Synthesis and antibacterial activity of novel pyrido[1,2,3-de][1,4] benzoxazine-6-carboxylic acid derivatives carrying the 3-cyclopropylaminomethyl- 4-substituted-1-pyrrolidinyl group as a C-10 substituent

Article abstract of DOI:10.1021/jm701428b

Novel pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid derivatives 5-9 carrying a 3-cyclopropylami-nomethyl-4-substituted-1-pyrrolidinyl moiety at the C-10 position were synthesized and their in vitro antibacterial activity, intravenous single-dose toxi

Full text of DOI:10.1021/jm701428b

3238
J. Med. Chem. 2008, 51, 3238–3249
Synthesis and Antibacterial Activity of Novel Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid
Derivatives Carrying the 3-Cyclopropylaminomethyl-4-substituted-1-pyrrolidinyl Group as a
C-10 Substituent
Yoshikazu Asahina,* Masaya Takei, Tetsuya Kimura, and Yasumichi Fukuda
DiscoVery Research Laboratories, Kyorin Pharmaceutical Co. Ltd., 2399-1, Nogi, Nogi-Machi, Shimotsuga-Gun, Tochigi, 329-0114, Japan
ReceiVed NoVember 13, 2007
Novel pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid derivatives 5-9 carrying a 3-cyclopropylami-
nomethyl-4-substituted-1-pyrrolidinyl moiety at the C-10 position were synthesized and their in vitro
antibacterial activity, intravenous single-dose toxicity, convulsion inductive ability, and phototoxicity were
evaluated. It appeared evident that compounds 5a, 6a, 8a, and 9a, which have a cis-oriented 4-methyl or
4-fluoro-3-cyclopropylaminomethyl-1-pyrrolidinyl moiety at the C-10 position, exhibited 2- to 16-fold more
potent in vitro antibacterial activity than clinafloxacin against quinolone-resistant Gram-positive clinical
isolates. Furthermore, it was obvious that introduction of a fluorine atom to the C-4 position of the
3-cyclopropylaminomethyl-1-pyrrolidinyl moiety reduced intraveneous single-dose acute toxicity and the convulsion
inductive ability, and introduction of a fluorine atom to the C-3 methyl group of the pyridobenzoxazine nucleus
eliminated the phototoxicity.
Introduction
derivatives such as 2 (PD117558),9 bearing the 3-ethylaminom-
ethylpyrrolidinyl group as the C-7 substituent show strong
cytotoxicity against mammalian cells.10 To find the more
effective fluoroquinolones against multidrug-resistant Gram-
positive bacteria, we focused on the fluoroquinolone derivatives
bearing the 3-alkylaminomethylpyrrolidinyl group as the C-7
(C-10 in the case of pyrido[1,2,3-de][1,4]benzoxazine deriva-
tives) substituent, especially 10-(3-cyclopropylamionomethyl-
1-pyrrolidinyl)pyrido[1,2,3-de][1,4]benzoxazine derivatives 4.
We found that compound 411 exhibited 32- to 64-fold more
potent in vitro antibacterial activity against QMRSA and VRE
Multidrug-resistant Gram-positive pathogens such as methi-
cillin-resistant Staphylococcus aureus (MRSA)a, vancomycin-
resistant Enterococcus (VRE), and penicillin-resistant Strepto-
coccus pneumoniae (PRSP), which are spread worldwide, have
become a serious problem in the treatment of infectious diseases.
In the field of fluoroquinolone antibacterial agents, since the
development of the first new quinolone, norfloxacin,1 a great
deal of effort has been put into developing more potent
quinolones for multidrug-resistant Gram-positive bacteria for
more than 10 years. As a result of these efforts, gatifloxacin,2
moxifloxacin,3 trovafloxacin,4 gemifloxacin,5 etc. have been
developed, and gatifloxacin and moxifloxacin in particular are
being used for the treatment of community-acquired pneumonia
caused by Streptococcus pneumoniae, including PRSP (Figure
1). However, the frequency of quinolone-resistant Gram-positive
bacteria, particularly, quinolone-resistant methicillin-resistant
Staphylococcus aureus (QMRSA), has been increasing.6 There-
fore, urgent development of more effective fluoroquinolones
against multidrug-resistant Gram-positive bacteria is still re-
quired.
A number of syntheses of fluoroquinolone derivatives have
been reported, together with the corresponding structure-activity
relationship (SAR) studies.7 As a result of these SAR studies,
it appears evident that the N-1, C-7, and C-8 substituents of
the fluoroquinolones play an important role in the antibacterial
potency, antibacterial spectrum, and toxicity of fluoroquinolones.
For example, a 3-ethylaminomethylpyrrolidinyl group, found
in the C-7 substituent of 1 (CI-934),8 showed enhancement of
antibacterial potency against Gram-positive bacteria. However,
it has also been reported that the 1-cyclopropylquinolone
than
10-(3-ethylamionomethyl-1-pyrrolidinyl)pyrido[1,2,3-
de][1,4]benzoxazine derivatives 3.9a Despite the pyrido[1,2,3-
de][1,4]benzoxazinecarboxylic acid derivative, in vitro antibac-
terial activity of 4 against QMRSA and VRE was superior to
that of clinafloxacin, which has been reported as one of the most
active fluoroquinolones against Gram-positive bacteria.9a,12
Thus, we designed and synthesized the compounds 5-9, into
which were introduced a methyl group or a fluorine atom to
the C-4 position of the 3-cyclopropylaminomethyl-1-pyrrolidinyl
moiety and a fluorine atom to a C-3 methyl group of the
pyrido[1,2,3-de][1,4]benzoxazine nucleus, with the intent of
increasing the in vitro antibacterial activity13 and reducing the
toxicity14 of 4 (Figure 2).
The results of the present study indicate that the introduction
of a fluorine atom to the C-4 position of a 3-cyclopropylami-
nomethylpyrrolidinyl moiety and a C-3 methyl group served to
reduce the phototoxicity of 4 without loss of highly potent
antibacterial activity. In this paper, we present the synthesis, in
vitro antibacterial activity, and toxicological properties of a
series of the pyrido[1,2,3-de][1,4]benzoxazinecarboxylic acid
derivatives 5-9 carrying a (3S)-3-cyclopropylaminomethyl-1-
pyrrolidinyl, (3S)-3-cyclopropylaminomethyl-4-methyl-1-pyr-
rolidinyl, or (3S)-3-cyclopropylaminomethyl-4-fluoro-1-pyrro-
lidinyl group at the C-10 position.
* To whom correspondence should be addressed. Phone: 81 (0)280 56
2201. Fax: 81 (0)280 57 1293. E-mail: yoshikazu.asahina@mb.kyorin-
pharm.co.jp.
a Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus;
VRE, vancomycin-resistant Enterococcus; PRSP, penicillin-resistant Strep-
tococcus pneumoniae; QMRSA, quinolone-resistant methicillin-resistant
Staphylococcus aureus; DAST, diethylaminosulfur trifluoride; MICs,
minimum inhibitory concentrations.
Chemistry
We planned to synthesize the compounds 5-9 using a
nucleophilic substitution reaction from diacetoxyboron chelate15
10.1021/jm701428b CCC: $40.75
2008 American Chemical Society
Published on Web 05/06/2008

Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3239
Figure 1. Structure of reference quinolones.
Figure 2
Scheme 1a
a Reagents and conditions: (a) (1) MsCl, Et3N, CH2Cl2; (2) NaCN, Bu4NCN, DMF, 69%; (b) LiAlH4, Et2O, 83%; (c) PhCHO, molecular sieves 4A,
MeOH, then BH3-pyridine, 78%; (d) [1-(ethoxycyclopropyl)oxy]trimethylsilane, NaBH3CN, AcOH, molecular sieves 3A, MeOH, 98%; (e) H2, Pd-C, EtOH,
67%.
of the (3S)-methyl- and (3R)-fluoromethyl-pyrido[1,2,3-
de][1,4]benzoxazinecarboxylic acid derivatives 10 and 11, and
appropriate (3S)-cyclopropylaminomethylpyrrolidine derivatives
12,16 13, and 14, followed by removal of the chelate (Figure
1).
The synthesis of cis-oriented (3R,4S)-3-cyclopropylaminom-
ethyl-4-methylpyrrolidine (13a) is shown in Scheme 1. Treat-
ment of (3S,4R)-1-benzyl-4-methyl-3-pyrrolidinol (15)17 with
methanesulfonyl chloride in the presence of triethylamine,
followed by sodium cyanide and tetrabutylammonium cyanide,
gave cyanopyrrolidine 16.18 Reduction of a cyano group of 16
with lithium aluminum hydride, followed by reductive benzy-
lation of the resulting amino group, provided the benzylami-
nomethyl derivative 18. Cyclopropanation of a benzylamino
group of 18 with [(1-ethoxycyclopropyl)oxy]trimethylsilane in
the presence of sodium cyanoborohydride,19 followed by
removal of two benzyl groups of 19 using catalytic hydrogena-
tion, gave the desired cis-oriented 13a.
The synthesis of trans-oriented (3R,4R)-3-cyclopropylami-
nomethyl-4-methylpyrrolidine (13b) is shown in Scheme 2.
Treatment of 2020 with excess cyclopropylamine gave N-
cyclopropylamide 21 directly. Reduction of a carbamoyl group
of 21 with a borane-dimethyl sulfide complex, followed by
removal of a benzyl group of 22 using catalytic hydrogenation
in the presence of trifluoroacetic acid, provided the desired trans-
oriented 13b.
The synthesis of cis-oriented (3R,4S)-3-cyclopropylaminom-
ethyl-4-fluoropyrrolidine (14a) and trans-oriented (3R,4R)-3-

3240 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Asahina et al.
Scheme 2a
a Reagents and conditions: (a) cyclopropylamine, 49%; (b) ΒΗ3-dimethyl sulfide, toluene, 94%; (c) H2, 10% Pd-C, CF3COOH, EtOH, then aqueous
NaOH, 76%.
Scheme 3a
a Reagents and conditions: (a) (1) HCOONH4, 10% Pd-C, aqueous MeOH; (2) ClCOOBn, Et3N, DMF, 91%; (b) (1) PhCOOH, DEAD, PPh3, THF, 99%;
(2) K2CO3, aqueous EtOH, 83%; (c) NaN3, CBr4, PPh3, DMF, 89% (for 25a), 85% (for 25b); (d) DAST, CH2Cl2, 60% (for 26a); (e) H2,PtO2, EtOH, 88%
(for 27a), 26% (for 27b from 25a), and 15% (for 31 from 25a); (f) PhCHO, molecular sieves 4A, MeOH, then BH3-pyridine, 82% (for 28a), 77% (for 28b);
(g) [1-(ethoxycyclopropyl)oxy]trimethylsilane, NaBH3CN, AcOH, molecular sieves 3A, MeOH, 99% (for 29a), 100% (for 29b); (h) H2, 10% Pd-C, EtOH,
82% (for 14a), 77% (for 14b).
Scheme 4a
a Reagents and conditions: (a) H3BO3, Ac2O, cat. ZnCl2, 88%; (b) 13a,b or 14a,b, Et3N, MeCN, then 5% aqueous AcOH, 47% (for 5a), 71% (for 5b),
71% (for 6a), 70% (for 6b).
cyclopropylaminomethyl-4-fluoropyrrolidine (14b) is illustrated
in Scheme 3. Removal of both of the two benzyl groups of
[(3R,4R)-1-benzyl-4-benzyloxypyrrolidin-3-yl]methanol (23)21
using the catalytic hydrogen transfer reaction, followed by
treatment with benzyl chloroformate in the presence of triethy-
lamine, afforded 24a. The selective azidation of the primary
alcohol group of 24a was achieved by treatment with sodium
azide, carbon tetrabromide, and triphenylphosphine to give 25a.
Fluorination of the secondary alcohol group of 25a using
diethylaminosulfur trifluoride (DAST) and catalytic hydrogena-
tion of an azide group of 26a in the presence of platinum dioxide
provided 27a. Reductive benzylation of an amino group of 27a,
cyclopropanation of the resulting benzylamino group of 28a with
[(1-ethoxycyclopropyl)oxy]trimethylsilane, and removal of both
of the benzyl group and benzyloxycarbonyl group of 29a by
catalytic hydrogenation afforded cis-oriented 14a. In contrast,
the synthesis of trans-oriented 14b was achieved by way of
inversion of the secondary alcohol of 24a. The Mitsunobu
reaction22 of 24a, followed by basic hydrolysis of the two
resulting benzoyl ester groups, gave cis-oriented 24b. After
conversion of 24b to azidomethyl derivative 25b, fluorination
of the secondary alcohol was performed by using DAST to give
26b. Unfortunately, opposite to fluorination of 25a, the fluori-
nated product 26b was obtained along with eliminated product
1
30 as an inseparable mixture. H NMR spectra of the mixture
indicated that the ratio of 26b and 30 was approximately 3:2.
After reduction of the azido group of the mixture by catalytic
hydrogenation, separation of 31 by silica gel column chroma-
tography provided the desired 27b. According to the method
for the synthesis of 14a, 27b was converted to trans-oriented
14b.
The reaction of the pyrrolidines 13 and 14 with diacetoxy-
boron chelate 10, which was prepared from the ester 3223,24 by
treatment with boric acid and acetic anhydride, followed by
removal of the chelate under acidic conditions, gave the
3-methyl derivatives 5 and 6 (Scheme 4).
Synthesis of the 3-fluoromethyl derivatives 7-9 is shown in
Scheme 5. Treatment of benzoylacetate 33 with triethyl ortho-

Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3241
Scheme 5a
a Reagents and conditions: (a) (1) HC(OEt)3, Ac2O; (2) (2R)-2-amino-3-(2-tetrahydropyranyl)oxypropanol (34), 83%; (b) KF, DMSO, 62%; (c) p-TsOH.H2O,
EtOH, 92%; (d) DAST, CH2Cl2, 56%; (e) H3BO3, Ac2O, cat. ZnCl2, 83%; (f) 12, 13a,b, or 14a,b, Et3N, MeCN, then 5% aqueous AcOH, 50% (for 7), 83%
(for 8a), 61% (for 8b), 72% (for 9a), 69% (for 9b).
Table 1. In Vitro Antibacterial Activity of Compounds 3-9 against
formate and acetic anhydride, followed by the reaction with
optically active aminopropanol 34, gave enaminoester 35.
Cyclization of 35 using potassium fluoride gave the optically
active pyrido[1,2,3-de][1,4]benzoxazine derivative 36. Removal
of a tetrahydropyranyl group under acidic conditions followed
by treatment with DAST gave the optically active 3-fluorom-
ethyl derivative 38.23 Compound 38 was converted to the
diacetoxyboron chelate 11, which reacted with the pyrrolidines
12-14, and removal of the chelate occurred in the same manner
described for the synthesis of the 3-methyl derivatives 5 and 6
to give the 3-fluoromethyl derivatives 7-9.
Standard Strains
MIC (µg/mL)a
Gram-positive organisms
Sa Spn Spy Ef
Gram-negative organisms
Ec Kpn Hi Pa
compd
4
0.008 0.016 0.016 0.063
0.008 0.016 0.016 0.063
0.002 0.016 0.016 0.031
0.008 0.016 0.031 0.063
0.016 0.063 0.125 0.125
0.008 0.016 0.031 0.063
0.008 0.016 0.031 0.063
0.008 0.031 0.031 0.063
0.008 0.016 0.031 0.063
0.016 0.063 0.125 0.125
0.125 0.063 0.063 0.25
0.008 0.063 0.016
0.016 0.063 0.004
0.016 0.125 0.008
0.008 0.031 0.004
0.016 0.125 0.008
0.004 0.031 0.004
0.016 0.063 0.004
0.016 0.125 0.008
0.008 0.031 0.004
0.031 0.125 0.008
0.031 NTc NTc
e0.001 0.004 0.008
e0.001 0.016 0.008
2
2
4
2
4
2
2
4
2
4
4
5a
5b
6a
6b
7
8a
8b
9a
9b
3
Results and Discussion
The minimum inhibitory concentrations (MICs) of the
compounds 3-9 against four Gram-positive standard strains
(Staphylococcus aureus Smith, Streptococcus pneumoniae Type
III, Streptococcus pyogenes IID692, and Enterococcus faecalis
IID682) and four Gram-negative standard strains (Escherichia
coli NIHJ JC-2, Klebsiella pneumoniae IID5209, Haemophilus
influenzae IID983, and Pseudomonas aeruginosa IID1210) are
shown in Table 1, along with those of ciprofloxacin, levofloxa-
cin, and clinafloxacin. The synthesized compounds 4-9, except
for 6b and 9b, exhibited 2- to 128-fold more potent in vitro
antibacterial activity against Gram-positive standard strains than
ciprofloxacin, levofloxacin, and the racemic 10-(3-ethylami-
nomethypyrrolidinyl) derivative 3. In contrast, against Gram-
negative strains such as E. coli and K. pneumoniae, the in vitro
antibacterial activity of 4-9 was 2- to 16-fold less potent than
those of ciprofloxacin, levofloxacin, and clinafloxacin. However,
4-9 exhibited comparable in vitro antibacterial activity to
ciprofloxacin and levofloxacin against H. influenzae, which is
an important pathogen of respiratory tract infection.
The MICs of 3-9 against three quinolone-resistant clinical
isolates (S. aureus OITI MR1-1002, S. pneumoniae No. 55, and
E. faecalis No. 15) and two VREs [E. faecalis KU1856 (VanA)
and E. faecalis KU1866 (VanB)], along with those for ciprof-
loxacin, levofloxacin, and clinafloxacin, are summarized in Table
2. These results indicate that the cis-oriented derivatives 5a,
6a, 8a, and 9a exhibited in vitro antibacterial activity against
quinolone-resistant clinical isolates and VREs, which was
superior to that of 4 and 7, as well as the trans-oriented
derivatives 5b, 6b, 8b, and 9b. In addition, these cis-oriented
CPFXb 0.25 0.5
LVFXb 0.125 0.5
0.5
0.5
0.5
1
0.125
0.5
CLFXb 0.016 0.031 0.063 0.063
0.002 0.004 e0.001 0.125
a Organisms selected for inclusion in the table: Sa, S. aureus Smith; Spn,
S. pneumoniae Type III; Spy, S. pyogenes IID692; Ef, E. faecalis IID682;
Ec, E. coli NIHJJC-2; Kpn, K. pneumoniae IID5209; Hi, H. influenzae
IID983; Pa, P. aeruginosa IID1210. b Abbreviations are as follows: CPFX
) ciprofloxacin; LVFX ) levofloxacin; CLFX ) clinafloxacin. c Not tested.
derivatives, 5a, 6a, 8a, and 9a, were found to show 2- to 16-
fold more potent activity than clinafloxacin.
We next evaluated intravenous single-dose acute toxicity,
convulsion inductive ability, and phototoxicity of 4, 5a,b, 6a,
7, 8a,b, and 9a. The results along with those of clinafloxacin
are summarized in Table 3. Concerning the single-dose acute
toxicity, 5a and 8a possessing the (3S,4S)-4-methyl-3-cyclo-
propylaminomethyl-1-pyrrolidinyl moiety were slightly more
toxic than 4. In contrast, 6a and 9a bearing the (3S,4S)-4-fluoro-
3-cyclopropylaminomethyl-1-pyrrolidinyl moiety were less toxic
than 4. In contrast, less convulsion inductive ability was
observed in compounds having a 3-methyl group rather than a
3-fluoromethyl group (4 vs 7 and 5 vs 8). However, 9a, which
has the (3S,4S)-4-fluoro-3-cyclopropylaminomethylpyrrolidinyl
moiety, showed less convulsion inductive ability, similar to the
3-methyl derivative 6a. Furthermore, the phototoxicity was
completely eliminated by the introduction of a fluorine atom to
a C-3 methyl group (4-6 vs 7-9). Introduction of a fluorine
atom to the C-4 position of a pyrrolidine ring was effective in
reducing the single-dose acute toxicity and convulsion inductive

3242 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Asahina et al.
Table 2. In Vitro Antibacterial Activity of Compounds 3-9 against Quinolone-Resistant Clinical Isolates and VRE
MIC (µg/mL)
quinolone-resistant clinical isolates
VRE
S. aureus OITI MR1-
S. pneumoniae
E. faecalis
no. 15
E. faecalis
KU1856
E. faecalis
KU1866
compd
1002
no. 55
4
5a
5b
2
0.5
2
0.25
0.125
0.5
1
1
1
0.5
1
1
0.5
1
1
6a
6b
7
8a
1
16
2
0.5
2
0.125
2
0.25
0.125
0.5
0.5
4
1
1
1
0.5
2
0.5
1
0.5
4
0.5
1
8b
1
1
9a
9b
3
1
0.25
2
2
64
32
1
4
32
64
64
8
0.5
2
16
64
32
2
0.5
4
16
64
32
4
16
32
64
>128
8
CPFXa
LVFXa
CLFXa
0.5
a Abbreviations are as follows: CPFX ) ciprofloxacin; LVFX ) levofloxacin; CLFX ) clinafloxacin.
Table 3. Intravenous Single-Dose Acute Toxicity, Convulsive Ability,
and Phototoxicity of Compounds 4, 5a,b, 6a, 7, 8a,b, and 9a
Table 5. In Vitro Antibacterial Activity of 9a against Sequentially
Acquired Quinolone-Resistant Mutants of S. aureus MS5935
intravenous single-dose
MIC (µg/mL)
CLFXe
acute toxicitya(mortality, convulsive activityb phototoxicity
strain
9a
LVFXe
dead/tested)
(µg/mouse)
(100 mg/kg, po)
wild
0.016
0.031
0.25
0.5
0.016
0.063
0.5
2
0.125
0.5
8
compd 100 mg/kg 200 mg/kg
-FB +FB
scorec
first-step mutanta
second-step mutantb
third-step mutantc
fourth-step mutantd
4
5a
5b
6a
7
0/5
3/3
0/5
0/5
0/5
1/5
0/5
0/5
0/5
3/3
NTd
3/3
0/5
4/5
3/3
4/5
0/5
0/5
20
>40
>40
>40
10
20
>40
>40
>40
10
2.0
2.0
1.3
1.7
0
32
>128
4
8
a The first-step mutant possessed a single grlA mutation. b The second-
step mutant possessed single grlA and single gyrA mutations. c The third-
step mutant possessed double grlA and single gyrA mutations. d The fourth-
step mutant possessed double grlA and double gyrA mutations. e Abbreviations
are as follows: LVFX ) levofloxacin; CLFX ) clinafloxacin.
8a
20
10
0
8b
9a
40
20
0
>40
10
>40
5
0
3.0
CLFXd
a Intravenous single-dose acute toxicity was characterized by a single-
dose toxicity after intravenous administration. b Convulsive activity was
characterized by a minimum inducible dose of behavioral convulsion after
intraventricular administration without Fenbufen (-FB) and with Fenbufen
(+FB). c Score is the mean value for three animals. Score is as follows: 0
) nil; 1 ) slight; 2 ) moderate; 3 ) marked change. d Abbreviations are
as follows: CLFX ) clinafloxacin.
Table 6. In Vivo Antibacterial Activity of Compound 9a against
Systemic Infection in Mice
ED50 (mg/kg)
infected bacteria
compd
sc
po
S. aureus Smith
9a
0.12
0.35
2.9
0.50
2.1
4.0
CLFXa
9a
S. pneumoniae Type III
Table 4. In Vitro Inhibition Potency of 9a against Topoisomerase IV
and DNA Gyrase Isolated from Wild and Resistant S. aureus MS5935
CLFXa
45
15
a Abbreviation is as follows: CLFX ) clinafloxacin.
IC50 (µg/mL)
DNA gyrase
topoisomerase IV
gyrase and topoisomerase IV compared with levofloxacin, which
has the same pyridobenzoxazine nucleus as 9a. Furthermore,
because the IC50 values of 9a against both the resistant type
DNA gyrase and topoisomerase IV were slightly smaller than
those of clinafloxacin, we can conclude that 9a more strongly
inhibited both of the target enzymes.
compd
wild
resistant
wild
resistant
9a
1.06
1.15
9.31
13.6
16.2
772
1.27
1.89
5.44
7.00
13.9
319
CLFXa
LVFXa
a Abbreviations are as follows: LVFX ) levofloxacin; CLFX )
clinafloxacin.
ability, and introduction of a fluorine atom to a C-3 methyl group
of a pyridobenzoxazine ring contributed to complete elimination
of the phototoxicity.
The in vitro antibacterial activity of 9a against sequentially
acquired quinolone-resistant mutants of S. aureus MS593526
along with those of clinafloxacin and levofloxacin is shown in
Table 5. Indicative of the strong in vitro inhibition potency
against both the target enzymes, 9a exhibited 2- to 4-fold more
potent in vitro antibacterial activity than clinafloxacin, with
particular potency being seen against third- and fourth-step
mutants.26
On the basis of the results regarding in vitro antibacterial
activity, intravenous single-dose acute toxicity, convulsion
inductive ability, and phototoxicity in this study, we selected
9a as the candidate compound and carried out further evaluation
of the in vitro and in vivo antibacterial activity of 9a. The results
regarding the inhibition potency of 9a against bacterial DNA
gyrase and bacterial topoisomerase IV isolated from a wild and
resistant strain of S. aureus MS593525 are shown in Table 4,
along with those of clinafloxacin and levofloxacin. The com-
pound 9a showed 4- to 9-fold smaller IC50 values against both
the wild-type DNA gyrase and topoisomerase IV, and over 40-
fold smaller IC50 values against both the resistant type DNA
Finally, we evaluated the in vivo efficacy of 9a by oral and
subcutaneous administration against experimental systemic
infections in mice caused by S. aureus Smith and S. pneumonia
Type III. The ED50 values of 9a along with those of clinafloxacin
are shown in Table 6. The in vivo efficacy of 9a against both
experimental systemic infections in mice caused by S. aureus

Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3243
stirred at 80 °C for 13 h and then concentrated in vacuo. After
addition of water (50 mL) to the residue, the resulting mixture was
extracted with diethyl ether (2 × 100 mL). The combined organic
extracts were washed with brine, dried over anhydrous Na2SO4,
filtered, and then concentrated in vacuo. Flash chromatography
Smith and S. pneumoniae Type III was superior to that of
clinafloxacin, especially by oral administration.
Conclusions
1
As described above, we synthesized a series of the novel
pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid derivatives
5-9 carrying a 3-cyclopropylaminomethyl-4-substituted-1-
pyrrolidinyl moiety at the C-10 position and evaluated the in
vitro antibacterial activity, the intravenous single-dose acute
toxicity, the convulsion inductive ability, and the phototoxicity
ofthecompounds5-9.Despitethepyrido[1,2,3-de][1,4]benzoxazine-
6-carboxylic acid derivatives, the in vitro antibacterial activity
of compounds 5-9 was superior to that of clinafloxacin due to
the replacement of the ethyl group of a 3-ethylaminomethylpyr-
rolidinyl moiety with a cyclopropyl group. In particular, the
cis-oriented derivatives 5a, 6a, 8a, and 9a exhibited 2- to 16-
fold more potent in vitro antibacterial activity than clinafloxacin
against quinolone-resistant Gram-positive clinical isolates. In
contrast, introduction of the cis-oriented fluorine atom to the
C-4 position of the 3-cyclopropylaminomethylpyrrolidinyl
moiety brought about a reduction in the intravenous single-dose
acute toxicity, similar to Inagaki’s study14. Furthermore, our
results revealed that the cis-oriented fluorine atom at the C-4
position of the 3-cyclopropylaminomethylpyrrolidinyl moiety
contributes to reduced convulsion inductive ability, and the
introduction of a fluorine atom to the C-3 methyl group of the
pyridobenzoxazine nucleus contributes to a complete elimination
of phototoxicity. As a result of these antibacterial and toxico-
logical evaluations, we selected the compound 9a, which has
cis-oriented (3S,4S)-3-cyclopropylaminomethyl-4-fluoropyrro-
lidinyl moiety at the C-10 position and a fluoromethyl group at
the C-3 position, as a candidate. Further investigations of 9a
for clinical trials are in progress.
(hexane/AcOEt ) 4:1) of the residue gave 16 (3.32 g, 79%). H
NMR (CDCl3) δ: 1.22 (d, J ) 7.3 Hz, 3H), 2.12 (dd, J ) 9.3, 8.3
Hz, 1H), 2.46-2.57 (m, 1H), 2.60-2.67 (m, 1H), 2.99 (dd, J )
9.3, 7.3 Hz, 1H), 3.09-3.19 (m, 2H), 3.62 (s, 2H), 7.25-7.35 (m,
5H). MS (CI+) m/z: 201 (M+ + H). HRMS (CI+) for C13H17N2
(M+ + H): calcd, 201.13918; found, 201.13734. [R]D23 -16.8° (c
0.742, CHCl3).
(3S,4S)-3-Aminomethyl-1-benzyl-4-methylpyrrolidine (17). A
solution of 16 (3.20 g, 16.0 mmol) in anhydrous diethyl ether (20
mL) was added dropwise to a suspension of LiAlH4 (2.43 g, 64.0
mmol) in anhydrous diethyl ether (66 mL) under cooling with ice,
and the whole mixture was stirred at room temperature for 1 h.
After quenching the reaction by adding saturated aqueous NaHCO3
solution (6.6 mL) under cooling with ice, the mixture was diluted
with diethyl ether (75 mL). The insoluble materials were filtered
off and washed with diethyl ether (75 mL). The combined filtrate
and the washing were dried over Na2SO4, filtered, and then
concentrated in vacuo. Flash chromatography (hexane/AcOEt )
1:1 f AcOEt/MeOH ) 10:1) of the residue gave 17 (2.98 g, 91%).
1H NMR (CDCl3) δ: 0.94 (d, J ) 7.3 Hz, 3H), 2.03 (dd, J ) 9.3,
1.5 Hz, 1H), 2.11-2.26 (m, 2H), 2.31-2.43 (m, 1H), 2.58 (dd, J
) 12.2, 8.8 Hz, 1H), 2.82 (dd, J ) 12.2, 5.9 Hz, 1H), 2.97-3.02
(m, 2H), 3.60 (s, 2H), 7.22-7.33 (m, 5H). MS (CI+) m/z: 205 (M+
+ H). HRMS (CI+) for C13H21N2 (M+ + H): calcd, 205.17048;
28
found, 205.16855. [R]D +10.2° (c 0.703, CHCl3).
(3R,4S)-1-Benzyl-3-benzylaminomethyl-4-methylpyrrolidine
(18). A mixture of 17 (2.80 g, 13.7 mmol), molecular sieves 4A
(1.23 g), and benzaldehyde (1.39 mL, 13.7 mmol) in anhydrous
MeOH (60 mL) was stirred at room temperature for 1 h.
Borane-pyridine complex (1.39 mL, 13.8 mmol) was added to the
above mixture, and the whole mixture was stirred at room
temperature for 3 h. After addition of 6 mol/L HCl (23.2 mL), the
whole mixture was stirred at room temperature for 1 h. After the
reaction mixture was adjusted to pH 14 by addition of 6 mol/L
aqueous NaOH solution, the resulting mixture was extracted with
diethyl ether (2 × 100 mL). The combined organic extracts were
washed with brine (2 × 50 mL), dried over anhydrous Na2SO4,
filtered, and then concentrated in vacuo. Flash chromatography
Experimental Section
Melting points were determined with a Yanagimoto micromelting
point apparatus and are uncorrected. Elemental analyses are within
(0.4% of the theoretical values and were determined by a Yanaco
CHN MT-5 instrument. Infrared spectra (IR) were recorded with a
JASCO FT/IR-5300 spectrometer. Measurements of mass spectra
(MS) and high resolution MS (HRMS) were performed with a JEOL
JMS SX-102A or a JEOL JMS-T100LP mass spectrometer. Proton
nuclear magnetic resonance (1H NMR) spectra were measured with
a JEOL EX-90 (90 MHz), a JEOL JMN-EX400 (400 MHz), or a
JEOL JMN-ECA-400 (400 MHz) spectrometer. The chemical shifts
are expressed in parts per million (δ value) downfield from
tetramethylsilane, using tetramethylsilane (δ ) 0) and/or residual
solvents such as chloroform (δ ) 7.26) as an internal standard.
Splitting patterns are indicated as follows: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br; broad peak. Column chroma-
tography was carried out with silica gel [silica gel 60 (Kanto)] as
an absorbent. Merck precoated thin layer chromatography (TLC)
plates (silica gel 60 F254, 0.25 mm, art. 5715) were used for the
TLC analysis. Solutions were dried over sodium sulfate and the
solvent was removed by rotary evaporation under reduced pressure.
(3R,4S)-1-Benzyl-4-methyl-3-pyrrolidinecarbonitrile (16). Meth-
anesulfonyl chloride (1.70 mL, 22.0 mmol) was added dropwise
to a solution of 15 (4.00 g, 20.9 mmol) and triethylamine (3.06
mL, 22.0 mmol) in anhydrous CH2Cl2 (40 mL) at -78 °C, and the
whole mixture was stirred at -78 °C for 1 h. After quenching the
reaction by adding water (40 mL), the CH2Cl2 layer was separated
and the aqueous layer was extracted with CH2Cl2 (40 mL). The
combined CH2Cl2 extracts were washed with water (2 × 20 mL),
dried over anhydrous Na2SO4, filtered, and then concentrated in
vacuo. To a solution of the residue in anhydrous DMF (120 mL),
sodium cyanide (2.05 g, 41.8 mmol) and tetrabutylammonium
cyanide (5.53 g, 20.9 mmol) were added; the whole mixture was
1
(hexane/AcOEt ) 4:1) of the residue gave 18 (3.49 g, 87%). H
NMR (CDCl3) δ: 0.92 (d, J ) 6.8 Hz, 3H), 2.00 (dd, J ) 8.8, 6.8
Hz, 1H), 2.13 (dd, J ) 9.3, 7.8 Hz, 1H), 2.29-2.40 (m, 2H), 2.51
(dd, J ) 11.2, 8.8 Hz, 1H) 2.71 (dd, J ) 11.2, 5.4 Hz, 1H),
2.96-3.02 (m, 2H), 3.59 (s, 2H), 3.78 (s, 2H), 7.21-7.34 (m, 10H).
MS (EI) m/z: 294 (M+). HRMS (EI) for C20H26N2 (M+): calcd,
24
294.2096; found, 294.2072. [R]D +6.1° (c 1.03, MeOH).
(3R,4S)-1-Benzyl-3-(N-benzyl-N-cyclopropyl)aminomethyl-4-me-
thylpyrrolidine (19). A mixture of 18 (3.40 g, 11.5 mmol),
molecular sieves 3A (3.53 g), AcOH (6.58 mL, 115 mmol), [(1-
ethoxycyclopropyl)oxy]trimethylsilane (9.25 mL, 46.0 mmol), and
NaBH3CN (2.17 g, 34.5 mmol) in anhydrous MeOH (40 mL) was
heated under reflux for 3 h. After the insoluble materials were
filtered off, the filtrate was adjusted to pH 14 by addition of 6 mol/L
aqueous NaOH solution, and the mixture was extracted by diethyl
ether (2 × 100 mL). The combined extracts were washed with brine
(2 × 50 mL), dried over anhydrous Na2SO4, filtered, and then
concentrated in vacuo. Flash chromatography (hexane/AcOEt )
1
4:1) of the residue gave 19 (3.72 g, 96%). H NMR (CDCl3) δ:
0.31-0.47 (m, 4H), 0.85 (d, J ) 7.3 Hz, 3H), 1.64-1.69 (m, 1H),
1.91 (dd, J ) 9.3, 7.3 Hz, 1H), 2.06 (t, J ) 9.3 Hz, 1H), 2.23-2.34
(m, 1H), 2.39 (dd, J ) 11.7, 9.3 Hz, 1H), 2.55 (dd, J ) 11.7, 5.9
Hz, 1H), 2.53-2.66 (m, 1H), 2.90-2.96 (m, 2H), 3.49 (d, J )
13.2 Hz, 1H), 3.59 (d, J ) 12.7 Hz, 1H), 3.63 (d, J ) 13.6 Hz,
1H), 3.74 (d, J ) 13.2 Hz, 1H), 7.21-7.32 (m, 10H). MS (FAB+)
m/z: 335 (M+ + H). HRMS (FAB+) for C23H31N2 (M+ + H): calcd,
25
335.2487; found, 335.2503. [R]D +11.2° (c 1.03, MeOH).

3244 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Asahina et al.
(3R,4S)-3-Cyclopropylaminomethyl-4-methylpyrrolidine (13a).
A suspension of 19 (3.60 g, 10.8 mmol), 10% Pd-C (3.60 g), and
CHCl3 (3.44 mL, 43.0 mmol) in EtOH (40 mL) was stirred at 50
°C for 10 h under H2 atmosphere (4 kg/cm2). After the insoluble
materials were filtered off, the filtrate was concentrated in vacuo.
The suspension of the residue and 10% Pd-C (3.60 g) in EtOH
(40 mL) was stirred at 50 °C for 6 h under H2 atmosphere (4 kg/
cm2). After the insoluble materials were filtered off, the filtrate was
concentrated in vacuo. The suspension of the residue and 10%
Pd-C (2.00 g) in EtOH (100 mL) was stirred at 50 °C for 10 h
under H2 atmosphere (4 kg/cm2). After the insoluble materials were
filtered off, the filtrate was concentrated in vacuo and then the
residue was dissolved in water (30 mL). After the mixture was
adjusted to pH 14 by addition of 6 mol/L aqueous NaOH solution,
the mixture was extracted with diethyl ether (4 × 50 mL). The
combined organic extracts were concentrated in vacuo. Flash
chromatography (CH2Cl2/MeOH ) 25:1) of the residue gave 13a
(1.29 g, 79%). 1H NMR (CDCl3) δ: 0.29-0.36 (m, 2H), 0.41-0.46
(m, 2H), 0.93 (d, J ) 7.3 Hz, 3H), 2.10-2.24 (m, 3H), 2.53-2.65
(m, 3H), 2.77 (dd, J ) 11.7, 6.3 Hz, 1H), 3.09 (dd, J ) 6.3, 2.9
Hz, 1H), 3.12 (dd, J ) 6.3, 2.9 Hz, 1H). MS (CI+) m/z: 155 (M+
+ H). HRMS (CI+) for C9H19N2 (M+ + H): calcd, 155.1548; found,
with solid NaOH, and the mixture was extracted with diethyl ether
(3 × 300 mL). The combined organic extracts were dried over
Na2SO4, filtered, and then concentrated in vacuo. Vacuum distil-
lation of the residue gave 13b (40.7 g, 87%); bp: 60-68 °C (0.7
mmHg). 1H NMR (CDCl3) δ: 0.30-0.37 (m, 2H), 0.41-0.45 (m,
2H), 1.04 (d, J ) 6.3 Hz, 3H), 1.68-1.75 (m, 2H), 2.08-2.13 (m,
1H), 2.46 (dd, J ) 10.7, 7.3 Hz, 1H), 2.57 (dd, J ) 11.7, 8.3 Hz,
1H), 2.64 (dd, J ) 10.7, 6.3 Hz, 1H), 2.81 (dd, J ) 11.7, 4.9 Hz,
1H), 3.11 (dd, J ) 10.7, 6.8 Hz, 1H), 3.15 (dd, J ) 10.7, 7.3 Hz,
1H). MS (CI+) m/z: 155 (M+ + H). HRMS (CI+) for C9H19N2
(M+ + H): calcd, 155.15483; found, 155.15414. [R]D25 -74.6° (c
0.648, MeOH). Anal. (C9H18N2 ·2CF3COOH) C, H, N.
(3R,4R)-1-Benzyloxycarbonyl-4-hydroxypyrrolidin-3-yl]metha-
nol (24a). To a solution of 23 (10.0 g, 33.6 mmol) in MeOH (200
mL), a suspension of 10% Pd-C (3.00 g) in water (60 mL) and
ammonium formate (21.2 g, 0.336 mol) was added; the whole
mixture was heated under reflux for 4 h. After the insoluble
materials were filtered off, the filtrate was concentrated in vacuo.
Benzyl chloroformate (6.00 mL, 40.6 mmol) was added to a solution
of the residue and triethylamine (9.40 mL, 63.4 mmol) in anhydrous
DMF (100 mL) under cooling with ice, the whole mixture was
stirred under the same condition for 1.5 h, and then concentrated
in vacuo. After dilution of the residue with AcOEt (400 mL), the
mixture was washed with brine (2 × 100 mL), dried over anhydrous
Na2SO4, filtered, and then concentrated in vacuo. Flash chroma-
tography (AcOEt/MeOH ) 20:1) of the residue gave 24a (7.66 g,
25
155.1539. [R]D -3.4° (c 1.14, MeOH).
(3R,4R)-1-Benzyl-N-cyclopropyl-4-methylpyrrolidine-3-carboxa-
mide (21). A mixture of 20 (150 g, 0.412 mol) and cyclopropy-
lamine (650 mL, 9.39 mol) was stirred at room temperature for
23 h and concentrated in vacuo. After the addition of diisopropyl
ether (800 mL) to the residue, the resulting precipitates were
collected by filtration. The obtained crystals were dissolved in
CH2Cl2 (800 mL), and the CH2Cl2 solution was extracted with 1
mol/L HCl (2 × 400 mL). The combined aqueous extracts were
adjusted to pH 13 by addition of 30% aqueous NaOH solution under
cooling with ice. The resulting precipitates were collected by
filtration, washed with water and diisopropyl ether, and then dried
in vacuo to give 21 (52.2 g, 49%); mp: 99-100 °C (hexane/AcOEt).
1H NMR (CDCl3) δ: 0.36-0.44 (m, 2H), 0.71-0.77 (m, 2H), 1.11
(d, J ) 6.8 Hz, 3H), 1.89 (dd, J ) 8.8, 7.3 Hz, 1H), 2.28-2.41
(m, 3H), 2.65-2.71 (m, 1H), 2.84 (dd, J ) 9.8, 2.0 Hz, 1H), 3.13
(t, J ) 8.8 Hz, 1H), 3.53 (d, J ) 12.7 Hz, 1H), 3.65 (d, J ) 12.7
Hz, 1H), 7.00 (br s, 1H), 7.25-7.35 (m, 5H). MS (EI) m/z: 258
1
91%). H NMR (CDCl3) δ: 1.91-2.16 (br, 1H), 2.30-2.39 (m,
1H), 2.44-2.60 (br, 1H), 3.20 (dd, J ) 11.0, 6.7 Hz, 1H), 3.32
(dt, J ) 11.0, 5.5 Hz, 1H), 3.59-3.77 (m, 4H), 4.23-4.35 (br,
1H), 5.12 (s, 2H), 7.29-7.36 (m, 5H). MS (CI+) m/z: 252 (M+
+
H). HRMS (CI+) for C13H18NO4 (M+ + H): calcd, 252.12358;
25
found, 252.12256. [R]D +11.6° (c 1.14, MeOH).
[(3R,4S)-4-Benzoyloxy-1-benzyloxycarbonylpyrrolidin-3-yl]meth-
anol (24b). Diethyl azodicarboxylate (40% toluene solution, 9.53
mL, 21.9 mmol) was added dropwise to a mixture of 24a (2.50 g,
9.95 mmol), triphenylphosphine (5.74 g, 21.9 mmol), and benzoic
acid (2.55 g, 20.9 mmol) in anhydrous THF (60 mL) under cooling
with NaCl-ice. The whole mixture was stirred at 0 °C for 1 h,
further stirred at room temperature for 2 h, and then concentrated
in vacuo. Flash chromatography (hexane/AcOEt ) 2:1) of the
residue gave pale-brown oil (4.52 g, 99%). To a solution of the
above oil (4.51 g, 9.82 mmol) in EtOH (60 mL) was added a
solution of K2CO3 (4.07 g, 29.4 mmol) in water (30 mL), the whole
mixture was heated under reflux for 3 h, and concentrated in vacuo.
After dilution of the residue with CH2Cl2 (200 mL), the mixture
was washed with brine (2 × 50 mL), dried over anhydrous Na2SO4,
filtered, and then concentrated in vacuo. Flash chromatography
(AcOEt/MeOH ) 10:1) of the residue gave 24b (2.04 g, 83%). 1H
NMR (CDCl3) δ: 2.29-2.40 (m, 1H), 2.52 (t, J ) 4.9 Hz, 0.5H),
2.58 (t, J ) 4.9 Hz, 0.5H), 2.95 (d, J ) 3.7 Hz, 0.5H), 3.00 (d, J
) 3.1 Hz, 0.5H), 3.41-3.47 (m, 1H), 3.53-3.61 (m, 3H),
3.82-3.96 (m, 2H), 4.43-4.56 (br, 1H), 5.09-5.17 (m, 2H),
7.29-7.36 (m, 5H). MS (CI+) m/z: 252 (M+ + H). HRMS (CI+)
for C13H18NO4 (M+ + H): calcd, 252.12358; found, 252.12085.
26
(M+). [R]D +26.8° (c 0.552, MeOH). Anal. (C16H22N2O) C, H,
N.
(3S,4R)-1-Benzyl-3-cyclopropylaminomethyl-4-methylpyrroli-
dine (22). Boran-dimethylsulfide complex (90%, 34.3 mL, 0.326
mol) was added dropwise to a suspension of 21 (70.0 g, 0.271 mol)
in anhydrous toluene (700 mL) under cooling with ice. The reaction
mixture was stirred for 15 min under cooling with ice and heated
under reflux for 5 h. After cooling to room temperature, 10%
aqueous Na2CO3 solution (400 mL) was added to the reaction
mixture and the whole mixture was stirred at 100 °C for 2 h. After
cooling to room temperature, the toluene layer was separated,
washed with water (2 × 250 mL), dried over anhydrous Na2SO4,
filtered, and then concentrated in vacuo. Vacuum distillation
(140-150 °C, 0.4 mmHg) of the residue gave 22 (62.1 g, 94%).
1H NMR (CDCl3) δ: 0.26-0.33 (m, 2H), 0.37-0.43 (m, 2H), 1.05
(d, J ) 6.3 Hz, 3H), 1.74-1.87 (m, 2H), 2.06-2.14 (m, 2H), 2.40
(dd, J ) 9.3, 5.4 Hz, 1H), 2.60-2.68 (m, 2H), 2.74-2.80 (m, 2H),
3.52 (d, J ) 13.2 Hz, 1H), 3.63 (d, J ) 13.0 Hz, 1H), 7.21-7.35
(m, 5H). MS (CI+) m/z: 245 (M+ + H). HRMS (CI+) for C16H25N2
(M+ + H): calcd, 245.20178; found, 245.20057. [R]D25 +41.2° (c
1.075, MeOH).
(3R,4R)-3-Cyclopropylaminomethy-4-methylpyrrolidine (13b).
A suspension of 22 (25.0 g, 0.102 mol), 10% Pd-C (12.5 g), and
trifluoroacetic acid (15.7 mL, 0.204 mol) in EtOH (200 mL) was
stirred at room temperature for 20 h under H2 atmosphere (4 kg/
cm2). After the insoluble materials ware filtered off, the filtrate was
concentrated in vacuo. After the addition of THF (100 mL) to the
residue, the resulting precipitates were collected by filtration,
washed with THF, and then dried in vacuo to give ditrifluoroacetic
acid salt of 13b (34.1 g, 87%). A solution of the above salt (116 g,
0.303 mol) in water (350 mL) was made strongly basic (pH >12)
25
[R]D +2.6° (c 0.835, MeOH).
(3R,4R)-3-Azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrro-
lidine (25a). A solution of CBr4 (4.34 g, 13.1 mmol) in anhydrous
CH2Cl2 (14 mL) was added dropwise to a mixture of 24a (3.00 g,
11.9 mmol), NaN3 (2.32 g, 35.7 mmol), and triphenylphosphine
(3.43 g, 13.1 mmol) in anhydrous DMF (60 mL) under cooling
with ice. The whole mixture was stirred at room temperature for
25 h and further stirred at 60 °C for 2 h. After quenching the
reaction by adding MeOH (5 mL), the mixture was concentrated
in vacuo. After dilution of the residue with AcOEt (200 mL), the
mixture was washed with brine (2 × 50 mL), dried over anhydrous
Na2SO4, filtered, and then concentrated in vacuo. Flash chroma-
tography (hexane/AcOEt ) 1:2) of the residue gave 25a (2.94 g,
1
89%). H NMR (CDCl3) δ: 2.07 (d, J ) 4.3 Hz, 1H), 2.29-2.40
(m, 1H), 3.24 (dd, J ) 11.0, 6.1 Hz, 1H), 3.30-3.48 (m, 3H),
3.68-3.77 (m, 2H), 4.09-4.26 (m, 1H), 5.13 (s, 2H), 7.31-7.37
(m, 5H). MS (CI+) m/z: 277 (M+ + H). HRMS (CI+) for

Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3245
24
C13H17N4O3 (M+ + H): calcd, 277.13007; found, 277.13275. [R]D
(3S,4R)-3-Benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropy-
rrolidine (28b). The compound 28b (329 mg, 77%) was prepared
from 27b (313 mg, 1.24 mmol) by the same method as that used
for 18. 1H NMR (CDCl3) δ: 2.26-2.47 (m, 3H), 3.40 (t, J ) 11.0
Hz, 1H), 3.52-3.81 (m, 5H), 5.02-5.20 (br, 1H), 5.13 (s, 2H),
7.24-7.37 (m, 5H). MS (FAB+) m/z: 343 (M+ + H). HRMS
(FAB+) for C20H24FN2O2 (M+ + H): calcd, 343.1822; found,
+13.3° (c 0.506, MeOH).
(3R,4S)-3-Azidomethyl-1-benzyloxycarbonyl-4-hydroxypyrroli-
dine (25b). The compound 25b (2.18 g, 85%) was prepared from
24b (2.33 g, 9.27 mmol) by the same method as that used for 25a.
1H NMR (CDCl3): δ 1.94-2.07 (br, 1H), 2.33-2.44 (m, 1H), 3.24
(t, J ) 11.0 Hz, 1H), 3.37-3.48 (m, 1H), 3.55-3.69 (m, 4H),
4.33-4.49 (br, 1H), 5.09-5.17 (m, 2H), 7.31-7.36 (m, 5H). MS
25
343.1815. [R]D -9.0° (c 0.519, MeOH).
(CI+) m/z: 277 (M+ + H). HRMS (CI+) for C13H17N4O3 (M+
+
(3S,4S)-3-(N-Benzyl-N-cyclopropyl)aminomethyl-1-benzyloxy-
carbonyl-4-fluoropyrrolidine (29a). The compound 29a (3.97 g,
99%) was prepared from 28a (3.59 g, 10.5 mmol) by the same
25
H): calcd, 277.13007; found, 277.12616. [R]D +3.6° (c 0.613,
MeOH).
1
method as that used for 19. H NMR (CDCl3) δ: 0.36-0.50 (m,
(3S,4S)-3-Aminomethyl-1-benzyloxycarbonyl-4-fluoropyrroli-
dine (27a). Diethylaminosulfur trifluoride (DAST, 1.20 mL, 9.08
mmol) was added dropwise to a solution of 25a (1.20 g, 4.34 mmol)
in anhydrous CH2Cl2 (40 mL) under cooling with NaCl-ice, and
the whole mixture was stirred at room temperature for 3 h.
Diethylaminosulfur trifluoride (0.57 mL, 4.31 mmol) was added
dropwise to the reaction mixture and then stirred at room temper-
ature additional 2 h. The reaction mixture was quenched by adding
saturated aqueous NaHCO3 solution (40 mL) under cooling with
ice. The organic layer was separated, washed with saturated aqueous
NaHCO3 solution (2 × 20 mL) and brine (20 mL), dried over
Na2SO4, filtered, and then concentrated in vacuo. Flash chroma-
tography (hexane/AcOEt ) 2:1) of the residue gave 26a (726 mg,
60%). A suspension of 26a (4.99 g, 17.9 mmol) and PtO2 (406
mg, 1.80 mmol) in EtOH (100 mL) was stirred at room temperature
for 7 h under H2 atmosphere (1 atm). After the insoluble materials
were filtered off, the filtrate was concentrated in vacuo. Flash
chromatography (AcOEt/MeOH ) 10:1) of the residue gave 27a
(3.99 g, 88%). 1H NMR (CDCl3) δ: 2.21-2.36 (m, 1H), 2.83-2.92
(m, 1H), 2.98-3.03 (m, 1H), 3.22 (t, J ) 11.0 Hz, 1H), 3.52-3.67
(m, 1H), 3.70-3.90 (m, 2H), 5.10-5.26 (m, 3H), 7.31-7.37 (m,
5H). MS (CI+) m/z: 253 (M+ + H). HRMS (CI+) for C13H18FN2O2
4H), 1.78-1.82 (m, 1H), 2.37-2.56 (m, 1H), 2.61-2.70 (m, 1H),
2.85 (dd, J ) 12.8, 7.3 Hz, 1H), 3.06 (dt, J ) 17.1, 11.0 Hz, 1H),
3.50 (ddt, J ) 40.0, 12.8, 3.7 Hz, 1H), 3.59-3.82 (m, 4H), 5.00
(ddt, J ) 53.2, 4.9, 3.1 Hz, 1H), 5.09-5.16 (m, 2H), 7.21-7.38
(m, 10H). MS (CI+) m/z: 383 (M+ + H). HRMS (CI+) for
C23H28FN2O2 (M+ + H): calcd, 383.21348; found, 383.21378.
24
[R]D -4.4° (c 0.462, MeOH).
(3S,4R)-3-(N-Benzyl-N-cyclopropyl)aminomethyl-1-benzyloxy-
carbonyl-4-fluoropyrrolidine (29b). The compound 29b (336 mg,
100%) was prepared from 28b (302 mg, 0.811 mmol) by the same
1
method as that used for 19. H NMR (CDCl3) δ: 0.34-0.56 (m,
4H), 1.76-1.80 (m, 1H), 2.04-2.37 (m, 2H), 2.74-2.85 (m, 1H),
3.06-3.32 (m, 2H), 3.47-3.68 (m, 3H), 3.79 (dd, J ) 13.4, 7.3
Hz, 1H), 4.94 (dd, J ) 52.6, 4.3 Hz, 1H), 5.09-5.16 (m, 2H),
7.19-7.36 (m, 10H). MS (FAB+) m/z: 383 (M+ + H). HRMS
(FAB+) for C23H28FN2O2 (M+ + H): calcd, 383.2135; found,
27
383.2119. [R]D -21.7° (c 0.409, MeOH).
(3S,4S)-3-Cyclopropylaminomethyl-4-fluoropyrrolidine (14a). A
suspension of 29a (1.22 g, 3.19 mmol) and 10% Pd-C (150 mg)
in EtOH (14 mL) was stirred at room temperature for 4 h under H2
atmosphere (1 atm). After the insoluble materials were filtered off,
the filtrate was concentrated in vacuo. Flash chromatography
(CH2Cl2/MeOH ) 10:1) of the residue gave 14a (414 mg, 82%).
1H NMR (CDCl3) δ: 0.30-0.46 (m, 4H), 2.12-2.18 (m, 1H),
2.19-2.35 (m, 1H), 2.76-2.82 (m, 2H), 2.96-3.03 (m, 2H),
3.07-3.12 (m, 2H), 3.23 (dd, J ) 25.1, 13.5 Hz, 1H), 5.10 (dt, J
) 55.0, 3.7 Hz, 1H). MS (CI+) m/z: 159 (M+ + H). HRMS (CI+)
24
(M+ + H): calcd, 253.13523; found, 253.13479. [R]D -0.8° (c
0.487, MeOH).
(3S,4R)-3-Aminomethyl-1-benzyloxycarbonyl-4-fluoropyrroli-
dine (27b). Diethylaminosulfur trifluoride (DAST, 2.10 mL, 15.9
mmol) was added dropwise to a solution of 25b (1.49 g, 5.39 mmol)
in anhydrous CH2Cl2 (30 mL) under cooling with ice, the whole
mixture was stirred at room temperature for 9 h, and then allowed to
stand at room temperature for overnight. The reaction mixture was
quenched by adding saturated aqueous NaHCO3 solution (30 mL)
under cooling with ice. The organic layer was separated, washed with
saturated aqueous NaHCO3 solution (10 mL) and brine (10 mL), dried
over Na2SO4, filtered, and then concentrated in vacuo. Flash chroma-
tography (hexane/AcOEt ) 2:1) of the residue gave a mixture of 26a
and 30 (929 mg). A suspension of the above mixture (925 mg) and
PtO2 (185 mg, 0.815 mmol) in EtOH (20 mL) was stirred at room
temperature for 4.5 h under H2 atmosphere (1 atm). After the insoluble
materials were filtered off, the filtrate was concentrated in vacuo. Flash
chromatography (AcOEt/MeOH ) 20:1) of the residue gave 27b (359
mg, 26%) and 31 (196 mg, 15%).
25
for C8H16N2 (M+ + H): calcd, 159.1298; found, 159.1316. [R]D
-26.6° (c 0.662, MeOH).
(3S,4R)-3-Cyclopropylaminomethyl-4-fluoropyrrolidine (14b).
The compound 14b (50.7 mg, 77%) was prepared from 29b (160
1
mg, 0.418 mmol) by the same method as that used for 14a. H
NMR (CDCl3) δ: 0.28-0.45 (m, 4H), 2.09-2.13 (m, 1H),
2.33-2.52 (m, 2H), 2.63 (ddd, J ) 12.2, 7.3, 1.8 Hz, 1H), 2.69
(dd, J ) 11.6, 7.9 Hz, 1H), 2.86 (ddd, J ) 35.5, 13.4, 3.7 Hz, 1H),
3.21 (ddd, J ) 22.0, 13.4, 1.2 Hz, 1H), 3.34 (dd, J ) 11.6, 7.9 Hz,
1H), 4.93 (dd, J ) 54.4, 3.7 Hz, 1H). MS (FAB+) m/z: 159 (M+
+ H). HRMS (FAB+) for C8H16FN2 (M+ + H): calcd, 159.1298;
25
found, 159.1286. [R]D +22.0° (c 0.239, MeOH).
(2R)-2-Amino-3-(tetrahydropyran-2-yl)oxypropanol (34). To a
suspension of L-serine methyl ester hydrochloride (195 g, 1.25 mol)
in anhydrous CH2Cl2 (973 mL) was added 3,4-dihydro-1H-pyran
(158 g, 1.88 mol) and p-toluenesulfonic acid monohydrate (4.76
g, 25.0 mmol), the whole mixture was stirred at room temperature
for 20 h. The resulting precipitates were collected by filtration,
washed with CH2Cl2, and dried in vacuo to give O3-(tetrahydro-
pyran-2-yl)-L-serine methyl ester hydrochloride (201 g, 67%).
To a suspension of LiAlH4 (91.1 g, 2.40 mol) in anhydrous THF
(2400 mL) was added O3-(tetrahydropyran-2-yl)-L-serine methyl
ester hydrochloride (288 g, 1.20 mol) portionwise under cooling
with ice, the whole mixture was stirred at room temperature for
0.5 h, and then heated under reflux for additional 2 h. After
quenching the reaction by adding aqueous KOH solution [KOH
(34.6 g) in water (173 mL)] under cooling with ice, the resulting
insoluble materials were filtered off and washed with THF (1000
mL). The combined filtrate and washing were concentrated in vacuo.
After dilution of the residue with CH2Cl2 (1000 mL), the mixture
was washed with aqueous 5 mol/L KOH solution (300 mL) and
brine (300 mL), dried over anhydrous Na2SO4, and then concen-
1
27b. H NMR (CDCl3) δ: 2.39-2.49 (m, 1H), 2.61-2.72 (m,
2H), 3.37-3.43 (m, 1H), 3.57-3.79 (m, 3H), 5.01-5.18 (m, 3H),
7.29-7.37 (m, 5H). MS (CI+) m/z: 253 (M+ + H). HRMS (CI+)
for C13H18FN2O2 (M+ + H): calcd, 253.1352; found, 253.1330.
25
[R]D -3.0° (c 0.717, MeOH).
31. 1H NMR (CDCl3) δ: 1.56-1.65 (m, 1H), 1.95-2.07 (m,
1H), 2.18-2.31 (m, 1H), 2.67-2.78 (m, 2H), 3.05-3.13 (m, 1H),
3.34-3.43 (m, 1H), 3.49-3.65 (m, 2H), 5.13 (s, 2H), 7.28-7.38
(m, 5H). MS (CI+) m/z: 235 (M+ + H).
(3S,4S)-3-Benzylaminomethyl-1-benzyloxycarbonyl-4-fluoropy-
rrolidine (28a). The compound 28a (3.64 g, 82%) was prepared
from 27a (3.26 g, 12.9 mmol) by the same method as that used for
18. 1H NMR (CDCl3) δ: 2.31-2.42 (m, 1H), 2.71-2.80 (m, 1H),
2.93 (ddd, J ) 11.6, 8.6, 3.7 Hz, 1H), 3.23 (dt, J ) 11.0, 4.3 Hz,
1H), 3.49-3.65 (m, 1H), 3.70-3.88 (m, 4H), 5.13 (d, J ) 2.4 Hz,
1H), 5.14 (d, J ) 2.4 Hz, 1H), 5.14 (ddd, J ) 53.2, 6.1, 3.1 Hz),
7.22-7.37 (m, 10H). MS (CI+) m/z: 343 (M+ + H). HRMS (CI+)
for C20H24FN2O2 (M+ + H): calcd, 343.18218; found, 343.18449.
24
[R]D -3.3° (c 0.516, MeOH).

3246 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Asahina et al.
trated in vacuo to give 34 (149 g, 71%). 1H NMR (CDCl3) δ:
1.47-1.87 (m, 6H), 3.05-3.11 (m, 1H), 3.46 (dd, J ) 9.8, 4.4 Hz,
1H), 3.50-3.56 (m, 1H), 3.53 (dd, J ) 10.8, 5.9 Hz, 1H), 3.67
(dd, J ) 10.8, 4.9 Hz, 1H), 3.74 (dd, J ) 9.8, 5.9 Hz, 1H),
3.83-3.89 (m, 1H), 4.58 (dd, J ) 4.9, 4.4 Hz, 1H). MS (CI+) m/z:
Hz, 1H), 5.06-5.13 (m, 1H), 7.64 (dd, J ) 10.6 Hz, 7.8 Hz, 1H),
8.64 (s, 1H). MS (EI) m/z 327 (M+). [R]D25 -101° (c 1.03, AcOH).
Anal. (C15H12F3NO4) C, H, N.
bis(Acetato-O)[(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-dihy-
dro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylato-
O6,O7]boron (10). A mixture of boric acid (23.9 g, 0.387 mol),
Ac2O (110 mL, 1.17 mol), and ZnCl2 (440 mg, 3.23 mmol) was
stirred at room temperature for 30 min. To the reaction mixture
was added 32 (40.0 g, 0.129 mol), the whole mixture was stirred
at 60 °C for 2 h, and then concentrated in vacuo. A solution of the
residue in AcOEt (1000 mL) was washed with saturated aqueous
NaHCO3 solution (2 × 500 mL) and water (500 mL), dried over
anhydrous Na2SO4, filtered, and then concentrated in vacuo. After
treatment, the residue was treated with diisopropyl ether (500 mL)
and the resulting precipitates were collected by filtration, washed
with diisopropyl ether, and then dried in vacuo to give 10 (46.8 g,
23
176 (M+ + H). [R]D -70.6° (c 1.09, MeOH).
Ethyl 3-[(2R)-1-Hydroxy-3-(tetrahydropyran-2-yloxy)prop-2-
ylamino]-2-(2,3,4,5-tetrafluorobenzoyl)acrylate (35). A mixture of
33 (220 g, 0.833 mol), triethyl orthoformate (208 mL, 1.25 mol),
and acetic anhydride (197 mL, 2.08 mol) was stirred at 115-120
°C for 3 h and concentrated in vacuo. To a solution of the residue
in EtOH (1000 mL) was added a solution of 34 (161 g, 0.916 mol)
in EtOH (300 mL) dropwise under cooling with ice, the whole
mixture was stirred at room temperature for 1 h, and then
concentrated in vacuo. Flash chromatography of the residue (CH2Cl2
f CH2Cl2/MeOH ) 10:1) gave 35 (312 g, 83%). 1H NMR (CDCl3)
δ: 0.84-1.26 (m, 3H), 1.33-2.12 (m, 6H), 2.40-2.79 (m, 1H),
3.19-4.31 (m, 9H), 4.46-4.78 (m, 1H), 6.84-7.15 (m, 1H),
8.12-8.32 (m, 1H), 9.36-9.93 (m, 0.2H), 10.5-11.4 (m, 0.8H).
1
88%); mp: >300 °C. H NMR (CDCl3) δ: 1.74 (d, J ) 6.8 Hz,
3H), 1.90 (s, 3H), 2.05 (s, 3H), 4.55 (dd, J ) 12.2, 2.4 Hz, 1H),
4.62 (dd, J ) 12.2, 2.4 Hz, 1H), 5.11-5.19 (m, 1H), 7.90 (dd, J )
9.8, 7.3 Hz, 1H), 9.25 (s, 1H). Anal. (C17H14BF2NO8) C, H, N.
bis(Acetato-O)[(3R)-9,10-difluoro-3-(2-fluoromethyl)-2,3-dihydro-
7-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylato-
O6,O7]boron (11). The compound 11 (24.5 g, 83%) was prepared
from 38 (22.6 g, 69.0 mmol) by the same method as that used for
10. 1H NMR (CDCl3) δ: 1.85 (s, 3H), 2.05 (s, 3H), 4.62 (ddd, J )
12.2, 3.9, 2.9 Hz, 1H), 4.74 (ddd, J ) 46.4, 10.3, 7.8 Hz, 1H),
4.90 (ddd, J ) 45.4, 10.3, 4.9 Hz, 1H), 4.92 (dd, J ) 12.7, 1.0 Hz
1H), 5.35-5.38 (m, 1H), 7.92 (dd, J ) 9.3, 7.3 Hz, 1H), 9.22 (s,
1H). Anal. (C17H13BF3NO8 ·0.75H2O) C, H, N.
24
MS (EI) m/z 449 (M+). [R]D -67.7° (c 0.514, MeOH).
Ethyl (3R)-9,10-Difluoro-2,3-dihydro-3-[(tetrahydropyran-2-
yloxy)methyl]-7-oxo-7H-pyrido[1,2,3-de]benzoxazine-6-carboxy-
late (36). A mixture of 35 (310 g, 0.690 mol), KF (spray dried,
140 g, 2.41 mol), and anhydrous DMSO (1200 mL) was stirred at
115-120 °C for 5 h. After cooling, MeOH (600 mL) was added to
the reaction mixture; the whole mixture was allowed to stand at
room temperature for 2 h. The resulting precipitates were collected
by filtration. The filtered precipitates were suspended in MeOH
(1000 mL) and then filtered. The filtered precipitates were
suspended in water (1000 mL), filtered, washed with MeOH (500
mL), and then dried in vacuo to give 36 (176 g, 62%); mp: 228-231
°C. 1H NMR (CDCl3) δ: 1.25-1.29 (m, 1H), 1.27 (t, J ) 6.8 Hz,
3H), 1.37-1.60 (m, 5H), 3.10-3.16 (m, 1H), 3.19-3.23 (m,1H),
3.73 (dd, J ) 10.3, 9.8 Hz, 1H), 3.82 (dd, J ) 10.3, 4.4 Hz, 1H),
4.20-4.27 (m, 2H), 4.52 (dd, J ) 11.7, 2.4 Hz, 1H), 4.71 (br, 1H),
4.83 (d, J ) 11.2 Hz, 1H), 4.94-4.96 (m, 1H), 7.65 (dd, J ) 10.8,
7.8 Hz, 1H), 8.61 (s, 1H). MS (EI) m/z 409 (M+). [R]D22 -103° (c
0.516, CHCl3). Anal. (C20H21F2NO6) C, H, N.
(3S)-10-[(3S)-3-Cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-
2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-
6-carboxylic Acid (4). A mixture of 10 (1.20 g, 2.93 mmol), 12
(493 mg, 3.51 mmol), and triethylamine (0.49 mL, 3.52 mmol) in
anhydrous CH3CN (20 mL) was stirred at 60 °C for 3 h and then
concentrated in vacuo. Flash chromatography (AcOEt/MeOH )
5:1) of the residue gave the product as a yellow foam. A mixture
of the above product in 5% aqueous AcOH solution (20 mL) was
stirred at 80 °C for 2 h. The reaction mixture was washed with
AcOEt (2 × 5 mL) and adjusted to pH 7 by addition of 2 mol/L
aqueous NaOH solution. The resulting precipitates were collected
by filtration and washed with water. Recrystallization of the
precipitates from EtOH gave 4 (719 mg, 61%); mp: 178-179 °C
Ethyl (3S)-9,10-Difluoro-2,3-dihydro-3-hydroxymethyl-7-oxo-
7H-pyrido[1,2,3-de]benzoxazine-6-carboxylate (37). A mixture of
36 (174 g, 0.424 mol), p-toluenesulfonic acid monohydrate (4.03
g, 21.2 mmol), and EtOH (3000 mL) was heated under reflux for
3 h and then allowed to stand at room temperature for 16 h. The
resulting precipitates were collected by filtration, washed with EtOH
(300 mL), and then dried in vacuo to give 37 (127 g, 92%); mp:
1
(EtOH). H NMR (CDCl3) δ: 0.33-0.36 (m, 2H), 0.44-0.49 (m,
2H), 1.60 (d, J ) 6.7 Hz, 3H), 1.63-1.65 (m, 1H), 2.06-2.12 (m,
1H), 2.13-2.18 (m, 1H), 2.36-2.47 (m, 1H), 2.81 (d, J ) 6.7 Hz,
2H), 3.57 (ddd, J ) 10.4 Hz, 7.9, 3.1 Hz, 1H), 3.67-3.73 (m, 1H),
3.77 (ddd, J ) 9.8, 7.9, 1.8 Hz, 1H), 3.84-3.91 (m, 1H), 4.27 (dd,
J ) 11.6 Hz, 2.4 Hz, 1H), 4.38-4.46 (m, 2H), 7.67 (d, J ) 14.1
1
235-237 °C. H NMR (DMSO-d6) δ: 1.27 (t, J ) 6.8 Hz, 3H),
3.53-3.59 (m, 1H), 3.73-3.78 (m, 1H), 4.16-4.28 (m, 2H), 4.44
(dd, J ) 11.7, 2.9 Hz, 1H), 4.62-4.66 (m, 1H), 4.77 (d, J ) 11.7
Hz, 1H), 5.32 (t, J ) 5.4 Hz, 1H), 7.62 (dd, J ) 11.2 Hz, 8.3 Hz,
Hz, 1H), 8.54 (s, 1H). MS (EI) m/z: 401 (M+). IR (KBr) cm-1
1709, 1622. [R]D -107° (c 0.512, 0.05 mol/L aqueous NaOH).
Anal. (C21H24FN3O4) C, H, N.
:
25
25
1H), 8.54 (s, 1H). MS (EI) m/z 325 (M+). [R]D -124° (c 1.03,
DMF). Anal. (C15H13F2NO5 ·H2O) C, H, N.
(3S)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-methyl-1-pyrro-
lidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (5a). The compound 5a (474
mg, 47%) was prepared from 10 (1.00 g, 2.44 mmol) and 13a (452
mg, 2.93 mmol) by the same method as that used for 4; mp:
Ethyl (3R)-9,10-Difluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-
pyrido[1,2,3-de]benzoxazine-6-carboxylate (38). To a suspension of
37 (40.7 g, 125 mmol) in anhydrous THF (1667 mL) was added
diethylaminosulfur trifluoride (19.8 mL, 150 mmol), and the whole
mixture was heated under reflux for 0.5 h. After cooling, the reaction
mixture was poured into saturated aqueous NaHCO3 solution (1000
mL). The resulting mixture was extracted with CH2Cl2 (700 mL). The
organic extracts were washed with water, dried over anhydrous
Na2SO4, and then concentrated in vacuo. A mixture of the residue
and THF (1500 mL) was heated under reflux for 1 h. The resulting
precipitates were collected by filtration to give crude 38 (17.6 g). The
filtrate was concentrated in vacuo. Flash chromatography (CH2Cl2/
acetone ) 10:1) of the residue gave additional crude 38 (12.5 g). A
mixture of the combined crude 38 (30.1 g) and EtOH (500 mL) was
heated under reflux for 0.5 h, and the resulting precipitates were
collected by filtration. The filtered precipitates were washed with EtOH
1
186-188 °C (EtOH). H NMR (CDCl3) δ: 0.30-0.38 (m, 2H),
0.42-0.49 (m, 2H), 1.03 (d, J ) 6.8 Hz, 3H), 1.60 (d, J ) 6.8 Hz,
3H), 2.12-2.17 (m, 1H), 2.32-2.44 (m, 2H), 2.73 (dd, J ) 11.7,
7.8 Hz, 1H), 2.84 (dd, J ) 11.7, 6.4 Hz, 1H), 3.41 (dt, J ) 10.7,
3.4 Hz, 1H), 3.72 (dd, J ) 7.3, 2.4 Hz, 1H), 3.98-4.03 (m, 1H),
4.26 (dd, J ) 11.2, 2.4 Hz, 1H), 4.34 (dd, J ) 11.2, 2.4 Hz, 1H),
4.40-4.45 (m, 1H), 7.66 (d, J ) 14.2 Hz, 1H), 8.53 (s, 1H). MS
(EI) m/z: 415 (M+). HRMS (EI) for C22H26FN3O4 (M+): calcd,
25
415.1907; found, 415.1892. IR (KBr) cm-1: 1719, 1621. [R]D
-115° (c 0.526, 0.05 mol/L aqueous NaOH). Anal.
(C22H26FN3O4 ·0.25H2O) C, H, N.
1
and dried in vacuo to give 38 (26.5 g, 65%); mp: 254-257 °C. H
(3S)-10-[(3S,4R)-3-Cyclopropylaminomethyl-4-methyl-1-pyrro-
lidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (5b). The compound 5b (362
mg, 71%) was prepared from 10 (500 mg, 1.22 mmol) and 13b
NMR (DMSO-d6) δ: 1.27 (t, J ) 7.3 Hz, 3H), 4.17-4.28 (m, 2H),
4.51 (ddd, J ) 12.2, 4.9, 2.9 Hz, 1H), 4.70 (ddd, J ) 46.5, 10.3, 7.8
Hz, 1H), 4.86 (ddd, J ) 46.0, 10.3, 4.9 Hz, 1H), 4.86 (d, J ) 12.3

Pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic Acid DeriVatiVes
(226 mg, 1.47 mmol) by the same method as that used for 4; mp:
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3247
2.31-2.42 (m, 2H), 2.72 (dd, J ) 11.7, 7.8 Hz, 1H), 2.84 (dd, J )
11.7, 6.4 Hz, 1H), 3.41 (dt, J ) 10.3, 3.4 Hz, 1H), 3.67-3.76 (m,
2H), 3.98 (ddd, J ) 10.3, 6.4 Hz, 3.4 Hz, 1H), 4.31 (ddd, J )
11.2, 4.9, 2.9 Hz, 1H), 4.59-4.86 (m, 4H), 7.66 (d, J ) 14.2 Hz,
1H), 8.54 (s, 1H). MS (EI) m/z: 433 (M+). HRMS (EI) for
C22H25F2N3O4 (M+): calcd, 433.1813; found, 433.1788. IR (KBr)
1
163-165 °C (EtOH). H NMR (CDCl3) δ: 0.32-0.39 (m, 2H),
0.45-0.51 (m, 2H), 1.13 (d, J ) 5.9 Hz, 3H), 1.61 (d, J ) 6.4 Hz,
3H), 1.89-1.99 (m, 2H), 2.13-2.18 (m, 1H), 2.67 (dd, J ) 11.7,
8.3 Hz, 1H), 2.98 (dd, J ) 11.7, 3.9 Hz, 1H), 3.53-3.59 (m, 1H),
3.67-3.84 (m, 3H), 4.26 (dd, J ) 11.2, 2.0 Hz, 1H), 4.40 (dd, J )
11.7, 2.0 Hz, 1H), 4.42-4.47 (m, 1H), 7.66 (d, J ) 14.2 Hz, 1H),
8.54 (s, 1H). MS (EI) m/z: 415 (M+). IR (KBr) cm-1: 1730, 1625.
24
cm-1: 1723, 1626. [R]D -162° (c 0.243, 0.05 mol/L aqueous
NaOH). Anal. (C22H25F2N3O4) C, H, N.
25
[R]D -123° (c 0.507, 0.05 mol/L aqueous NaOH). Anal.
(3R)-10-[(3S,4R)-3-Cyclopropylaminomethyl-4-methyl-1-pyrro-
lidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (8b). The compound 8b (620
mg, 61%) was prepared from 11 (1000 mg, 2.34 mmol) and 13b
(397 mg, 2.57 mmol) by the same method as that used for 4; mp:
(C22H26FN3O4) C, H, N.
(3S)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrro-
lidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (6a). The compound 6a (217
mg, 71%) was prepared from 10 (300 mg, 0.733 mmol) and 14a
(128 mg, 0.806 mmol) by the same method as that used for 4; mp:
1
183-184 °C (EtOH). H NMR (CDCl3) δ: 0.32-0.38 (m, 2H),
0.44-0.49 (m, 2H), 1.12 (d, J ) 6.4 Hz, 3H), 1.90-1.98 (m, 2H),
2.12-2.17 (m, 1H), 2.65 (dd, J ) 11.7, 8.3 Hz, 1H), 2.98 (dd, J )
11.7, 3.9 Hz, 1H), 3.52-3.58 (m, 1H), 3.66-3.72 (m, 1H),
3.73-3.84 (m, 2H), 4.30 (ddd, J ) 11.7, 4.4, 2.4 Hz, 1H),
4.59-4.87 (m, 4H), 7.66 (d, J ) 14.2 Hz, 1H), 8.55 (s, 1H). MS
(EI) m/z: 433 (M+). HRMS (EI) for C22H25F2N3O4 (M+): calcd,
1
202-204 °C (EtOH). H NMR (CDCl3) δ: 0.33-0.51 (m, 4H),
1.61 (d, J ) 6.7 Hz, 3H), 2.15-2.20 (m, 1H), 2.37-2.55 (m, 1H),
2.93 (dd, J ) 12.2, 6.7 Hz, 1H), 3.09 (dd, J ) 12.2, 7.9 Hz, 1H),
3.73-3.86 (m, 2H), 3.93 (dt, J ) 10.5, 3.3 Hz, 1H), 4.22-4.48
(m, 4H), 5.21 (dt, J ) 54.0, 3.2 Hz, 1H), 7.70 (d, J ) 14.1 Hz,
1H), 8.55 (s, 1H). MS (FAB+) m/z: 420 (M+ + H). HRMS (FAB+)
for C21H24F2N3O4 (M+ + H): calcd, 420.1735; found, 420.1778.
24
433.1813; found, 433.1824. IR (KBr) cm-1: 1721, 1625. [R]D
-143° (c 0.449, 0.05 mol/L aqueous NaOH). Anal. (C22H25F2N3O4)
C, H, N.
24
IR (KBr) cm-1: 1728, 1625. [R]D -196° (c 0.257, 0.05 mol/L
aqueous NaOH). Anal. (C21H23F2N3O4) C, H, N.
(3R)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrro-
lidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (9a). The compound 9a
(1399 mg, 72%) was prepared from 11 (1900 mg, 4.45 mmol) and
14a (774 mg, 4.89 mmol) by the same method as that used for 4;
mp: 197-198 °C (EtOH). 1H NMR (CDCl3) δ: 0.33-0.39 (m, 2H),
0.46-0.50 (m, 2H), 2.15-2.20 (m, 1H), 2.37-2.54 (m, 1H), 2.92
(dd, J ) 12.2, 6.7 Hz, 1H), 3.08 (dd, J ) 12.2, 7.9 Hz, 1H),
3.71-3.85 (m, 2H), 3.91 (dt, J ) 10.4, 3.1 Hz, 1H), 4.20-4.36
(m, 2H), 4.61-4.87 (m, 2H), 5.20 (dt, J ) 53.8, 3.1 Hz, 1H), 7.69
(3S)-10-[(3S,4R)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrro-
lidinyl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (6b). The compound 6b (50.8
mg, 70%) was prepared from 10 (71.0 mg, 0.174 mmol) and 14b
(30.0 mg, 0.190 mmol) by the same method as that used for 4;
mp: 201-203 °C (EtOH). 1H NMR (CDCl3) δ: 0.30-0.35 (m, 2H),
0.45-0.48 (m, 2H), 1.62 (d, J ) 6.7 Hz, 3H), 2.11-2.16 (m, 1H),
2.58-2.80 (m, 3H), 3.48 (dt, J ) 10.4, 2.4 Hz, 1H), 3.83 (dd, J )
25.1, 12.2 Hz, 1H), 4.04-4.19 (m, 2H), 4.31 (dd, J ) 11.6, 2.4
Hz, 1H), 4.41 (dd, J ) 11.6, 2.4 Hz, 1H), 4.44-4.47 (m, 1H),
5.10 (dt, J ) 53.2, 2.4 Hz, 1H), 7.70 (d, J ) 13.4 Hz, 1H), 8.56 (s,
1H). MS (FAB+) m/z: 420 (M+ + H). HRMS (FAB+) for
C21H24F2N3O4 (M+ + H): calcd, 420.1735; found, 420.1719. IR
(d, J ) 14.1 Hz, 1H), 8.57 (s, 1H). MS (FAB+) m/z: 438 (M+
+
H). HRMS (FAB+) for C21H23F3N3O4 (M+ + H): calcd, 438.1641;
found, 438.1656. IR (KBr) cm-1: 1733, 1625. [R]D -207° (c
28
0.260, 0.05 mol/L aqueous NaOH). Anal. (C21H22F3N3O4) C, H,
N.
26
(KBr) cm-1: 1707, 1625. [R]D -7.9° (c 0.242, 0.05 mol/L
aqueous NaOH). Anal. (C21H23F2N3O4) C, H, N.
(3R)-10-[(3S,4R)-3-Cyclopropylaminomethyl-4-fluoro-1-pyrro-
lidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (9b). The compound 9b (71.0
mg, 69%) was prepared from 11 (100 mg, 0.234 mmol) and 14b
(40.6 mg, 0.257 mmol) by the same method as that used for 4;
mp: 198-200 °C (EtOH). 1H NMR (CDCl3) δ: 0.31-0.34 (m, 2H),
0.45-0.48 (m, 2H), 2.10-2.15 (m, 1H), 2.58-2.78 (m, 3H), 3.48
(dt, J ) 10.4, 2.4 Hz, 1H), 3.81 (dd, J ) 24.4, 12.2 Hz, 1H),
4.04-4.19 (m, 2H), 4.35 (ddd, J ) 12,2, 4.3, 2.4 Hz, 1H),
4.58-4.86 (m, 4H), 5.10 (dt, J ) 53.8, 2.4 Hz, 1H), 7.72 (d, J )
13.4 Hz, 1H), 8.56 (s, 1H). MS (FAB+) m/z: 438 (M+ + H). HRMS
(FAB+) for C21H23F3N3O4 (M+ + H): calcd, 438.1641; found,
(3R)-10-[(3S)-3-Cyclopropylaminomethyl-1-pyrrolidinyl]-9-fluoro-
3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-de][1,4]benzo-
xazine-6-carboxylic Acid (7). A mixture of 11 (22.6 g, 53.0 mmol),
12 (8.41 g, 60.0 mmol), and triethylamine (7.59 g, 75.0 mmol) in
anhydrous CH2Cl2 (273 mL) was allowed to stand at room
temperature for 13 h. The reaction mixture was washed with water
(200 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered,
and then concentrated in vacuo. Flash chromatography (CH2Cl2/
MeOH ) 15:1) of the residue gave the product as a yellow foam.
A mixture of the above product in 5% aqueous AcOH solution
(100 mL) was stirred at 80 °C for 3 h. After washing of the reaction
mixture with AcOEt (100 mL), the aqueous solution was adjusted
to pH 7 by addition of 1 mol/L aqueous NaOH solution. The
resulting precipitates were collected by filtration and washed with
water. Recrystallization of the precipitates from EtOH gave 7 (11.2
g, 50%); mp: 198-199 °C (EtOH). 1H NMR (DMSO-d6) δ:
0.15-0.26 (m, 2H), 0.28-0.40 (m, 2H), 1.50-1.59 (m, 1H),
1.95-2.00 (m, 1H), 2.02-2.07 (m, 1H), 2.25-2.35 (m, 1H),
2.58-2.67 (m, 2H), 3.31 (br, 1H), 3.44-3.49 (m, 1H), 3.60-3.80
(m, 3H), 4.34 (ddd, J ) 12.2, 4.9, 2.9 Hz, 1H), 4.73 (ddd, J )
47.0, 9.8, 8.3 Hz, 1H), 4.77 (d, J ) 12.2 Hz, 1H), 4.89 (ddd, J )
45.5, 9.8, 4.9 Hz, 1H), 5.12-5.19 (m, 1H), 7.53 (d, J ) 12.2 Hz,
1H), 8.80 (s, 1H). MS (EI) m/z: 419 (M+). IR (KBr) cm-1: 1717,
25
438.1664. IR (KBr) cm-1: 1709, 1624. [R]D -38.9° (c 0.118,
0.05 mol/L aqueous NaOH). Anal. (C21H22F3N3O4) C, H, N.
In Vitro Antibacterial Activity. The MIC (µg/mL) was
determined by the agar dilution method27 with Muller-Hinton agar
(Difco Laboratories, Detroit, MI). The MIC was defined as the
lowest concentration of an antibacterial agent that inhibited visible
growth after incubation at 35 °C for 18 h.
In Vivo Antibacterial Activity. Male ICR mice (4 weeks)
weighting about 20 g were infected intraperitoneally with bacterial
suspensions. The bacteria used for infection were S. aureus Smith
(2.7 × 106 CFU per mouse) and S. pneumoniae Type III (660 CFU
per mouse). The test compounds were administered orally or
subcutaneously 1 h after bacterial challenge. Untreated and treated
groups at each dose were composed of five mice each. The 50%
effective dose (ED50s) were calculated by the least-squares method
or the probit method on the basis of the number of survivals at 7
days after infection.
Inhibitory Activity against DNA Gyrase and Topoisomerase IV
of S. aureus. The supercoiling activity of DNA gyrase and the
decatenation activity of topoisomerase IV were determined by a
previous reported procedure.25 The inhibitory activity was assayed
25
1623. [R]D -139° (c 0.518, 0.05 mol/L aqueous NaOH). Anal.
(C21H23F2N3O4) C, H, N.
(3R)-10-[(3S,4S)-3-Cyclopropylaminomethyl-4-methyl-1-pyrro-
lidinyl]-9-fluoro-3-fluoromethyl-2,3-dihydro-7-oxo-7H-pyrido[1,2,3-
de][1,4]benzoxazine-6-carboxylic Acid (8a). The compound 8a (422
mg, 83%) was prepared from 11 (500 mg, 1.17 mmol) and 13a
(199 mg, 1.29 mmol) by the same method as that used for 4; mp:
1
200-201 °C (EtOH). H NMR (CDCl3) δ: 0.30-0.37 (m, 2H),
0.42-0.50 (m, 2H), 1.02 (d, J ) 6.4 Hz, 3H), 2.12-2.18 (m, 1H),

3248 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Asahina et al.
(8) Domagala, J. M.; Heifetz, C. L.; Mich, T. F.; Nichols, J. B. 1-Ethyl-
7-[3-[(ethylamino)methyl]-1-pyrrolidinyl]-6,8-difluoro-1,4-dihydro-3-
quinolinecarboxylic Acid. New Quinolone Antibacterial with Potent
Gram-Positive Activity. J. Med. Chem. 1986, 29, 445–448.
(9) (a) Sanchez, J. P.; Domagala, J. M.; Hagen, S. E.; Heifetz, C. L.;
Hutt, M. P.; Nichols, J. B.; Trehan, A. K. Quinolone Antibacterial
Agents. Synthesis and Structure-Activity Relationships of 8-Substi-
tuted Quinolone-3-carboxylic Acids and 1,8-Naphthyridine-3-carboxy-
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Heifetz, C. L.; Hutt, M. P.; Mich, T. F.; Nichols, J. B.; Solomon, M.;
Worth, D. F. 1-Substituted 7-[3-[(Ethylamino)methyl]-1-pyrrolidinyl]-
6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic Acids. New Quan-
titative Structure-Activity Relationships at N1 for the Quinolone
Antibacterials. J. Med. Chem. 1988, 31, 991-1001.
by determining the concentration required to inhibit 50% of the
enzyme reaction.
Intravenous Single-Dose Toxicity. The test compounds were
dissolved in 0.1 mol/L NaOH in saline at different concentrations.
The solution was administered intravenously to three or five four-
week-old male ICR mice at the speed of 0.1 mL/1 min. The total
volume of administration was adjusted 10 mL/kg of body weight.
The number of dead mice was counted 7 days after administration.
Convulsive Activity Test. Ten µL of the test compounds solution
was intracerebroventricularly injected into four-week-old male ICR
mice. In experiments with concurrent NSAID, 20 min before
intracerebroventricular injection of the test article solution, γ-oxo-
(1,1′-bipenyl)-4-butanoic acid (fenbufen) was orally administered
at a dose of 200 mg/kg. Development of clonic convulsion was
examined until 2 h after injection.
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M. A. Fluoroquinolones: Relationships between Structural Variations,
Mammalian Cell Cytotoxicity, and Antimicrobial Activity. J. Med.
Chem. 1992, 35, 4745–4750.
Phototoxicity Test. The back of mice was shaved and then
ultraviolet A was irradiated for 3 h (4.59 J/cm2) 60 min after oral
administration of the test compounds. The erythema of skin was
evaluated 72 h after completion of the irradiation by scoring (0,
nil; 1, slight; 2, moderate; 3, marked change).
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thesis and Structure-Activity Relationships of 5-Amino-6-fluoro-1-
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Acknowledgment. We thank Drs. Keiji Hirai and Takayoshi
Ishizaki, Kyorin Pharmaceutical Co. Ltd., for their encourage-
ment and support of this project. We also thank Hinako Gomori,
Kyorin Pharmaceutical Co. Ltd., for providing the antibacterial
data.
Supporting Information Available: Purity data for compounds
4-11, 13b, 21, 36-38. This material is available free of charge
via the Internet at http://pubs.acs.org.
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