline-treated by the addition of 10 µL of 1 N NaOH
with incubation at 37°C for 1 h. Intact and alkaline-treated samples were diluted with HPLC
mobile phase and spiked with an internal standard. After centrifugation, portions of the
supernatants were analyzed by HPLC [LC-10A System, Shimadzu; Kyoto, Japan; analytical
column, ODS-80TM, 5 µm, 4.6 × 150 mm, Tosoh, Tokyo, Japan; flow rate, 1 mL/min; column
temperature, 40°C; mobile phase, A (MeCN), B (distilled water containing 20 mM sodium
1-decansulfonate, 40 mM phosphoric acid, and 0.2% (v/v) triethylamine), isocratic A/B (40/60);
detection wavelength, 290 nm]. The extent of excretion (% of dose) was calculated by dividing
the amount of 10a-e excreted in the urine and bile by the administered dose. The oral
absorption rate (%) was calculated by dividing the total urinary and biliary excretions of 10a-e
(p.o.) by the total excretion (i.v.).
6.4 Solubility in JP2
Suspensions of 10a-e were prepared by adding excess amounts of 10a-e into 0.6 mL of JP2,
which is a 50 mM potassium dihydrogenphosphate solution at a pH of 6.8. Each suspension
was incubated at 25°C for 24 h with shaking to prepare a thermodynamically equilibrated
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421
422
423
424
425
426
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430
solution, which was followed by filtration through a 0.45 µm Millex HV membrane filter
(Millipore, MA, USA). The concentrations of 10a-e in the filtrate were measured with HPLC
and defined as the solubilities. HPLC separation was achieved on a UPLC BEH C18 column
(1.7 µm 2.1 × 100 mm, Waters) using gradient elution at a flow rate of 0.3 mL/min. The mobile
phases consisted of 10% acetonitrile/90% water containing 0.1% formic acid (A) and
acetonitrile containing 0.1% formic acid (B). The following HPLC gradient program was used:
starting at 15% mobile phase B, a linear gradient was run beginning at 0.5 min to reach 40%
B at 3 min, with the solvent composition being returned to 15% in 0.2 min and then
re-equilibration for 1.8 min. The column temperature was set at 40°C, and UV detection was
applied at 290 nm.
431
432
433
434
435
436
437
438
439
6.5 Membrane permeability in PAMPA
The passive membrane permeabilities of 10a-e were evaluated using a PAMPA plate system
(BD Biosciences, CA, USA). Ten micromolar solutions of 10a-e in 300 µL of phosphate buffered
saline (PBS) containing 5% MeOH were added into the donor plate. The acceptor plate filled
with 200 µL of PBS was placed on the donor plate, which was followed by incubation at 25°C
for 5 h. The solutions of both plates were analyzed by HPLC as described above (section 6.4).
The permeability coefficients (Peff) of 10a-e were calculated by following formula [30]:
Peff = −ln[1−Ca(t)/Cequ]/[A×(1/Vd+1/Va) ×t]
Cequ = [Cd(t) ×Vd+Ca(t)+ Va]/[Vd+Va]
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443
where Cd(t) and Ca(t) are the concentrations of 10a-e in the solutions of the donor and acceptor
plates at time t, respectively. Vd and Va are the volumes of the solutions in the donor and
acceptor plates, respectively,
(18,000 s).
A
is the filter surface area (0.3 cm2), and
t
is the incubation time
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