Design, Synthesis, and Biological Evaluation of Novel 7-[(3 aS,7 aS)-3 a-Aminohexahydropyrano[3,4- c]pyrrol-2(3 H)-yl]-8-methoxyquinolines with Potent Antibacterial Activity against Respiratory Pathogens

Article abstract of DOI:10.1021/acs.jmedchem.8b00644

Novel 7-[(3aS,7aS)-3a-aminohexahydropyrano[3,4-c]pyrrol-2(3H)-yl]-6-fluoro-1-[(1R,2S)-2- fluorocyclopropyl]-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 5 (DS21412020) was designed and synthesized to obtain potent antibacterial drugs for the tre

Full text of DOI:10.1021/acs.jmedchem.8b00644

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Article
Design, Synthesis, and Biological Evaluation of Novel 7-[(3aS,7aS)-3a-
Aminohexahydropyrano[3,4-c]pyrrol-2(3H)-yl]-8-methoxyquinolines
with Potent Antibacterial Activity Against Respiratory Pathogens
Takashi Odagiri, Hiroaki Inagaki, Masatoshi Nagamochi,
Takahiro Kitamura, satoshi komoriya, and Hisashi Takahashi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00644 • Publication Date (Web): 31 Jul 2018
Downloaded from http://pubs.acs.org on August 1, 2018
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Journal of Medicinal Chemistry
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Design, Synthesis, and Biological Evaluation of
Novel 7ꢀ[(3aS,7aS)ꢀ3aꢀAminohexahydropyrano[3,4ꢀ
c]pyrrolꢀ2(3H)ꢀyl]ꢀ8ꢀmethoxyquinolines with Potent
Antibacterial Activity Against Respiratory
Pathogens
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Takashi Odagiri,†,* Hiroaki Inagaki,† Masatoshi Nagamochi,† Takahiro Kitamura,‡ Satoshi
Komoriya,† and Hisashi Takahashi†
†R&D division, Daiichi Sankyo CO., Ltd, 1ꢀ2ꢀ58 Hiromachi, Shinagawaꢀku, Tokyo 140ꢀ8710,
Japan
‡R&D division, Daiichi Sankyo RD Novare CO., Ltd, 1ꢀ16ꢀ13 Kitakasai, Edogawaꢀku, Tokyo
134ꢀ8630, Japan
.
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ABSTRACT.
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Novel
7ꢀ[(3aS,7aS)ꢀ3aꢀaminohexahydropyrano[3,4ꢀc]pyrrolꢀ2(3H)ꢀyl]ꢀ6ꢀfluoroꢀ1ꢀ[(1R,2S)ꢀ2ꢀ
9
fluorocyclopropyl]ꢀ8ꢀmethoxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic acid 5 (DS21412020)
was designed and synthesized to obtain potent antibacterial drugs for the treatment of respiratory
tract infections. Compound 5 possessing a transꢀfused pyranose ring on the pyrrolidine moiety at
the Cꢀ7 position of the quinolone scaffold exhibited potent in vitro antibacterial activity against
respiratory pathogens, including quinoloneꢀresistant and methicillinꢀresistant Staphylococcus
aureus (QRꢀMRSA) and quinoloneꢀresistant Escherichia coli (QRꢀE. coli). Furthermore,
compound 5 showed in vivo activity against the experimental murine pneumonia model due to
penicillinꢀresistant Streptococcus pneumoniae (PRSP) and favorable profiles in preliminary
toxicological and nonclinical pharmacokinetic studies. In particular, the reduced lipophilicity and
basicity of compound 5 compared to those of the previously synthesized carbaꢀtype compound 4
resulted in a significant reduction in the human etherꢀaꢀgoꢀgo (hERG) related gene channel
inhibition which have the potential to prolong the QT interval.
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INTRODUCTION
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After various antibiotics have been successfully used for decades, the emergence of antibiotic
resistance among Gramꢀpositive and Gramꢀnegative bacteria makes a serious threat for public
health. Recently, the innovation rate of novel antibiotics is unable to keep up with the emergence
of antibioticꢀresistant bacteria. In the United States, It is estimated that the impact of antibioticꢀ
resistant bacteria causes over 23,000 deaths, 2 million illnesses, and costs amounting to 20
billion dollars each year.1 Because of the increasing levels of resistance to βꢀlactams and
macrolides exhibited by communityꢀacquired pathogens, such as the multidrugꢀresistant
Streptococcus pneumoniae (MDRSP),2,3 newer quinolones are increasingly being used as firstꢀ
line antibacterial therapy for respiratory tract infections in clinical settings. Respiratory
fluoroquinolones, including levofloxacin (LVFX) and moxifloxacin (MFLX), have been
important therapeutic options for communityꢀacquired bacterial pneumoniae (CABP) due to their
enhanced activity against Streptococcus pneumoniae, compared to ciprofloxacin (CPLX),
coupled with favorable pharmacokinetic (PK) and safety profiles.4ꢀ6 However, the antibacterial
activity of these newer quinolones may be insufficient to prevent the emergence of strains such
as quinoloneꢀresistant S. pneumoniae (QRSP) and communityꢀacquired methicillinꢀresistant
Staphylococcus aureus (CAꢀMRSA).7,8 Due to their insufficient activity against MRSA, US
Food and Drug Administration (FDA) doesn’t recommend using fluoroquinolones in the
treatment of MRSAꢀrelated infections.9 Furthermore, clinical adverse events (e.g., Torsades de
Pointes or fatal liver injury) have been shown to be associated with some quinolones.10ꢀ14
Therefore, novel quinolone antibiotics exhibiting improved activity with minimal adverse effects
are required.
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As previously reported,15,16 compound 1 shows potent activity against respiratory pathogens,
including PRSP, together with a superior safety profile similar to that of LVFX. As a result of
further research,17 we found that compound 4, which has a transꢀfused bicyclic structure at the
Cꢀ7 position, also has potent activity against respiratory pathogens; however, it inhibits hERG
potassium channels.
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Generally, the degree of hERG inhibition closely relates to the lipophilicity of the compound,
which is expressed as LogD or P’.18 Therefore, we planned to introduce an oxygen atom into the
structure of compound 4 at the Cꢀ7 pyrrolidine substituent, in order to decrease its P’ value.
Moreover, it is known that the degree of hERG inhibition correlates with the basicity of the
compound and that reducing the basicity of a compound is effective in preventing hERG
inhibition.19 According to a report by Morgenthaler et al, the γꢀposition (Figure 1, γ1 and γ2)
from the amino group on the Cꢀ7 side chain would be appropriate for the introduction of an
oxygen atom.19 Based on the knowledge that the introduction of an heteroatom into the γ2ꢀ
position reduces antibacterial activity,20 compound 5 bearing an oxygen atom at the γ1ꢀposition
from the amino group at the Cꢀ7 side chain was designed.
In this article, we describe the details of the synthesis, in vitro and in vivo antibacterial
activities, safety profile, and pharmacokinetic profile of the designed compound 5. Since the
absolute stereochemistry of the amino group on the Cꢀ7 side chain greatly affects the strength of
the antibacterial activity,15,16 the synthesis was carried out only for the enantiomer having more
potent activity.
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Figure 1. Design of novel compound 5.
CHEMISTRY
As shown in Scheme 1, we planned to synthesize the desired 7ꢀsubstituted 8ꢀ
methoxyquinolone derivative 5 by an aromatic nucleophilic substitution reaction from amine 6
and 6,7ꢀdifluoroꢀ1ꢀ[(1R,2S)ꢀ2ꢀfluorocyclopropanꢀ1ꢀyl]ꢀ8ꢀmethoxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ
3ꢀcarboxylic acid BF2 chelate 7,21 in which the BF2 chelate was known to improve the reactivity
at the Cꢀ7 position.22 We planned to prepare amine 6 by the hydrogenation of conjugated amide
8, which would be synthesized from known compound 9.23
Scheme 1. Synthetic Route to Compound 5
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The synthesis of intermediates 14 and 15 is illustrated in Scheme 2. Hydroxymethylation of
known compound 9 gave 10 as the major product. The minor product obtained in trace amounts
could not be isolated due to contamination with an impurity. Deoxygenation of 10 gave
methylate 11, whose 1HꢀNMR spectrum matched that of the reported compound.23 Therefore, the
absolute stereochemistry of 10 was determined to be the desired (3S). After alkylation of 10,
intramolecular cyclization was accomplished by employing lithium hexamethyldisilazide
(LHMDS). Subsequent reduction with NaBH4 gave alcohol 13 as a diastereomeric mixture,
which was converted to key intermediate 8 by sequential oneꢀpot mesylation and elimination
with the use of DBU. Hydrogenation of conjugate amide 8 gave cis and trans diastereomers 14
and 15 in approximately 4:1 ratio. Compound 14 was obtained as prisms, and its absolute
stereochemistry was determined to be 7aS by XꢀRay crystallographic analysis (Figure 2).
Unfortunately, the undesired cisꢀisomer 14 was the major product in the hydrogenation step.
Scheme 2. Synthesis of Key Intermediate 14a
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O
O
O
O
OH
COOtBu
Ph
O
O
a
b
O
10
=
O
N
O
O
N
N
N
Ph
Ph
Ph
11
10
9
9
O
O
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COOtBu
OH
COOtBu
O
HO
c
COOEt
e
COOtBu
d
COOtBu
O
O
N
O
N
O
N
N
Ph
Ph
O
Ph
Ph
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12
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8
O
O
COOtBu
f
COOtBu
COOtBu
O
O
O
N
N
N
+
Ph
Ph
Ph
8
14
15
aReagents and conditions: (a) (HCHO)n, NaH, DMF, 23%; (b) (1) PhSSPh, Bu3P, THF, 65 °C,
(2) H2, RaneyꢀNi, EtOH, 12%; (c) NaH, ethyl bromoacetate, Bu4NI, DMF, 84%; (d) (1)
LHMDS, THF, –40 °C, (2) NaBH4, (e) (1) MsCl, triethylamine, CH2Cl2, –10 °C, (2) DBU, 40
°C, 36% from 12, (f) H2, Pd/C (wet), EtOAc, 14 (79%), 15 (21%).
Figure 2. ORTEP of compound 14.
As described above, the desired transꢀisomer 15 was the minor product in the Pdꢀcatalyzed
reduction process. Therefore, we attempted to investigate the reaction conditions to increase the
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ratio of 15. As a chelate reagent, MgBr2 was selected as the additive with reference to the report
by Nagano et al.24 As shown in Table 1, when MgBr2 (2.5eq.) was added, the reaction did not
proceed at all under an atmospheric pressure of hydrogen (entry 2). We speculated that MgBr2
was effectively chelated on the surface of 8. Thus, increasing the amount of catalyst and
hydrogen pressure up to 50 atm facilitated the progress of the reaction, and 15 was obtained as
the main product (entry 3). When ethanol was mixed with the reaction solvent for reducing the
amount of the catalyst and accelerating the hydrogenation, the reaction proceeded at atmospheric
pressure, although the selectivity was slightly reduced (entry 4). There was a positive correlation
between the hydrogen pressure and the selectivity, consistent with Le Chatelier’s law.
Specifically, the equilibrium between the chelated form (single molecule) and the dechelated
form (two molecules) was shifted toward the chelate side upon increasing the hydrogen pressure.
Finally, an effective synthesis of the oxa transꢀform 15 was achieved under the conditions shown
in entry 6.
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Table 1. Reaction Conditions for Hydrogenation
Additive
(MgBr2)
Solvent
Time
Pressure
(atm)
1a
Pd/C (50%wet)
14 (cis)b/
15 (trans)b
79/21 %
(wt%)
50
1
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4
5
6
none
2.5 eq.
1 eq.
1 eq.
1 eq.
1 eq.
EtOAc
EtOAc
EtOAc
1 day
1 day
1 day
1a
50
No Reaction.
23/77 %
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200
50
EtOAc:EtOH=2:1 3 days
1a
ꢀc/71 %
EtOAc:EtOH=2:1
14 h
14 h
10
50
ꢀc/83 %
EtOAc:EtOH=2:1
30
50
ꢀc/89 %d
b
c
d
aBalloon pressure. Isolated yield. Not isolated. Experimental details described in the
experimental section.
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Oxa cisꢀfused compound 19 was synthesized as shown in Scheme 3. After the reduction of 14
with a BH3–THF complex, the phenylethyl group was converted to the benzyloxycarbonyl
group. Then, the tꢀBu group of 16 was removed, and the carboxylic acid was subsequently
converted to the carbamoyl group. Furthermore, the carbamoyl group was subjected to Hoffman
rearrangement conditions, and Bocꢀprotected amine 17 was obtained as a single product. The
benzyloxycarbonyl group of 17 was then removed by catalytic hydrogenolysis, and the resultant
crude substance was heated with quinolonecarboxylic acid BF2 chelate 7 and triethylamine in
DMSO; finally oxa cisꢀfused compound 19 was obtained after dechelation and deprotection of
the Boc group.
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Scheme 3. Synthesis of Cisꢀfused Compound 19a
aReagents and conditions: (a) (1) BH3–THF complex, THF, (2) CbzCl, 1,2ꢀdichloroethane, 40
°C, 68%; (b) (1) TFA, CH2Cl2, (2) 1,1'ꢀcarbonylbisꢀ1Hꢀimidazole, CH3CN, 0 °C, then NH3 gas,
(3) Pb(OAc)4, tꢀBuOH, 80 °C, 65%; (c) H2, Pd/C (wet), MeOH; (d) (1) 7, TEA, DMSO, (2)
TEA, 80% aqueous EtOH, reflux, (3) concentrated aqueous HCl, 67% from 17. TEA =
triethylamine.
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Oxa transꢀfused isomer 15 was converted to Cbzꢀprotected amine 20 by a similar method as
that for cisꢀfused isomer 14 (Scheme 4). Initially, we attempted the Hoffmann rearrangement as
in the case of the cisꢀfused isomer, but the reaction did not proceed at all. Although the reasons
are unknown, the bulkiness of the transꢀform scaffold is presumed to prevent the reaction.
Instead, the Curtius rearrangement proceeded with high yield, and the desired Bocꢀprotected
amine 21 was obtained as a single product. The subsequent steps were carried out in the same
manner as in the case of the cisꢀfused isomer, and the targeted oxa transꢀfused compound 5
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1
(DS21412020) was synthesized. Finally, the HꢀNMR spectrum of 5 was confirmed to be
different from that of 19.
Scheme 4. Synthesis of Transꢀfused Compound 5a
aReagents and conditions: (a) (1) BH3–THF complex, THF, 40 °C, (2) CbzCl, 1,2ꢀ
dichloroethane, 35 °C, 82%; (b) (1) TFA, CH2Cl2, (2) diphenylphosphorylazide, TEA, toluene,
110 °C, (3) 6N aqueous HCl, 1,4ꢀdioxane, 60 °C, (4) Boc2O, 50 °C, 83%; (c) H2, Pd/C (wet),
MeOH; (d) (1) 7, TEA, DMSO, (2) TEA, 80% aqueous EtOH, reflux, (3) concentrated aqueous
HCl, 72% from 21.
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RESULTS AND DISCUSSION
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The minimum inhibitory concentrations (MICs) of the synthesized compounds 5 and 19
against several representative Gramꢀpositive and Gramꢀnegative bacteria are summarized in
Table 2, along with the corresponding data for LVFX, MFLX, and the previously reported
compounds 1–4 for comparison. Oxa transꢀfused compound 5 showed a broad antibacterial
spectrum against Gramꢀpositive and Gramꢀnegative bacteria. In particular, the activity of 5
against Gramꢀpositive bacteria was 2ꢀ to 4ꢀfold stronger than that of oxa cisꢀfused compound 19
and control compound 1, although the activity of 5 against some of the bacteria was slightly
weaker than that of carba transꢀfused compound 4.
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Table 2. Antibacterial Activities MIC (ꢀg/mL) of Synthesized Compounds and Reference
Quinolones against Gramꢀpositive and Gramꢀnegative Bacteriaa
Compounds
5
19
2
3
4
LVFX MFLX 1
0.025 0.012
Organisms
S. aureus SMITH
S. pneumoniae J24b
S. pneumoniae J41b
0.006 0.012 <0.003 0.006 <0.003 0.1
0.1
0.2
0.2
0.2
0.05
0.1
0.05
0.05
0.025 0.78
0.025 1.56
0.025 0.39
0.1
0.1
0.2
0.05
0.1
0.05
S. pyogenes ATCC 12344 0.05
0.025 0.1
0.2
E. faecalis ATCC 19433
0.2
0.39 0.1
0.2
0.1
0.78
0.2
0.2
B. subtilis ATCC 6633
E. coli NIHJ
0.006 0.025 0.012 0.012 0.006 0.05
0.006 0.05 0.025 0.025 0.006 0.012 0.012 0.012
0.05 0.2 0.1 0.1 0.05 0.05 0.1 0.05
0.025 0.012
K. pneumoniae TYPE 1
H. influenzae ATCC49247 0.006 0.012 0.006 0.025 0.012 0.012 0.012 0.012
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M(B).
ATCC25238
catarrhalis
0.025 0.1
0.025 0.1
1.56
0.025 0.025 0.05
0.39 0.39 0.78
0.05
0.78
P. aeruginosa PAOꢀ1
0.78 3.13 0.39
7
aAntibacterial activities were determined using a standard microbroth dilution method, MIC
8
9
ꢁg/mL. bPenicillinꢀsusceptible S. pneumoniae (PSSP).
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The antibacterial activities against several resistant bacteria and mutant strains are shown in
Table 3. Oxa transꢀfused compound 5 exhibited potent antibacterial activity against MRSA,
resistant E. coli, and mutant E. coli. The antibacterial activity of 5 was stronger than that of 1 and
slightly weaker than that of 4; the same tendency was observed against representative bacteria
pathogens.
Table 3. Antibacterial Activities MIC (ꢀg/mL) of Synthesized Compounds and Reference
Quinolones against Resistant Bacteria and Mutant Strainsa
Compounds
5
19
2
3
4
LVFX MFLX 1
>6.25 0.78 0.78
Organisms
MRSA 870307a
MRSA 890325ꢀ1a
PRSP 033806b
E. coli DNS5101c
E. coli 5ꢀ037042 '98d
0.2
0.78
0.78
ꢀ
0.78 0.39 0.1
0.78 1.56 0.1
0.2
6.25
1.56
1.56
0.25
0.78
0.12
0.12
ꢀ
ꢀ
ꢀ
1.56 >6.25 6.25 >6.25 1.56 >6.25 >6.25 6.25
0.05 0.39 0.1 0.1 0.025 0.1 0.1 0.05
b
aLevofloxacinꢀresistant and methicillinꢀresistant S. aureus (levofloxacinꢀrꢀMRSA). Penicillinꢀ
resistant S. pneumoniae. cQuinoloneꢀresistant E. coli. dGyrase A mutation: Asp87→Gly.
Finally, the MICs against clinically isolated MRSA of compound 5 and reference quinolones are
shown in Table 4. The MIC90 of compound 5 was 0.5 ꢀg/mL, superior to the other quinolones.
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Thus, compound 5 was considered to have potent activity against not only representative
5
6
respiratory pathogens but also resistant strains.
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Table 4. Antibacterial Activities MIC (ꢀg/mL) against MRSA (44 strains)a,b
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MICrange
0.008 ꢀ 8
0.25 ꢀ >64
0.015 ꢀ 16
0.008 ꢀ 8
MIC50
0.015
0.5
MIC90
5
0.5
8
LVFX
MFLX
GRNXc
1
0.06
0.03
0.03
2
1
0.015 ꢀ 4
1
b
a Clinical Isolates collected in Japan. S. aureus ATCC 29213 was used as the quality control
strains for MRSA. The quality control strains were included as internal controls throughout the
study and the control results were within acceptable ranges defined by CLSI (M100ꢀS16).
c
GRNX : garenoxacin.
Table 5 shows the result of the synthesized compounds on the hERG potassium current in
hERGꢀtransfected cells along with the apparent partition coefficient value (P’).
Electrocardiogram studies revealed that compounds inhibit hERG potassium channels have the
potential to prolong the QT interval and increase the risk of fatal cardiac arrhythmia.25,26 As
mentioned above, carba transꢀfused compound 4 inhibited hERG currents at a concentration of
30 ꢀM, and the hERG inhibition of 4 was almost equal to that of MFLX, which is known to
cause QT prolongation syndrome at clinical dosage.27 On the other hand, oxa compounds 5 and
19 showed almost no hERG inhibitory activity, as was anticipated on the basis of their structures.
Although the P’ value (lipophilicity) of 5 was reduced compared to that of carba transꢀfused
compound 4, it was almost the same as that of MFLX. Therefore, the decrease in lipophilicity is
not the only factor causing the suppression of hERG inhibition. Instead, the reduction in basicity,
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achieved by the introduction of an oxygen atom at the γ1ꢀposition from the amino group of 5,
may be responsible for this change. We had established an inhibition threshold within 10% at a
concentration of 30 ꢀM, so compound 5 and 19 meet this criterion.
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Table 5. Effects on hERG Potassium Current in hERG Transfected Cellsa and Apparent Partition
Coefficient (P’)b
Concentration (ꢁM)
Compound
P’
30
ꢀ2.2
ꢀ6.4
19
100
ꢀ2.5
ꢀ7.5
36
5c
19d
49
37
4d
>68
5.1
54
LVFXc
MFLXc
1c
ꢀ0.9
22
4.2
42
1.9
2.7
19
aData represent % inhibition.28,29 bApparent partition coefficient, CHCl3/0.1M phosphate buffer
(pH 7.4).30 cCHOꢀK1 cells. dHEK293 cells.
The metabolic stability, protein binding, and mechanismꢀbased inhibition (MBI) against
CYP3A4 are shown in Table 6. Both 5 and 19 showed high metabolic stability in human
microsomes. The protein binding rate was very low for both compounds. In particular, the
protein binding rate for compound 5 was almost 0%. Furthermore, the potential inhibitory effects
of compound 5 on the MBI against CYP3A4 was investigated. Compound 5 did not show any
inhibitory effect on the MBI against CYP3A4.
Table 6. Metabolic Stability, Protein Binding, and MBI against CYP3A4a
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Protein
CYP3A4
Metabolic
Compound
Stabilitya
Bindingb MBIc
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9
100
100
95
0
97.4
ꢀ
5
12
14
1.3
5.4
19
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LVFX
MFLX
1
104
97.9
78.4d
92
97
aThe % remaining value using human microsomes. 1 ꢀM, 30 min. Protein binding (%) to
human albumin The % remaining value at a 10 ꢀM concentration of compounds reacted with
b
c
d
CYP3A4 probe substrates after 30 min preincubation in human liver microsomes. As a result of
detailed examination, it was judged to be negative.15
The pharmacokinetics (PK) profiles of the synthesized compounds, LVFX, MFLX, and 1
following single oral administration in rats are shown in Table 7. The maximum drug
concentration (Cmax) and area under the time–concentration curve (AUC) for compound 5 were
almost equal to those of compound 1. Furthermore, investigation of the PK profile in monkeys
revealed that compound 5 had a higher blood concentration than compound 1, LVFX, and
MFLX (Table 8). The urinary recovery rate of 5 was as high as that of LVFX. Thus, compound 5
is expected to be effective in the treatment of urinary tract infections.
Table 7. Pharmacokinetic Parameters of Synthesized Compounds and Reference Quinolones in
Rats After an Oral Dose of 5 mg/kg (n = 3)a
PK parameters
LVFX
1.47
MFLXb
1.49
5
2
4
1
Cmax (ꢁg/mL)
0.88
0.049
1.28
1.22
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3.14
0.21
4.76
3.41
4.46
3.08
AUC0ꢀ8h (ꢁg·h/mL)
5
6
7
aAnimal: 7ꢀweekꢀold male Crj:CD rats. bMoxifloxacin hydrochloride hydrate was
administered.
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Table 8. Pharmacokinetic Parameters of Synthesized Compounds and Reference Quinolones in
Monkeys after a Dose of 5 mg/kg (n = 3)a
Route
p.o.
PK parameters
Cmax (ꢁg/mL)
5b
LVFX
1.92
MFLXc
1.03
1
3.7
2.2
AUC0ꢀ24h
(ꢁg·h/mL)
Serum
19.8
16.9
15.2
6.63
t
1/2 (h)
4.6
64.1
75
4.8
61.3
71
3.5
73.9
ꢀ
5.3
8.1
Urinary recovery0ꢀ24h (%)d
Bioavailability (%)
C5min (ꢁg/mL)
61
7.0
3.66
4.17e
2.36
AUC0ꢀ8h
Serum
22.9
23.8
15.5e
10.9
(ꢁg·h/mL)
i.v.
3.5
4.8
3.2f
4.8
t
1/2α (h)
Urinary recovery0ꢀ24h (%)d
65.0
69.8
80.7f
11.0
b
aAnimal: female cynomolgus monkeys. p.o.: MsOH salt (0.5 H2O), see experimental section
c
d
(5a), i.v.: free (5). Moxifloxacin hydrochloride hydrate was administered. The values were
e
calculated by using the concentrations of unchanged quinolones. The values result from the
division of data for 10 mg/kg intravenous administration by levofloxacin. The values are the
f
results for a 10 mg/kg intravenous administration.
The therapeutic efficacies of oxa transꢀfused compound 5 in experimental murine pneumonia
models due to penicillinꢀresistant S. pneumoniae (PRSP033806) are compared with the efficacies
of moxifloxacin and compound 1 in Figure 3. The therapeutic efficacy of subcutaneously
administrated 5 was superior to that of moxifloxacin and equal to that of compound 1. Based on
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the PK/PD theory of antibacterial drugs,31 the efficacy of compound 5 after oral administration
can be expected sufficiently for clinical treatment.
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Figure 3. Therapeutic effects on pneumococcal pneumonia (PRSP) in mice. Animals:
CBA/JNCrj mice (n = 4). Bacteria: S. pneumoniae 033806 (PRSP). Test drugs were administered
subcutaneously twice a day (2 h and 8 h after infection).
The result of convulsion incidence test was shown in Table 9. As a representative side effect of
quinolone drugs, it is known that there is a case of rarely enhancing convulsion induction when
used in combination with NSAIDs32. The convulsion inducing activity of compound 5 itself was
weaker than the other quinolones. Furthermore, when BPAA was used in combination, 5 did not
show the effect of enhancing convulsion induction as much as compound 1.
Table 9. Effect of Ciprofloxacin (CPFX), LVFX and compounds on Convulsion Incidence in
Mice
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6
Dose
Incidence of Convulsion (Death)
Compound
(ꢁg/5ꢁl/mouse
, i.cist.)
Without BPAA
With BPAAa
7
8
9
5
0/6ꢀ1/6 (0/6)
4/6ꢀ6/6 (1/6ꢀ0/6)
6/6 (2/6ꢀ5/6)
0/6
6/6 (6/6)
6/6 (6/6)
6/6 (6/6)
1/6
CPFX
LVFX
1
15
50
5
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5
1/6
6/6
0/6 (0/6)
2/6 (0/6)
6/6 (0/6)
0/6 (0/6)
0/6 (0/6)
4/6 (0/6)
0/6 (0/6)
3/6 (0/6)
5/6 (0/6)
0/6 (0/6)
0/6 (0/6)
1/6 (0/6)
15
50
5
5
15
50
aBPAA : 4ꢀBiphenylacetic Acid (reactive metabolite of fenbufen). 30 minutes before the drug solution administration, BPAA 400
mg/kg was orally administered at a volume of 10 mL/kg.
CONCLUSION
Novel oxa transꢀfused compound 5 (DS21412020), a 6ꢀfluoroꢀ8ꢀmethoxyquinolone possessing
a [(3aS,7aS)ꢀ3aꢀaminohexahydropyrano[3,4ꢀc]pyrrolꢀ2(3H)ꢀyl] group at the Cꢀ7 position of the
scaffold, was designed, synthesized, and evaluated. Compound 5 exhibited about 2ꢀ to 4ꢀfold
better antibacterial activity in vitro against Gramꢀpositive bacteria as compared with the
previously reported compound 1.15 Compound 5 also showed high AUCs in rats and monkeys,
and stronger in vivo antibacterial activity against penicillinꢀresistant S. pneumoniae infection in
mice than that of MFLX. Furthermore, oxa compound 5, which was designed to present lower
lipophilicity and basicity than does carba compound 4, solved the problem of strong hERG
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inhibition. Finally, we developed an efficient method for the synthesis of the oxa transꢀfused
5
6
intermediate by chelate control using MgBr2.
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EXPERIMENTAL SECTION
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General. All melting points were recorded on a Yanaco MPꢀ500D melting point apparatus and
reported uncorrected. Optical rotations were measured in a 0.5 dm cell at 25 °C at 589 nm with a
1
HORIBA SEPAꢀ300 polarimeter. H NMR spectra were recorded on a JEOL JNMꢀEX400
spectrometer. Chemical shifts are reported in parts per million relative to tetramethylsilane or
1
sodium 2,2ꢀdimethylꢀ2ꢀsilapentaneꢀ5ꢀsulfonate as internal standards. Significant H NMR data
are tabulated in the following order: number of protons, multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet), coupling constant(s) in hertz. Mass spectra were obtained on a
JEOL JMSꢀ700 mass spectrometer under electron impact ionization conditions (EI), electron
spray ionization conditions (ESI), or fast atom bombardment ionization conditions (FAB). Highꢀ
resolution mass (HRMS) spectra were recorded on a JEOL JMSꢀ100LP spectrometer. Elemental
analyses are indicated only by the symbols of the elements; analytical results were within 0.4%
of the theoretical values. Purities of ≥95% were determined by elemental analysis (all tested
compounds). Column chromatography refers to flash column chromatography conducted on
Merck silica gel 60, 230–400 mesh ASTM. Thinꢀlayer chromatography (TLC) was performed
with Merck silica gel 60 F254 TLC plates, and compound visualization was effected with a 5%
solution of molybdophosphoric acid in ethanol, UV lamp, iodine, or Wako ninhydrin spray. All
experimental procedures for the animals were performed in accordance with the guidelines of the
Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd.
In Vitro Antibacterial Activity. The MICs of the compounds tested in this study were
determined by the twoꢀfold micro dilution method using Mueller–Hinton broth (Difco
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Laboratories, Detroit, MI) with an inoculum size of approximately 105 CFU per well. The MIC
was defined as the lowest concentration that prevented visible bacterial growth after incubation
at 35 °C for 18 h.
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In Vivo Efficacy against S. pneumoniae Infection in Mice. Threeꢀweekꢀold male CBA/JNCrj
mice (4 mice/group) were used. The bacteria used for infection was quinoloneꢀsensitive S.
pneumoniae 033806 (penicillinꢀresistant, QSꢀPRSP), which was inoculated by nasal drops
(Inoculam Size : 10^6 CFU/mouse, Inoculation Volume : 0.05 mL). One day after the infection,
drugs were administered subcutaneously twice a day (2 h and 8 h after infection, administration
volume : 0.1 mL / 10 g body weight). The number of bacteria in the lungs was examined on the
day after the final administration of drug. The lungs were removed aseptically and weighed, and
then the viable bacterial counts were determined. The detection limit was ≥2.30 log10 CFU/g of
lung.
7ꢀ[(3aS,7aS)ꢀ3aꢀAminohexahydropyrano[3,4ꢀc]pyrrolꢀ2(3H)ꢀyl]ꢀ6ꢀfluoroꢀ1ꢀ[(1R,2S)ꢀ2ꢀ
fluorocyclopropyl]ꢀ8ꢀmethoxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic Acid (5)
To a solution of 21 (610 mg, 1.62 mmol) in MeOH (20 mL) was added 10% Pd/C (200 mg,
containing 50% water), and the mixture was stirred for 3 h at ambient temperature under a
hydrogen atmosphere. The catalyst was filtered off, and the filtrate was concentrated in vacuo.
The residue was mixed with 7 (566 mg, 1.57 mmol), TEA (0.621 mL, 4.48 mmol), and DMSO
(8 mL). The mixture was stirred for 18 h at 40 °C. To this solution were added 90% aqueous
EtOH (150 mL) and TEA (15 mL), and the resultant mixture was stirred at 80 °C for 2.5 h, and
then concentrated in vacuo to give the residue, which was diluted with EtOAc. The organic layer
was washed with 10% aqueous citric acid solution, water, and brine dried over anhydrous
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Na2SO4, and concentrated in vacuo. To the residue was added concentrated aqueous HCl (4 mL)
at 0 °C, and the mixture was stirred for 20 min at ambient temperature. The aqueous solution was
washed with CHCl3 and made alkaline with saturated aqueous NaOH at 0 °C. The pH of the
solution was adjusted to 7.4 with concentrated aqueous HCl and then with diluted aqueous HCl.
The resultant solution was extracted with CHCl3 (3 ×), the combined organic solution dried over
anhydrous Na2SO4 and concentrated in vacuo. The resultant solid was recrystallized from EtOH
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1
to yield 5 (470mg, 72%) as a pale yellow powder, m.p. 190–192 °C. H NMR (0.1 N
NaOD/D2O): δ 1.32–1.36 (1H, m), 1.48–1.54 (1H, m), 1.71–1.88 (2H, m), 2.11–2.14 (1H, m),
3.24 (1H, d, J = 10.3 Hz), 3.51–3.67 (8H, m), 3.95–4.02 (2H, m), 4.09 (1H, dd, J = 11.4, 4.0 Hz),
13
5.06 (1H, dd, J = 64.1, 3.5 Hz), 7.65 (1H, d, J = 14.4 Hz), 8.36 (1H, d, J = 3.7 Hz). C NMR
(0.1N NaOD/D2O): δ 13.92, 14.01, 22.80, 38.08, 39.59, 39.66, 42.88, 52.49, 52.55, 54.87, 58.88,
58.92, 60.49, 66.68, 71.67, 73.44, 74.43, 106.5, 106.7, 115.9, 119.6, 119.7, 133.9, 136.0, 136.1,
139.8, 139.9, 149.7, 151.8, 153.8, 172.2, 174.9, 175.0. MS (ESI) m/z: 436(M + H)+. [α]D = –
25
160.882° (c 0.27, 0.1N aqueous NaOH). Anal. calcd for C21H23F2N3O5·0.25H2O, C 57.33, H
5.38, F 8.64, N 9.55; found, C 57.28, H 5.39, F 8.48, N 9.27. IR (ATR): 3502, 3374, 3091, 2948,
2881, 2850, 1716, 1617, 1513 cmꢀ1.
7ꢀ[(3aS,7aS)ꢀ3aꢀAminohexahydropyrano[3,4ꢀc]pyrrolꢀ2(3H)ꢀyl]ꢀ6ꢀfluoroꢀ1ꢀ[(1R,2S)ꢀ2ꢀ
fluorocyclopropyl]ꢀ8ꢀmethoxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic
Methanesulfonic acid salt (5a·MsOH salt)
acid,
To a solution of 21 (1.79 g, 4.75 mmol) in MeOH (40 mL) was added 10% Pd/C (900 mg,
containing 50% water), and the mixture was stirred for 7 h at ambient temperature under a
hydrogen atmosphere. The catalyst was filtered off, and the filtrate was concentrated in vacuo.
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The residue was mixed with 7 (1.67 g, 4.64 mmol), TEA (1.84 mL, 13.3 mmol), and DMSO (6.5
mL). The mixture was stirred for 4 h at 40 °C. To this solution were added water (30 mL), and
the mixture was stirred for 0.5 h at ambient temperature. Then, the precipitate was filtered and
washed with water (20 mL). To a suspension of the resultant solid in 90% aqueous EtOH (100
mL) was added TEA (10 mL), and the mixture was stirred for 2 h at 80 °C. After concentration
in vacuo, EtOAc and 10% aqueous citric acid solution were added to the residue. The organic
solution was washed with water and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. To the residue were added isopropanol (4.7 mL) and methanesulfonic acid (847 mg, 8.81
mmol) at ambient temperature, and the mixture was stirred for 3 h at 50 °C and at 40 °C for 18 h.
After the mixture was stirred for 1 h at 0 °C, the precipitate was filtered and washed with cold
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1
isopropanol (10 mL) to yield 5a (2.06 g, 88%) as a pale yellow powder, m.p. >280°C. H NMR
(0.1N NaOD/D2O): δ 1.41–1.51 (2H, m), 1.78–1.93 (2H, m), 2.53–2.56 (1H, m), 2.70 (3H, s),
3.49–3.53 (1H, m), 3.53 (3H, s), 3.56 (1H, d, J = 12.7 Hz), 3.71 (1H, dd, J = 11.0, 5.4 Hz), 3.77
(1H, d, J = 12.2 Hz), 3.84 (1H, t, J = 9.0 Hz), 3.89 (1H, d, J = 12.2 Hz), 4.02–4.07 (1H, m), 4.12
(1H, dd, J = 11.5, 4.6 Hz), 4.17 (1H, d, J = 12.2 Hz), 5.02 (1H, d, J = 64.0 Hz), 7.20 (1H, d, J =
13.7 Hz), 8.60 (1H, d, J = 3.7 Hz). MS (ESI) m/z: 436(M + H)+. Anal. calcd for
C21H23F2N3O5·MsOH·0.5H2O, C 48.88, H 5.22, F 7.03, N 7.77, S 5.93; found, C 48.93, H 5.17,
F 7.07, N 7.59, S 6.12..
tertꢀButyl
(3aS)ꢀ1ꢀOxoꢀ2ꢀ[(1R)ꢀ1ꢀphenylethyl]ꢀ1,2,3,6ꢀtetrahydropyrano[3,4ꢀc]pyrroleꢀ
3a(4H)ꢀcarboxylate (8)
To a mixture of LHMDS (0.835mL, 1.3M in THF) and THF (3mL) was added a solution of 12
(200 mg, 493 mmol) in THF (3 mL) at –40 °C under a nitrogen atmosphere. The mixture was
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stirred for 20 min at –40 °C and at 0 °C for 30 min, and then saturated aqueous NH4Cl (30 mL)
and EtOAc (30 mL) were added. The resulting mixture was stirred for 30 min at ambient
temperature, then NaBH4 (224 mg, 594 mmol) was added, and the reaction mixture was stirred
for another 10 h at ambient temperature. The organic solution was washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The resultant residue was purified by
silica gel column chromatography, eluting with hexane/EtOAc = 33:67 to yield 13 as a colorless
oil (98.0 mg, diastereomixture ca 4 : 1).
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*NMR data of diastereomixture 13: 1H NMR (CDCl3): δ 1.45 (9H, s), 1.53 (3H, d, J = 7.1 Hz),
2.82 (0.8H, d. J = 10.8 Hz), 2.89 (0.2H, d, J = 5.4 Hz), 2.97 (0.8H, d, J = 10.7 Hz), 3.08 (0.8H,
s), 3.30ꢀ3.25 (1.4H, m), 3.45 (0.8H, d, J = 12.4 Hz), 3.83 (0.2H, m), 3.92 (0.8H, J = 12.2 Hz),
3.95–4.02 (0.2H, m), 4.06–4.13 (3H, m), 5.52 (1H, q, J = 7.2 Hz), 7.26–7.38 (5H, m). MS (ESI)
m/z: 362 (M + H)+.
To a solution of 13 and TEA (0.056 mL, 407 mmol) in CH2Cl2 (4 mL) was added
methanesulfonyl chloride (0.030 mL, 352 mmol) at –10 °C under a nitrogen atmosphere. After
stirring for 30 min, MeOH (0.030 mL) was added, and the reaction mixture stirred for 10 min at
ambient temperature. 1,8ꢀDiazabicyclo[5.4.0]ꢀ7ꢀundecene (0.202 mL, 1.36 mmol) was then
added, and the resultant mixture was stirred for 10 h at 40 °C. Afterwards, 10 wt% citric acid
aqueous solution and EtOAc were added. The organic solution was washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The crude was purified by silica gel
column chromatography, eluting with hexane/EtOAc = 33:67 to yield 8 (60.5 mg, 36 % from 12)
as a colorless solid, mp 132–134 °C.1H NMR (CDCl3): δ 1.27 (9H, s), 1.51 (3H, d, J = 7.1 Hz),
3.13 (1H, d, J = 10.0 Hz), 3.21 (1H, d, J = 10.0 Hz), 3.26 (1H, d, J = 10.3 Hz), 4.24 (1H, dd, J =
18.6, 2.4 Hz), 4.45–4.50 (2H, m), 5.57 (1H, d, J = 7.2 Hz), 6.60 (1H, t, J = 2.3 Hz), 7.32–7.29
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(5H, m). MS (ESI) m/z: 344 (M + H)+. [α]D = –4.124° (c 1.0, CHCl3). Anal. calcd for
5
6
C20H25NO4ꢀ0.25·H2O: C 69.04, H 7.39, N 4.03; found, C 69.01, H 7.42, N 4.03.
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tertꢀButyl
3ꢀ(Hydroxymethyl)ꢀ5ꢀoxoꢀ1ꢀ[(1R)ꢀ1ꢀphenylethyl]pyrrolidineꢀ3ꢀcarboxylate
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(10). To a solution of 9 (4.0g, 13.8 mmol) in DMF (40 mL) were added paraformaldehyde (0.83
g, 27.7 mmol) and NaH (60% in oil, 0.60 g, 13.8 mmol) at ambient temperature. After stirring
for 0.5 h, the mixture was poured into 10% citric acid aqueous solution at 5 °C. After dilution
with EtOAc, the organic solution was washed with water (3×) and brine. The organic layer was
dried over anhydrous Na2SO4 and concentrated in vacuo. The resultant residue was purified by
silica gel column chromatography, eluting with hexane/EtOAc = 1:1 to yield 10 (1.0 g, 23%) as a
colorless solid, mp 88–90 °C. Another stereoisomer was obtained as a mixture with an impurity.
1H NMR (CDCl3): δ 1.35 (9H, s), 1.53 (3H, d, J = 7.3 Hz), 2.40 (1H, d, J = 17.3 Hz), 2.51 (1H,
dd, J = 7.8, 5.4 Hz), 2.78 (1H, d, J = 17.1 Hz), 3.21 (1H, d, J = 10.3 Hz), 3.39 (1H, d, J = 10.5
Hz), 3.61 (1H, dd, J = 11.2, 7.8 Hz), 3.77 (1H, dd, J = 11.2, 5.4 Hz), 5.51 (1H, q, J = 7.2 Hz),
7.28–7.34 (5H, m). MS (ESI) m/z: 320 (M + H)+. [α]D25 = 95.350° (c 1.0, CHCl3). HRMS (ESI)
calcd for C18H26NO4: 320.1862. Found: 320.1860.; Anal. calcd for C18H25NO4, C 67.69, H 7.89,
N 4.39; found, C 67.34, H 7.82, N 4.47.
tertꢀButyl (3S)ꢀ3ꢀMethylꢀ5ꢀoxoꢀ1ꢀ[(1R)ꢀ1ꢀphenylethyl]pyrrolidineꢀ3ꢀcarboxylate (11)
To a solution of 10 (165 mg, 0.516 mmol) in THF (10 mL) were added diphenyl disulfide (169
mg, 0.775 mmol) and Bu3P (0.26 mL, 1.03 mmol) at ambient temperature. The mixture was
stirred at 65 °C for 15 h. The reaction mixture was allowed to cool to ambient temperature, and
diluted with EtOAc; then, the organic solution was washed with 1N aqueous NaOH solution,
dried over anhydrous MgSO4, and concentrated in vacuo. The resultant residue was purified by
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silica gel column chromatography, eluting with hexane/EtOAc = 3:7. To a solution of resultant
residue (31 mg) in EtOH (6 mL) was added Raney nickel (about 50%, 1 mL) and the mixture
was stirred at ambient temperature for 6 h under a hydrogen atmosphere. The resultant
suspension was filtered through Celite, and the filtrate was concentrated in vacuo. The residue
was purified by silica gel column chromatography, eluting with hexane/EtOAc = 2:1 to yield 11
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(16.4 mg, 12% from 10) as a colorless solid. The HꢀNMR spectrum data of 11 corresponded
with the reported data.20
1H NMR (CDCl3): δ ppm: 1.34 (12H, s), 1.52 (3H, d, J = 7.10 Hz), 2.27 (1H, d, J = 17.0 Hz),
2.93 (1H, d, J = 17.0 Hz), 3.05 (1H, d, J = 10.1 Hz), 3.32 (1H, d, J = 10.1 Hz), 5.50 (1H, q, J =
7.1 Hz), 7.23ꢀ7.38 (5H, m).
tertꢀButyl
(3S)ꢀ3ꢀ[(2ꢀEthoxyꢀ2ꢀoxoethoxy)methyl]ꢀ5ꢀoxoꢀ1ꢀ[(1R)ꢀ1ꢀ
phenylethyl]pyrrolidineꢀ3ꢀcarboxylate (12)
To a solution of 10 (10.0 g, 31.3 mmol), ethyl bromoacetate (7.8 g, 47.0 mmol), and tetraꢀnꢀ
butylammonium iodide (0.58 g, 1.6 mmol) in THF (300mL) was added NaH (60% in oil, 1.6 g,
38 mmol) at 0 °C. The mixture was stirred for 0.5 h at 0 °C and then at ambient temperature for
18 h. After the reaction mixture was diluted with EtOAc, the organic solution was washed with
saturated aqueous NH4Cl and brine. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The resultant residue was purified by silica gel column chromatography,
eluting with hexane/EtOAc = 4:6 to yield 12 (10.6 g, 84%) as a colorless oil.1H NMR (CDCl3): δ
1.28 (3H, t, J = 7.2 Hz), 1.35 (9H, s), 1.53 (3H, d, J = 7.3 Hz), 2.56 (1H, d, J = 17.1 Hz), 2.80
(1H, d, J = 17.1 Hz), 3.31 (1H, d, J = 10.2 Hz), 3.46 (1H, d, J = 10.2Hz), 3.68 (2H, brs), 4.08
(2H, s), 4.21 (2H, q, J = 7.2Hz), 5.49 (1H, q, J = 7.0 Hz), 7.28–7.34 (5H, m). MS (ESI) m/z: 406
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(M + H)+. HRMS (ESI) calcd for C22H32NO6: 406.2229. Found: 406.2240. [α]D = 56.560° (c
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0.531, CHCl3).
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tertꢀButyl
(3aS,7aS)ꢀ1ꢀOxoꢀ2ꢀ[(1R)ꢀ1ꢀphenylethyl]hexahydropyrano[3,4ꢀc]pyrroleꢀ
(14) and tertꢀButyl (3aS,7aR)ꢀ1ꢀOxoꢀ2ꢀ[(1R)ꢀ1ꢀ
3a(4H)ꢀcarboxylate
phenylethyl]hexahydropyrano[3,4ꢀc]pyrroleꢀ3a(4H)ꢀcarboxylate (15)
To a solution of 8 (231 mg, 0.67 mmol) in EtOAc (12 mL) was added 10% Pd/C (50% wet,
116 mg), and the mixture was stirred for 4 h under a hydrogen atmosphere. After removal of the
catalyst by filtration, the filtrate was concentrated in vacuo. The resultant residue was purified by
silica gel column chromatography, eluting with hexane/EtOAc = 4:1–1:1 to yield 14 (183 mg,
79%) and 15 (48.5 mg, 21%) as colorless solids, respectively.
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Compound 14: m.p. 88–90 °C. H NMR (CDCl3): δ 1.41 (9H, s), 1.53 (3H, d, J = 7.1 Hz),
2.01–2.03 (2H, m), 2.91 (2H, dd, J = 18.3, 10.2 Hz), 3.02 (1h, t, J = 4.6 Hz), 3.29 (1H, d, J =
11.7 Hz), 3.39 (1H, m), 3.77–3.82 (1H, m), 4.12 (1H, J = 11.7 Hz), 5.54 (1H, d, J = 7.1 Hz),
13
7.26–7.36 (5H, m). C NMR (CDCl3): δ 16.00, 21.80, 27.89, 41.44, 45.67, 45.89, 49.06, 65.66,
70.23, 82.09, 127.1, 127.7, 128.7, 139.4, 171.9, 173.0. MS (ESI) m/z: 346(M + H)+. [α]D
=
25
106.722° (c 1.0, CHCl3). Anal. calcd for C20H27NO4, C 69.54, H 7.88, N 4.06; found, C 69.30, H
7.84, N 4.08.
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Compound 15: m.p. 183–185 °C. H NMR (CDCl3): δ 1.23 (9H, s), 1.46 (3H, d, J = 7.3 Hz),
1.92–1.96 (1H, m), 2.19–2.28 (2H, m), 3.08 (1H, d, J = 10.0 Hz), 3.17 (1H, d, J = 9.8 Hz), 3.33–
13
3.38 (2H, m), 4.10 (1H, dd, J = 11.0, 4.4 Hz), 5.48 (1H, d, J = 7.2 Hz), 7.24–7.29 (5H, m). C
NMR (CDCl3): δ 15.98, 23.07, 27.67, 47.12, 48.45, 48.99, 50.40, 68.16, 73.61, 81.78, 127.4,
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127.5, 128.5, 139.9, 170.6, 172.7. MS (ESI) m/z: 346(M + H)+. [α]D25 = 83.118° (c 1.0, CHCl3).
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Anal. calcd for C20H27NO4, C 69.54, H 7.88, N 4.06; found, C 69.40, H 7.91, N 4.10.
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Xꢀray Crystallographic Analysis of 14. A colorless prismꢀshaped crystal was formed from
Et2O: C20H27NO4; FW = 345.44; sample dimensions, 0.38 mm × 0.30 mm × 0.20 mm. The
lattice parameters and intensities were measured on a Rigaku AFC7R diffractometer (CuKα
radiation, λ = 1.54178 Å, graphite monochromator, ω–2θ scans, 2θmax = 120.1°); monoclinic,
space group P21(#4); a = 6.486(1), b = 10.763(3), c = 13.994(2), V = 970.8(3)Å3, Z = 2; Dcalcd
=
1.18 g/cm3; F000 = 372; ꢀ = 6.62 cmꢀ1. The structure was solved by direct methods with the
program Sir92.33 The final cycle of fullꢀmatrix leastꢀsquares refinement was based on 1684
observed reflections and 254 variable parameters and converged at R = 0.086 (Rw = 0.161).
These data can be obtained free of charge from The Cambridge Crystallographic Data Centre
via
http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?,
CCDC1834361.
Improved method Using MgBr2 (Table 1, entry 6)
To a solution of 8 (400 mg, 1.16 mmol) in EtOAc (7 mL) and EtOH (3.5 mL) was added
MgBr2 (225 mg, 1.22 mmol). The mixture was stirred until a clear solution was obtained. The
resultant solution was introduced in a stainlessꢀsteel autoclave, and 10% Pd/C (50%wet, 200 mg)
was added. The autoclave was closed, purged with hydrogen gas, and then pressurized with
hydrogen up to 30 atm. After stirring the mixture at ambient temperature for 14 h, the pressure
was released and water (3.5 mL) was added to the reaction solution. After stirring for 15 min, the
catalyst was removed by filtration and washed with EtOAc. The filtrate was washed with 1N
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aqueous NaOH and brine, dried over anhydrous Na2SO4, and concentrated in vacuo. To the
resultant solid were added diisopropyl ether (3 mL) and nꢀhexane (2 mL), and the mixture was
stirred for 15 min. The mixture was filtered to yield pure 15 (357 mg, 89%) as a colorless solid.
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2ꢀBenzyl
3aꢀtertꢀButyl
(3aS,7aS)ꢀtetrahydropyrano[3,4ꢀc]pyrroleꢀ2,3a(3H,4H)ꢀ
dicarboxylate (16)
To a solution of 14 (3.36 g, 9.73 mmol) in THF (50 mL) was added BH3ꢀTHF complex (48.6
mL, 1 M in THF) at ambient temperature under a nitrogen atmosphere. After stirring for 3 days,
EtOH (27 mL), water (3 mL), and TEA (3 mL) were added, and the reaction mixture was stirred
at 80 °C for 2 h. After evaporation, the residue was diluted with EtOAc. The organic solution
was washed with saturated aqueous NH4Cl, water, and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The resultant residue was purified by on a short silica gel column, eluting
with hexane/EtOAc = 65:35.
The residue was dissolved in 1,2ꢀdichloroethane (18 mL) and benzyloxycarbonyl chloride
(3.40 g, 19.9 mmol) was added. The mixture was stirred at 40 °C for 24 h. The mixture was
purified by silica gel column chromatography, eluting with hexane/EtOAc = 65:35 to yield 16
(2.39 g, 68%) as a colorless oil.1H NMR (CDCl3): δ 1.44 (9H, s), 1.80–1.93 (1H, m), 2.70–2.81
(1H, m), 3.33–3.41 (1H, m), 3.48 (1H, t, J = 10.5 Hz), 3.54–3.77 (6H, m), 3.88 (1H, dd, J = 20.6,
11.8 Hz), 5.14 (2H, s), 7.24–7.39 (5H, m). MS (ESI) m/z: 384 (M + Na)+. HRMS (ESI) calcd for
C20H28NO5: 362.1969. Found: 362.1985. [α]D25 = 21.676° (c 0.95, CHCl3).
Benzyl (3aS,7aR)ꢀ3aꢀ[(tertꢀButoxycarbonyl)amino]hexahydropyrano[3,4ꢀc]pyrroleꢀ2(3H)ꢀ
carboxylate (17)
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To a solution of 16 (2.30 g, 6.94 mmol) in CH2Cl2 (20 mL) was added TFA (10 mL). The
mixture was stirred for 24 h at ambient temperature. After evaporation, to the residue was added
1N aqueous NaOH. The aqueous solution was washed with CHCl3, and acidified by adding
concentrated aqueous HCl. After extraction from CHCl3 (2×), the organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo. The resultant residue was dissolved in CH3CN (40
mL) and 1,1'ꢀcarbonylbisꢀ1Hꢀimidazole (1.55g, 9.53 mmol) was added at 0 °C under a nitrogen
atmosphere. After stirring for 1 h, NH3 gas was bubbled into the solution for 30 min. The
reaction solution was then diluted with EtOAc; the organic layer washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The resultant residue was purified by
short silica gel column chromatography, eluting with EtOAc. The resultant residue was dissolved
in tertꢀBuOH (50 mL) and lead (IV) acetate (3.98 g, 8.97 mmol) was added at ambient
temperature under a nitrogen atmosphere. Then, the reaction mixture was warmed up to 80 °C
and stirred for 1 h. To the solution were added Na2CO3 (3.52 g, 41.9 mmol) and EtOAc. After
filtration through Celite, the filtrate was washed with saturated aqueous Na2CO3 and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The resultant residue was purified by silica
gel column chromatography, eluting with hexane/EtOAc = 70:30 to yield 17 (1.70 g, 65%) as a
colorless oil.1H NMR (CDCl3): δ 1.43 (9H, s), 1.50–1.54 (1H, m), 1.79–1.87 (1H, m), 2.55–2.63
(1H, m), 3.34–3.38 (1H, m), 3.57–3.86 (7H, m), 4.66 (1H, d, J = 7.6 Hz), 5.13 (2H, s), 7.31–7.36
(5H, m). MS (ESI) m/z: 399 (M + Na)+. [α]D25 = 36.895° (c 1.00, CHCl3). HRMS (ESI) calcd for
C20H28NO5: 377.2078. Found: 377.2099.
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7ꢀ[(3aS,7aR)ꢀ3aꢀAminohexahydropyrano[3,4ꢀc]pyrrolꢀ2(3H)ꢀyl]ꢀ6ꢀfluoroꢀ1ꢀ[(1R,2S)ꢀ2ꢀ
fluorocyclopropyl]ꢀ8ꢀmethoxyꢀ4ꢀoxoꢀ1,4ꢀdihydroquinolineꢀ3ꢀcarboxylic Acid (19)
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