Metallo-β-lactamase inhibitory activity of 3-alkyloxy and 3-amino phthalic acid derivatives and their combination effect with carbapenem

Article abstract of DOI:10.1016/j.bmc.2013.07.006

3-Alkyloxy and 3-amino phthalic acid derivatives were found to have metallo-β-lactamase inhibitory activity. Among them, 3-amino phthalic acid derivatives showed both potent activity against metallo-β-lactamase, IMP-1 inhibitory activity and a strong combination effect with biapenem (BIPM), carbapenem antibiotic. In particular, the 4′-hydroxy-piperidine derivative showed strong IMP-1 inhibitory activity and a combination effect with various antibiotics.

Full text of DOI:10.1016/j.bmc.2013.07.006

Bioorganic & Medicinal Chemistry 21 (2013) 5841–5850
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Metallo-b-lactamase inhibitory activity of 3-alkyloxy and 3-amino
phthalic acid derivatives and their combination effect with
carbapenem
,
⇑
Yukiko Hiraiwa , Akihiro Morinaka, Takayoshi Fukushima, Toshiaki Kudo
Pharmaceutical Research Center, Meiji Seika Pharma, Co., Ltd, 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Alkyloxy and 3-amino phthalic acid derivatives were found to have metallo-b-lactamase inhibitory
activity. Among them, 3-amino phthalic acid derivatives showed both potent activity against
metallo-b-lactamase, IMP-1 inhibitory activity and a strong combination effect with biapenem (BIPM),
carbapenem antibiotic. In particular, the 40-hydroxy-piperidine derivative showed strong IMP-1
inhibitory activity and a combination effect with various antibiotics.
Received 24 May 2013
Accepted 4 July 2013
Available online 18 July 2013
Keywords:
Metallo-b-lactamase
Pseudomonas aeruginosa
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
against Gram-negative bacteria. Moreover, it is reported that
MDRP strains produce MBLs at high frequency.4
Owingto their efficacy and safety, b-lactam antibioticsare widely
used for the treatment of bacterial infection in the clinical field. It is
well known, however, that mechanisms of resistance to b-lactam
antibiotics exist. In this regard, inactivation by b-lactamase is the
major mechanism of resistance against b-lactam antibiotics.
b-Lactamases are classified as four types A–D, based on their
amino acid sequence homology by Amber classification.1 Due to
serine-catalysed hydrolysis of the b-lactam ring, class A, C, and D
b-lactamases are called serine b-lactamases. On the other hand,
class B b-lactamase is a metallo-b-lactamase (MBL) having one or
two zinc ions in the active site.2 Various types of metallo-b-lacta-
mases, for instance, IMP, VIM, and SPM types etc., have been re-
ported. In Japan, the IMP-1 metallo-b-lactamase has been
reported so far.3 MBLs can hydrolyze almost all b-lactam antibiot-
ics, including penicillins, cephalosporins and carbapenems.
As a result, we are interesting in developing a MBL inhibitor. In
a previous study, we found that phthalic acids substituted with a
bulky group at the 3-position had potent IMP-1 inhibitory activ-
ity.5 In particular, compound 1, which is substituted with a
40-hydroxyphenyl group at the 3-position, was found to have the
most potent IMP-1 inhibitory activity (IC50 = 1.55 lM) (Fig. 1). Fur-
thermore, compound 1 showed a combination effect with biape-
nem (BIPM), a carbapenem antibiotic, against IMP-1 P. aeruginosa
strains producing IMP-1. As a result, here we have continued to de-
velop a more potent MBL inhibitor.
2. Results and discussion
2.1. Chemistry
Carbapenems play an important role in the clinical field because
they are effective against both Gram-positive and Gram-negative
bacteria including Pseudomonas aeruginosa. Therefore, MBL-pro-
ducing pathogens are a significant problem in the clinical field. In
addition, multi-drug resistant P. aeruginosa (MDRP) strains are a
significant problem because of their resistance against aminogly-
cosides, carbapenems and quinolones, which are normally effective
Scheme 1 shows the synthesis of 3-alkyloxy phthalic acid deriv-
atives from commercially available 3-hydroxyphthalic anhydride.
Esterification of 3-hydroxyphthalic anhydride, followed by alkyl-
ation of the hydroxyl group, gave 4. Hydrolysis of the diethylester
4 under alkaline conditions gave the phthalic acid 5 in good yield.
Scheme 2 shows the synthesis of 3-aminophthalic acid deriva-
tives from commercially available 3-nitrophthalic acid. Two step
esterification of the 3-nitrophthalic acid 6 gave the diethylester
7. Hydrogenation of 7, followed by alkylation of the 3-amino group
of 8, afforded 9. Hydrolysis of the diethyl ester 9 under alkaline
conditions afforded 10.
⇑
Corresponding author. Tel.: +81 45 680 1663; fax: +81 45 680 1664.
E-mail address: yukiko.hiraiwa@meiji.com (Y. Hiraiwa).
3-Dimethylamino phthalic acid and its cyclic amine derivatives
were synthesized from 3-fluorophthalic acid (Scheme 3).
Esterification of the 3-fluorophthalic acid 11 afforded the diethyl
Present address: Yokohama Branch, Marketing Division, Meiji Seika Pharma, Co.,
Ltd, 2th Fl Minato Fantajia-Building, 32-1, 3-Chome, Minaminakadori, Naka-ku,
Yokohama-shi, Kanagawa 231-0006, Japan.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.006

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Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
Figure 1. Lead compound of IMP-1 inhibitor
Scheme 1. Synthesis of 3-alkoxyphthalic acid derivatives. Reagents and conditions: (a) H2SO4, EtOH, reflux; (b) CH3I, K2CO3, DMF, rt (4b); benzylbromide, K2CO3, DMF, rt (4c);
nBuI, K2CO3, DMF, rt (4d); (3-bromopropyl)cyclohexane, K2CO3, DMF, rt (4e); 3-phenylpropylbromide, K2CO3, DMF, rt (4f); 2-bromoethanol, K2CO3, DMF, rt (4h); 3-bromo-1-
propanol, K2CO3, DMF, rt (4i); (c) ethyl 5-bromovalerate, K2CO3, DMF, rt; (d) (1) NaOH, H2O, 1,4-dioxane, 80 °C; (2) HCl, H2O; (e) (1) NaOH, H2O, rt; (2) HCl, H2O.
5c showed about a 20-fold increase in activity as compared with
5b. The derivatives 5d, 5e and 5f with longer carbon chains showed
potent IMP-1 inhibitory activity. In compounds 5g, 5h and 5i, the
hydrophilic hydroxyl or carboxyl group was introduced to the C2,
C3 or C4 alkyl chain. These derivatives (5g–i) showed weak IMP-
1 inhibitory activity (IC50 = 18.8–47.8
Next, we evaluated the combination effect with BIPM, a carba-
penem antibiotic, against P. aeruginosa KG50026/pMS3637 (
Mex-
lM).
D
AB) and PAO1/pMS363 strains that produce IMP-1. The 3-alkyloxy
phthalic acids 5e and 5f, which were strong IMP-1 inhibitors,
showed no combination effect with BIPM against P. aeruginosa
PAO1/pMS363. Although the IMP-1 inhibitory activity of 5g, 5h
and 5i was weaker than that of 5e or 5f, the derivatives 5g–i
showed potent combination effect with BIPM against P. aeruginosa
PAO1/pMS363. On the other hand, 5f showed a strong combination
effect with BIPM against P. aeruginosa KG5002/pMS363 (
Interestingly, the hydroxyl derivatives 5h and 5i showed a combi-
nation effect against P. aeruginosa KG5002/pMS363 ( MexAB) that
was equal to that against P. aeruginosa PAO1/pMS363. These re-
sults indicated that 5f was affected by the efflux system of Mex-
AB-OprM, whereas the hydroxyl derivatives 5h and 5i were not
affected by this efflux system.
Table 2 shows the activities of the 3-aminophthalic acid deriv-
atives. No substituted 3-aminiophthalic acid showed weak IMP-1
inhibitory activity, but the n-butyl (10) and N,N-dimethyl amino
(14) derivatives showed improved IMP-1 inhibitory activity.
Although the N,N-dimethyl amino derivative 14 had weak IMP-1
inhibitory activity, this compound showed a potent combination
effect with BIPM against P. aeruginosa strains that produced IMP-
1. On the other hand, cyclic amine compounds showed potent
IMP-1 inhibitory activity and a combination effect with BIPM
against P. aeruginosa strains producing IMP-1. In particular, the
DMexAB).
Scheme 2. Synthesis of 3-aminophthalic acid derivatives. Reagents and conditions:
(a) (1) cH2SO4, EtOH, reflux; (2) EtI, K2CO3, DMF; (b) H2, 10% Pd–C, EtOH, rt; (c) n-
butylaldehyde, acetic acid, triacetoxyborohydride, 1,2-dichloroethane, rt; (d) (1)
NaOH, H2O, 1,4-dioxane, 80 °C; (2) HCl, H2O.
D
ester derivative 12. Next, dimethyl amine or various cyclic amines
were reacted with 12 to give diethyl esters of the 3-aminophthalic
acid derivatives. Hydrolysis under alkaline conditions gave 3-
aminophthalic acid derivatives.
2.2. Structure and activity relationship (biological activity)
The IMP-1 inhibitory activities of the 3-alkyloxy phthalic acid
derivatives were shown in Table 1. Although the hydroxyl deriva-
tive 5a showed no IMP-1 inhibitory activity, the methoxy
derivative 5b showed weak activity. The 3-benzyloxy phthalic acid

Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
5843
Scheme 3. Synthesis of cyclic amine derivatives and N,N-dimethyl amino derivatives. Reagents and conditions: (a) (1) H2SO4, EtOH, reflux; (2) EtI, K2CO3, DMF, rt; (b)
dimethylamine, THF, ,sealed tube, 90 °C; (c) NaOH, H2O, 1,4-dioxane, 80 °C; (d) pyrolidine, 80 °C (15a); piperidine, 80 °C (15b); 3-hydroxypiperidine, DMSO, 80 °C (15c); 3-
(hydroxylmethyl)piperidine, DMSO, 80 °C (15d); 4-hydroxypiperidine, DMSO, 80 °C (15e); 4-(hydroxylmethyl)piperidine, DMSO, 80 °C (15f); 4-(hydroxylethyl)piperidine,
DMSO, 80 °C (15g); 4-hydroxy-4-phenylpiperidine, DMSO, 80 °C (15h); (c) (1) NaOH, H2O, 1,4-dioxane, 80 °C; (2) HCl, H2O; (e) NaH, MeI, THF, rt.
Table 1
IMP-1 inhibitory activity of 3-alkyloxy phthalic acid derivatives and their combination effect with BIPM
O
OH
OH
O
O
R1
Compd
R1
IMP-1 inhibitory activity IC50
(
lM)
Combination effect (50
MIC of BIPM ( g/mL)
lg/mL) with BIPM
l
P. aeruginosa KG5002 /pMS363 (
D
mexAB)
P. aeruginosa PAO1/pMS363
5a
5b
–H
–CH3
>300
142
NT
NT
NT
NT
5c
5d
5e
7.40
5.10
2.00
NT
NT
4
NT
NT
64
CH3
5f
1.70
0.5
64
COOH
OH
OH
5g
5h
5i
18.8
47.8
21.4
2
8
16
16
16
16
(BIPM alone)
64–128
64–128
piperidine derivative 16b showed an excellent combination effect
with BIPM.
Next, we aimed to optimize the piperidine derivatives. The IMP-
1 inhibitory activity and combination effect of piperidine deriva-
tives were shown in Table 3. As compared with 16b, the IMP-1
inhibitory activities were mainly improved. The 40-hydroxyethyl
compound 16g showed low potent IMP-1 inhibitory activity. In
addition, this compound showed a weak effect of combination
with BIPM. The 40-hydroxy-40-phenyl compound 16h and the 40-
methoxy compound 16i showed potent IMP-1 inhibitory activity
and a potent combination effect against P. aeruginosa KG5002/
pMS363 (DmexAB); however, these compounds showed a weak

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Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
Table 2
IMP-1 inhibitory activity of 3-amino phthalic acid derivatives and their combination effect with BIPM
O
OH
OH
N
O
R2 R3
Compd
R2
R3
IMP-1 inhibitory activity IC50
(
lM)
Combination effect (50
MIC of BIPM ( g/mL)
lg/mL) with BIPM
l
P.aeruginosa KG5002 /pMS363 (
D
mexAB)
P.aeruginosa PAO1/pMS363
–H
–H
300
NT
NT
CH3
10
14
–H
–CH3
13.1
94.5
1
4
64
4
–CH3
N
N
16a
16b
2.80
0.5
4
10.8
50.5
4
(BIPM alone)
64–128
64–128
Table 3
IMP-1 inhibitory activity of piperidine derivatives and their combination effect with BIPM
O
OH
OH
N
O
R4
Combination effect (50
MIC of BIPM ( g/mL)
P. aeruginosa KG5002/pMS363 (
lg/mL) with BIPM
N
R4
Compd
IMP-1 inhibitory activity IC50
(lM)
l
D
mexAB)
P. aeruginosa PAO1/pMS363
16b
16c
10.8
2.70
50.5
4
N
N
OH
50.5
1
2
OH
16d
2.10
0.5
N
N
16e
16f
2.70
2.60
50.25
1
2
OH
OH
1
N
OH
16g
25.6
2.30
8
32
16
N
N
OH
16h
16i
50.5
3.70
50.25
2
N
OCH3
(BIPM alone)
64–128
64–128
combination effect against P. aeruginosa PAO1/pMS363. From these
results, it is likely that these compounds were effluxed by the Mex-
AB system.
As compared with 16c and 16d, or 16e and 16f and 16g, exten-
sion of the carbon chain resulted in a decreased combination effect
against P. aeruginosa producing IMP-1. The presence of a hydroxyl
group at the 30 or 40 position of the piperidine ring led to potent
IMP-1 inhibitory activity. In particular, the 40-hydroxy piperidine
derivative 16e showed the most potent effect of combination with
BIPM.

Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
5845
Figure 2.
From the above results, we selected the 40-hydroxy piperidine
derivative 16e, and carried out more detailed tests. We tested
the combination effect 16e with various antibiotics, including
BIPM, meropenem (MEPM) and ceftazidime (CAZ), against clinical
isolates of P. aeruginosa producing IMP-1 (Fig. 2).
BIPM is the favored antibiotic for combination with the MBL-
inhibitor 16e.
3. Conclusion
All antibiotics were shown to have
a combination effect
In this study, we found that the 3-alkyloxy and 3-amino phtha-
lic acid derivatives had potent IMP-1 inhibitory activity. Regarding
the structure–activity relationship of 3-alkyloxy phthalic acids,
although the long carbon chain compounds had potent IMP-1
inhibitory activity, their combination effect with BIPM against P.
aeruginosa strains producing IMP-1 was not parallel. In contrast,
the hydroxy derivatives 5h and 5i had low IMP-1 inhibitory
with 16e, and this effect was dose-dependent on 16e. These
results indicate that the MBL-inhibitor 16e is effective against
clinical isolates of P. aeruginosa producing IMP-1. For CAZ
and MEPM, however, some strains were not sensitive. It is
considered that CAZ and MEPM were affected not only by
IMP-1 but also by another mechanism of resistance. Thus,

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Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
activity, but showed a strong combination effect with BIPM against
P. aeruginosa without being subject to efflux pump. In the case of
the 3-aminophtahalic acid derivatives, the piperidine derivatives
showed strong IMP-1 inhibitory activity; in particular, polar substi-
tution increased this activity.
4.3. 3-Benzyloxyphthalic acid (5c)
To a DMF (5.0 mL) solution of 3 (100 mg, 0.420 mmol), K2CO3
(120 mg, 0.860 mmol) and benzylbromide (0.060 mL, 0.500 mmol)
were added. The mixture was stirred at room temperature for
overnight. Water was added to the reaction mixture, and the mix-
ture was extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under
reduced pressure to give 4c (90.0 mg, 65.3%).
The 40-hydroxypiperidine compound 16e showed ideal activity.
This compound showed a combination effect with various antibiot-
ics against P. aeruginosa strains producing IMP-1. In this study,
BIPM was the most promising antibiotic for combination with the
MBL inhibitor 16e against clinical isolates of P. aeruginosa produc-
ing IMP-1. On the basis of these findings, further structure–activity
relationship studies of MBL inhibitors are currently in progress.
ESIMS: m/z 329 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 4c (90.0 mg, 0.274 mmol), 5.0
mol/L NaOH solution (5.0 mL) was added. The mixture was stirred at
80 °C for overnight. The reaction mixture was extracted with EtOAc
three times. 1.0 mol/L HCl solution was added to aqueous solution
to adjustthe pHuntil pH3, and then extracted withEtOAc three times.
The organic layers were dried over anhydrous MgSO4 and concen-
trated under reduced pressure to give 5c (64.0 mg, 85.8%).
1H NMR (400 MHz, CD3OD) d 5.09 (2H, s, benzyl), 7.27 (7H, m,
phenyl), 7.51 (1H, dd, J = 0.98, 7.6 Hz, phenyl), ESIMS: m/z 271
(MꢀH)ꢀ.
4. Experimental section
1H NMR spectra were recorded on JNM-LA400 spectrometers
with chemical shifts reported in ppm with internal tetramethylsil-
ane as a basis. Electron ionization (EI) mass spectra were recorded
on a Hitachi M-80B instrument. Fast-atom bombardment (FAB)
mass spectra were recorded on a JEOL JMS-700 instrument. Ther-
mospray (TSP) mass spectra were recorded on a Hewlett–Packard
5989A instrument. Electrospray ionization (ESI) mass spectra were
recorded on a Hewlett–Packard 5989A instrument. Atmospheric
pressure chemical ionization (APCI) mass spectra were recorded
on a Hewlett–Packard 5989A instrument. High-resolution mass
spectra (HRMS) were recorded under FAB conditions.
4.4. 3-Buthoxyphthalic acid (5d)
To a DMF (5.0 mL) solution of 3 (60.0 mg, 0.252 mmol), K2CO3
(87.0 mg, 0.578 mmol) and 1-iodobutane (0.034 mL, 0.299 mmol)
were added. The mixture was stirred at room temperature for
overnight. Water was added to the reaction mixture, and the mix-
ture was extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under
reduced pressure. Purification with preparative TLC (hexane/
EtOAc = 2:1) afforded 4d (52.0 mg, 70.2%).
4.1. 3-Hydroxy phthalic acid (5a)
3-Hydroxyphthalic anhydride 2 (60.0 mg, 0.360 mmol) was
solved in water (2.0 mL) and 1.0 mol/L NaOH (2.0 mL) solution.
The solution was stirred at room temperature for 15 h. 1.0 mol/L
HCl solution was added to adjust the pH until pH2, and the mixture
was extracted three times with EtOAc. The organic layers were
combined and dried over anhydrous MgSO4. It was then concen-
trated under reduced pressure to give 5a (14.0 mg, 21.4%).
1H NMR (400 MHz, CDCl3) d 7.07 (2H, m, phenyl), 7.44 (1H, dd,
J = 7.6, 8.2 Hz, phenyl); ESIMS: m/z 181 (MꢀH)ꢀ.
ESIMS: m/z 295 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 4d (50.0 mg, 0.170 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for 3 h. The reaction mixture was extracted with EtOAc
three times. 1.0 mol/L HCl solution was added to the aqueous solu-
tion to adjust the pH until pH2, and then extracted with EtOAc three
times. The organic layers were dried over anhydrous MgSO4 and
concentrated under reduced pressure to give 5d (40.0 mg, 98.9%).
1H NMR (400 MHz, CDCl3) d 0.95 (3H, t, J = 7.3 Hz, methyl), 1.48
(2H, m, CH2), 1.77 (2H, m, CH2), 4.04 (2H, t, J = 6.5 Hz, CH2), 7.13
(1H, d, J = 8.0 Hz, phenyl), 7.38 (1H, dd, J = 8.0 Hz, phenyl), 7.60
(1H, d, J = 8.0 Hz, phenyl), EIMS: m/z 238 (M+).
4.2. 3-Methoxyphthalic acid (5b)
conc. H2SO4 (3.0 mL) was added to a solution of 3-hydroxyphta-
lic anhydride (650 mg, 3.96 mmol) in EtOH (15 mL). The mixture
was refluxed for overnight and then concentrated under reduced
pressure. Water was added to the residue and the mixture was ex-
tracted three times with EtOAc. The combined organic layers were
dried over anhydrous MgSO4 and concentrated under reduced
pressure to give 3 (930 mg, 98.7%). ESIMS: m/z 237 (MꢀH)ꢀ.
To a DMF (5.0 mL) solution of 3 (100 mg, 0.420 mmol), K2CO3
(120 mg, 0.868 mmol) and methyliodide (0.031 mL, 0.500 mmol)
were added. The mixture was stirred at room temperature for
overnight. Water was added to the reaction mixture, and the mix-
ture was extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under
reduced pressure to give 4b (35.0 mg, 33.1%).
4.5. (3-Cyclohexylpropoxy)phthalic acid (5e)
To a DMF (5.0 mL) solution of 3 (290 mg, 1.22 mmol), K2CO3 (500
mg, 3.62 mmol) and (3-bromopropyl)cyclohexane (500 mg, 2.44
mmol) were added. The mixture was stirred at room temperature
for two days. Water was added to the reaction mixture, and the mix-
ture was extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under
reduced pressure. The residue was purified with silica–gel column
chromatography (hexane/EtOAc = 2:1) to give 4e (430 mg, 97.3%).
EIMS: m/z 362 (M+).
To a 1,4-dioxane (2.0 mL) solution of 4e (430 mg, 1.19 mmol), 5.0
mol/L NaOH solution (10 mL) was added. The mixture was stirred at
80 °C for overnight. The reaction mixture was extracted with EtOAc
three times. 1.0 mol/L HCl solution was added to the aqueous solu-
tion to adjust the pH until pH2, and then extracted with EtOAc three
times. The organic layers were dried over anhydrous MgSO4 and
concentrated under reduced pressure to give 5e (283 mg, 77.5%).
1H NMR (400 MHz, CDCl3) d 0.90 (2H, m), 1.09–1.37 (6H, m),
1.67 (5H, m), 1.83 (2H, m), 4.06 (2H, t, J = 6.7 Hz, CH2), 7.19 (1H,
ESIMS: m/z 253 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 4b (34.0 mg, 0.135 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. The reaction mixture was extracted with
EtOAc three times. 1.0 mol/L HCl solution was added to the aqueous
solution to adjust the until pH3, and then extracted with EtOAc three
times. The organic layers were dried over anhydrous MgSO4 and
concentrated under reduced pressure to give 5b (14.0 mg, 52.9%).
1H NMR (400 MHz, CD3OD) d 3.08 (3H, s, methyl), 7.21 (1H, dd,
J = 0.97, 8.3 Hz, phenyl), 7.51 (1H, dd, J = 8.0, 8.3 Hz, phenyl), 7.49
(1H, dd, J = 0.97, 8.0 Hz, phenyl); ESIMS: m/z 195 (MꢀH)ꢀ.
d, J = 8.3 Hz, phenyl), 7.46 (1H, dd, J = 7.8, 8.3 Hz, phenyl), 7.65
(1H, d, J = 7.8 Hz, phenyl), ESIMS: m/z 307 (M+H)
+
.

Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
5847
4.6. (3-Phenylpropoxy)phthalic acid (5f)
1H NMR (400 MHz, CDCl3+CD3OD) d 3.88 (2H, t, J = 4.3 Hz, CH2),
4.16 (2H, t, J = 4.3 Hz, CH2), 7.17 (1H, d, J = 8.5 Hz, phenyl), 7.42
(1H, dd, J = 7.8, 8.5 Hz, phenyl), 7.64 (1H, d, J = 7.8 Hz, phenyl).
ESIMS: m/z 225 (MꢀH)ꢀ.
To a DMF (3.0 mL) solution of 3 (82.0 mg, 0.345 mmol), K2CO3
(120 mg, 0.868 mmol) and 3-phenylpropylbromide (81.0 mg, 0.407
mmol) were added. The mixture was stirred at room temperature
for overnight. Waterwas added to the reaction mixture, and the mix-
ture was extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under re-
duced pressure. The residue was purified with silica-gel column
chromatography (hexane/EtOAc = 1:1) to give 4f (90.0 mg, 73.3%).
EIMS: m/z 356 (M+).
4.9. 3-(3-Hydroxypropoxy)phthalic acid (5i)
To a DMF (4.0 mL) solution of 3 (100 mg, 0.420 mmol), K2CO3
(170 mg, 1.23 mmol) and ethyl 3-bromo-1-propanol (120 mg,
0.863 mmol) were added. The mixture was stirred at room temper-
ature for overnight. Water was added to the reaction mixture, and
the mixture was extracted with EtOAc three times. The combined
oreganic layers were dried over anhydrous MgSO4 and concen-
trated under reduced pressure. Purification with preparative TLC
(hexane/EtOAc = 1:1) afforded 4i (110 mg, 88.5%).
To a 1,4-dioxane (1.0 mL) solution of 4f (90.0 mg, 0.253 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for 3 h. The reaction mixture was extracted with EtOAc
three times. 1.0 mol/L HCl solution was added to the aqueous solu-
tion to adjust the pH until pH2, and then extracted with EtOAc
three times. The organic layers were dried over anhydrous MgSO4
and concentrated under reduced pressure to give 5f (54.0 mg,
71.1%).
EIMS: m/z 296 (M+).
To a 1,4-dioxane (1.0 mL) solution of 4i (110 mg, 0.371 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for 3 h. 1.0 mol/L HCl solution was added to the reac-
tion mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. Purification with synthetic adsorption re-
sin SP-207 (H2O–CH3CN) gave 5i (35.0 mg, 39.3%).
1H NMR (400 MHz, CDCl3 + CD3OD) d 2.11 (2H, m, CH2), 2.81
(2H, t, J = 7.6 Hz, CH2), 4.02 (2H, t, J = 6.2 Hz, CH2), 7.08 (1H, d,
J = 8.3 Hz, phenyl), 7.23 (5H, m, phenyl), 7.38 (1H, dd, J = 7.8, 8.3
Hz, phenyl), 7.62 (1H, d, J = 7.8 Hz, phenyl),
1H NMR (400 MHz, CDCl3+CD3OD) d 2.02 (2H, m, CH2), 3.80 (2H,
t, J = 5.7 Hz, CH2), 4.18 (2H, t, J = 5.7 Hz, CH2), 7.16 (1H, d, J = 8.3 Hz,
phenyl), 7.41 (1H, dd, J = 7.9, 8.3 Hz, phenyl), 7.64 (1H, d, J = 7.9 Hz,
phenyl).
ESIMS: m/z 299 (MꢀH)ꢀ.
4.7. (3-Carboxybuthoxy)phthalic acid (5g)
EIMS: m/z 240 (M+).
To a DMF (5.0 mL) solution of 3 (100 mg, 0.420 mmol), K2CO3
(150 mg, 1.09 mmol) and ethyl 5-bromopentanoate (100 mg,
0.478 mmol) were added. The mixture was stirred at room temper-
ature for overnight. Water was added to the reaction mixture, and
the mixture was extracted with EtOAc three times. The combined
organic layers were dried over anhydrous MgSO4 and concentrated
under reduced pressure. Purification with silica gel column chlo-
matography (hexane/EtOAc = 7:3) afforded 4g (149 mg, 96.9%).
EIMS: m/z 366(M+).
To a 1,4-dioxane (1.0 mL) solution of 4g (140 mg, 0.383 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. The reaction mixture was extracted with
EtOAc. 1.0 mol/L HCl solution was added to the aqueous solution to
adjust the pH until pH2, and then extracted with EtOAc three times.
The organic layers were dried over anhydrous MgSO4 and concen-
trated under reduced pressure to give 5g (67.7 mg, 62.7%).
1H NMR (400 MHz, CDCl3+CD3OD) d 1.48 (4H, m, CH2), 2.39 (2H,
m, CH2), 2.51 (2H, m, CH2), 7.13 (1H, d, J = 8.0 Hz, phenyl), 7.39 (1H,
4.10. 3-(Butylamino)phthalic acid (10)
conc. H2SO4 (10 mL) was added to a solution of 3-nitrophthalic
acid (5.00 g, 23.7 mmol) in EtOH (100 mL). The mixture was re-
fluxed for 6 h. Water was added to the reaction mixture, and the
mixture was extracted with EtOAc three times. The combined or-
ganic layers were dried over anhydrous MgSO4 and evaporated.
The residue was solved in DMF (200 mL) and then K2CO3 (9.78 g,
70.8 mmol) and Ethyliodide (2.9 mL, 36.3 mmol) were added.
The mixture was stirred at room temperature for overnight. Water
was added to the reaction mixture and the mixture was extracted
with EtOAc three times. The combined organic layers were dried
over anhydrous MgSO4 and concentrated under reduced pressure.
The residue was purified with silica-gel column chromatography
(hexane/EtOAc) to give 7 (5.68 g, 89.9%).
ESIMS: m/z 268 (M+H)+.
dd, J = 8.0, 8.0 Hz, phenyl), 7.62 (1H, d, J = 8.0 Hz, phenyl).
To a EtOH (40 mL) solution of 7 (2.68 g, 10.0 mmol), 10% Pd–C
(530 mg) was added and the mixture was hydrogenated under bal-
loon pressure of hydrogen at room temperature for 5 h. The mix-
ture was purified with silica-gel column chromatography
(hexane/EtOAc) to give 8 (2.38 g, 100%).
+
ESIMS: m/z 283 (M+H)
.
4.8. 3-(2-Hydroxyethoxy)phthalic acid(5h)
ESIMS: m/z 238 (M+H)+.
To a DMF (4.0 mL) solution of 3 (100 mg, 0.420 mmol), K2CO3
(170 mg, 1.23 mmol) and ethyl 2-bromoethanol (100 mg, 0.800
mmol) were added. The mixture was stirred at room temperature
for overnight. Water was added to the reaction mixture, and the
mixture was extracted with EtOAc three times. The combined or-
ganic layers were dried over anhydrous MgSO4 and concentrated
under reduced pressure. Purification with silica gel column chlo-
matography (hexane/EtOAc = 7:3) afforded 4h (45.0 mg, 38.0%).
EIMS: m/z 282 (M+).
To a 1,4-dioxane (0.6 mL) solution of 4h (45.0 mg, 0.160 mmol),
5.0 mol/L NaOH solution (3.0 mL) was added. The mixture was stir-
red at 80 °C for 3 h. 1.0 mol/L HCl solution was added to the reac-
tion mixture until pH2, and then concentrated under reduced
pressure. Purification with synthetic adsorption resin SP-207
(Mitsubishi Chemical Co., Ltd,) (H2O–CH3CN) gave 5h (18.2 mg,
50.3%).
To a 1,2-dichloroethane (10 mL) solution of 8 (237 mg, 1.00
mmol), n-butylaldehyde (0.13 mL, 1.44 mmol), acetic acid (0.11
mL, 1.92 mmol) and triacetoxyborohydride (420 mg, 2.22 mmol)
were added. The mixture was stirred at room temperature for
overnight. Saturated NaHCO3 solution was added to the reaction
mixture and stirred, and then extracted with EtOAc three times.
The combined organic layers were dried over anhydrous MgSO4
and evaporated. The residue was purified with preparative TLC
(hexane/EtOAc) afforded 9 (190 mg, 64.8%).
ESIMS: m/z 294 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 9 (190 mg, 0.648 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was
stirred at 80 °C for overnight. The reaction mixture was extracted
with EtOAc. 1.0 mol/L HCl solution was added to the aqueous
solution to adjust the pH until pH2, and then extracted with

5848
Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
EtOAc three times. The organic layers were dried over anhydrous
MgSO4 and concentrated under reduced pressure to give 10 (112
mg, 72.9%).
was added to the reaction mixture, and then extracted with EtOAc
three times. The combined organic layers were dried over anhy-
drous MgSO4 and concentrated under reduced pressure. The resi-
due was purified with preparative TLC (hexane/EtOAc) to give
15b (166 mg, 99.7%).
1H NMR (400 MHz, CDCl3+CD3OD) d 0.96 (3H, t, J = 7.4 Hz, CH3),
1.45 (2H, m, CH2), 1.64 (2H, m, CH2), 3.16 (2H, t, J = 7.1 Hz, CH2),
6.78 (2H, m, phenyl), 7.30 (1H, dd, J = 7.9, 7.9 Hz, phenyl).
ESIMS: m/z 236 (MꢀH)ꢀ.
EIMS: m/z 305 (M+).
To a 1,4-dioxane (1.0 mL) solution of 15b (150 mg, 0.492 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. 1.0 mol/L HCl solution was added to the
reaction mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. The residue was purified with synthetic
adsorption resin (Sepabeads SP-207) (H2O–CH3CN) to give 16b
(70.0 mg, 57.1%).
4.11. 3-Dimethylaminophthalic acid (14)
To a EtOH (200 mL) solution of 11 (9.35 g, 50.8 mmol), conc.
H2SO4 (20 mL) was dropped, and then refluxed for 6 h. The reaction
mixture was concentrated under reduced pressure. Water was
added to the residue, and then extracted with EtOAc three times.
The combined organic layers were dried over anhydrous MgSO4
and concentrated under reduced pressure. The residue was solved
in DMF (150 mL), K2CO3 (21.0 g, 152 mmol) and iodoethane (6.1
mL, 75.6 mmol) were added. The mixture was stirred at room tem-
perature for two days. Water was added to the reaction mixture
and then extracted with EtOAc three times. The combined organic
layers were dried over anhydrous MgSO4 and concentrated under
reduced pressure. The residue was purified with silica-gel column
chromatography (hexane/EtOAc) afforded 12 (9.13 g, 74.8%).
EIMS: m/z 240 (M+).
FABMS: m/z 250 (M+H)+; FAB-HMS (M+H)+ calcd for C13H16NO4:
250.1079, found: 250.1079.
4.14. 3-(30-Hydroxypiperidine-1-yl)phthalic acid (16c)
To a DMSO (3.0 mL) solution of 12 (375 mg, 1.56 mmol), 3-
hydroxypiperidine (1.26 g, 12.4 mmol) was added. The mixture
was stirred at 80 °C for overnight. Water was added to the reaction
mixture, and then extracted with EtOAc three times. The combined
organic layers were dried over anhydrous MgSO4, and concentrated
under reduced pressure. The residue was purified with silica-gel col-
umn chromatography (hexane/EtOAc) to give 15c (410 mg, 81.9%).
EIMS: m/z 321 (M+).
To a 1,4-dioxane (1.0 mL) solution of 15c (400 mg, 1.25 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. 1.0 mol/L HCl solution was added to the
reaction mixture until pH2, and then concentrated under reduced
pressure. The residue was purified with synthetic adsorption resin
Sepabeads SP-207 (H2O–CH3CN) to give 16c (150 mg, 45.4%).
1H NMR (400 MHz, D2O) d 1.80–2.15 (4H, m, piperidine), 3.28
(1H, dd, J = 4.2, 12.4 Hz, piperidine), 3.39 (2H, m, piperidine), 3.66
(1H, dd, J = 1.6, 12.4 Hz, piperidine), 4.21 (1H, m, piperidine),
7.46 (1H, dd, J = 1.0, 7.6 Hz, phenyl), 7.64 (1H, dd, J = 7.6, 8.1 Hz,
phenyl), 7.70(1H, dd, J = 1.0, 8.1 Hz, phenyl);FABMS: m/z 266
(M+H)+; FAB-HMS (M+H)+ calcd for C13H16NO4: 266.1028, found:
266.1028.
Compound 12 (120 mg, 0.500 mmol) was added to 2.0 M
dimethylamine in THF solution (3.0 mL) and then stirred at 90 °C
in sealed tube for overnight. The reaction mixture was evaporated,
and then purified with preparative TLC (hexane–EtOAc) to give 13
(130 mg, 98.1%).
FABMS: m/z 266 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 13 (13 0 mg, 0.49 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for 3 h. 1.0 mol/L HCl solution was added to the reac-
tion mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. The residue was purified with synthetic
adsorption resin, Sepabeads SP-207 (H2O–CH3CN) to give 14
(23.0 mg, 22.5%).
1H NMR (400 MHz, D2O) d 3.12 (6H, s, CH3), 7.47 (1H, dd, J = 1.0,
7.6 Hz, phenyl), 7.62 (1H, dd, J = 7.6, 8.3 Hz, phenyl), 7.73 (1H, dd,
J = 1.0, 8.3 Hz, phenyl).
FABMS: m/z 210 (M+H)+.
4.15. 3-(30-Hydroxymethyl)piperidine-1-yl)phthalic acid(16d)
4.12. 3-(Pyrolidine-1-yl) phthalic acid (16a)
To a DMSO (3.0 mL) solution of 12 (200 mg, 0.833 mmol), 3-
(hydroxylmethyl)piperidine (960 mg, 8.33 mmol) was added. The
mixture was stirred at 80 °C for overnight. The reaction mixture
was purified with silica-gel column chromatography (hexane/
EtOAc) to give 15d (220 mg, 78.8%).
Compound 12 (120 mg, 0.500 mmol) was added to pyrolidine
(1.0 mL, 12.0 mmol) and then stirred at 80 °C for overnight. The
reaction mixture was concentrated under reduced pressure. The
residue was purified with silica-gel column chromatography (hex-
ane/EtOAc) to give 15a (27.0 mg, 18.6%).
EIMS: m/z 335 (M+).
To a 1,4-dioxane (1.0 mL) solution of 15d (220 mg, 0.657
mmol), 5.0 mol/L NaOH solution (5.0 mL) was added. The mixture
was stirred at 80 °C for overnight. 1.0 mol/L HCl solution was
added to the reaction mixture to adjust the pH until pH2, and then
concentrated under reduced pressure. The residue was purified
with synthetic adsorption resin Sepabeads SP-207 (H2O–CH3CN)
to give 16d (150 mg, 81.8%).
FABMS: m/z 292 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 15a (27 mg, 0.0927 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The reaction mixture
was stirred at 80 °C for overnight. 1.0 mol/L HCl solution was
added to the reaction mixture to adjust the pH until pH2, and then
concentrated under reduced pressure. The residue was purified
with synthetic adsorption resin (sepabeads SP-207) (H2O–CH3CN)
to give 16a (21.0 mg, 96.4%).
1H NMR (400 MHz, D2O) d 1.36 (1H, m), 1.83 (2H, m), 2.06 (2H,
m), 3.21–3.55 (6H, m), 7.44 (1H, d, J = 7.3 Hz, phenyl), 7.62–7.69
(2H, m, phenyl).
1H NMR (400 MHz, D2O) d 2.79 (4H, m, pyrolidine), 4.24 (4H, m,
pyrolidine), 8.03(1H, dd, J = 2.7, 5.8 Hz, phenyl), 8.20 (1H, d, J = 5.8
Hz, phenyl), 8.21 (1H, d, J = 2.7 Hz, phenyl).
FABMS: m/z 280 (M+H)+; FAB-HMS (M+H)+ calcd for C14H18NO5:
280.1185, found: 280.1185.
ESIMS: m/z 236 (M+H)+.
4.16. 3-(40-Hydroxypiperidine-1-yl)phthalic acid (16e)
4.13. 3-(Piperidine-1-yl) phthalic acid (16b)
To a DMSO (10 mL) solution of 12 (1.0 g, 4.17 mmol), 4-hydroxy-
piperidine (4.2 g, 41.5 mmol) was added. The mixture was stirred at
80 °C for overnight. Water was added to the reaction mixture, and
Compound 12 (120 mg, 0.500 mmol) was added to piperidine
(0.82 mL, 8.30 mmol) and then stirred at 80 °C for overnight. Water

Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
5849
then extracted with EtOAc three times. The combined organic layers
were dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified with silica-gel column chroma-
tography (hexane/EtOAc) to give 15e (1.35 g, 99.4%).
EIMS: m/z 321 (M+).
mixture was stirred at 80 °C for overnight. Water was added to
the reaction mixture, and then extracted with EtOAc three times.
The combined organic layers were dried over anhydrous MgSO4,
and then concentrated under reduced pressure. The residue was
purified with preparative TLC (hexane/EtOAc) to give 15h (41.0
mg, 20.7%).
To a 1,4-dioxane (5.0 mL) solution of 15e (1.0 g, 3.12 mmol), 5.0
mol/L NaOH solution (25.0 mL) was added. The mixture was stirred
at 80 °C for overnight. 1.0 mol/L HCl solution was added to the
reaction mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. The residue was purified with synthetic
adsorption resin Sepabeads SP-207 (H2O–CH3CN) to give 16e
(500 mg, 60.5%).
ESIMS: m/z 398 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 15h (60.0 mg, 0.151
mmol), 5.0 mol/L NaOH solution (5.0 mL) was added. The mixture
was stirred at 80 °C for overnight. 1.0 mol/L HCl solution was
added to the reaction mixture to adjust the pH until pH2, and then
concentrated under reduced pressure. The residue was purified
with SP-207 (H2O/CH3CN) to give 16h (20.0 mg, 38.8%).
1H NMR (400 MHz, CDCl3+CD3OD) d 2.05 (2H, m, piperidiene),
2.64 (2H, m, piperidiene), 3.14 (2H, m, piperidiene), 3.66 (2H, m,
piperidiene), 7.33 (1H, d, J = 7.6 Hz, phenyl), 7.41 (1H, dd,
J = 7.3,7.6 Hz, phenyl), 7.54 (1H, d, J = 7.3 Hz, phenyl), 7.66 (5H,
br s, phenyl).
1H NMR (400 MHz, D2O) d 1.88 (2H, m, piperidiene), 2.16 (2H,
m, piperidiene), 3.44 (2H, m, piperidiene), 3.58 (2H, m, piperidi-
ene), 4.05 (1H, m, piperidiene), 7.46 (1H, dd, J = 0.97, 7.5 Hz, phe-
nyl), 7.65 (1H, dd, J = 7.5, 8.1 Hz, phenyl), 7.70(1H, dd, J = 0.97,
8.1 Hz, phenyl).
FABMS: m/z 266 (M+H)+; FAB-HMS (M+H)+ calcd for C13H16NO5:
266.1028, found: 266.1028.
FABMS: m/z 342 (M+H)+.
4.17. 3-(40-(Hydroxymethyl)piperidine-1-yl)phthalic acid (16f)
4.20. 3-(40-(Methoxy)piperidine-1-yl)phthalic acid (16i)
4-(Hydroxymethyl)piperidine (960 mg, 9.50 mmol) was added
to 12 (200 mg, 0.833 mmol), and then stirred at 80 °C for overnight.
The reaction mixture was purified with preparative TLC (hexane/
EtOAc) go give 15f (50.0 mg, 17.9%).
To a THF (5.0 mL) solution of 15e (200 mg, 0.623 mmol), so-
dium hydride (75.0 mg, 3.12 mmol), methyl iodide (0.15 mL,
2.40 mmol) were added. The mixture was stirred at room temper-
ature for overnight. Water was added to the reaction mixture, and
then extracted with EtOAc three times. The combined organic lay-
ers were dried over anhydrous MgSO4, and then concentrated un-
der reduced pressure. The residue was purified with silica-gel
column chromatography (hexane/EtOAc) to give 15i (58.0 mg,
27.8%).
EIMS: m/z 335 (M+).
To a 1,4-dioxane (1.0 mL) solution of 15f (50.0 mg, 0.149 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. 1.0 mol/L HCl solution was added to the
reaction mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. The residue was purified with HP-20
(H2O–CH3CN) to give 16f (30 mg, 72.2%).
ESIMS: m/z 336 (M+H)+.
To a 1,4-dioxane (1.0 mL) solution of 15i (58.0 mg, 0.173 mmol),
5.0 mol/L NaOH solution (5.0 mL) was added. The mixture was stir-
red at 80 °C for overnight. 1.0 mol/L HCl solution was added to the
reaction mixture to adjust the pH until pH2, and then concentrated
under reduced pressure. The residue was purified with synthetic
adsorption resin Sepabeads SP-207 (H2O/CH3CN) to give 16i (40.0
mg, 82.9%).
1H NMR (400 MHz, D2O) d 1.55 (2H, m, piperidiene), 1.85 (1H,
m, piperidiene), 2.06 (2H, m, piperidiene), 3.48 (6H, m, piperidiene,
CH2), 7.37 (1H, dd, J = 2.5, 6.5 Hz, phenyl), 7.60 (1H, d, J = 6.5 Hz,
phenyl), 7.61 (1H, d, J = 2.5 Hz, phenyl).
FABMS: m/z 280 (M+H)+; FAB-HMS (M+H)+ calcd for C14H18NO5:
280.1185, found: 280.1185.
1H NMR (400 MHz, D2O) d 1.95 (2H, m, piperidiene), 2.16 (2H,
m, piperidiene), 3.33 (3H, s, methyl), 3.40 (2H, m, piperidiene),
3.56 (2H, m, piperidiene), 3.70 (1H, m, piperidiene), 7.41 (1H, d,
J = 6.6 Hz, phenyl), 7.62 (2H, m, phenyl).
4.18. 3-(40-(Hydroxyethyl)piperidine-1-yl)phthalic acid (16g)
To a DMSO (5.0 mL) solution of 12 (152 mg, 0.633 mmol), 4-
(hydroxyethyl)piperidine (810 mg, 6.26 mmol) was added. The mix-
ture was stirred at 80 °C for overnight. Water was added to the reac-
tion mixture, and then extracted with EtOAc three times. The
combined organic layers were dried over anhydrous MgSO4, and
then concentrated under reduced pressure. The residue was purified
with preparative TLC (hexane/EtOAc) to give 15g (52.0 mg, 23.5%).
ESIMS: m/z 350 (M+H)+.
ESIMS: m/z 278 (MꢀH)ꢀ; FAB-HMS (M+H)+ calcd for C14H18NO5:
280.1185, found: 280.1185.
4.21. MBL inhibitory activity
MBL inhibitory activity was determined spectrophotometrically
using nitrocefin (Oxoid, Basingstoke, England) as the substrate.
IMP-1 was PCR amplified from plasmid DNA prepared from a car-
bapenems-resistant P. aeruginosa MSC15369. The PCR product was
cloned into pTrcHis2 TOPO vector (Invitrogen, Carlsbad, CA) and
To a 1,4-dioxane (1.0 mL) solution of 15g (50.0 mg, 0.143
mmol), 5.0 mol/L NaOH solution (5.0 mL) was added. The mixture
was stirred at 80 °C for overnight. 1.0 mol/L HCl solution was
added to the reaction mixture to adjust the pH until pH2, and then
concentrated under reduced pressure. The residue was purified
with HP-20 (H2O/CH3CN) to give 16g (16.0 mg, 38.2%).
1H NMR (400 MHz, D2O) d 1.55 (2H, m, piperidiene), 1.85 (1H,
m, piperidiene), 2.06 (2H, m, piperidiene), 3.35 (8H, m, piperidiene,
CH2), 7.37(1H, dd, J = 2.5, 6.5 Hz, phenyl), 7.60 (1H, d, J = 6.5 Hz,
phenyl), 7.61 (1H, d, J = 2.5 Hz, phenyl).
expressed in Escherichia coli DH5
induction with 0.5 mM isopropyl-b-
(Wako, Osaka, Japan) for 3 h at room temperature. Soluble IMP-1
was purified from cell extracts by Ni-NTA slurry (Qiagen, Valencia,
CA). The IC50 of inhibitors were determined following incubation
for 20 min at room temperature with IMP-1 (1.0 nM in 50 mM
a
(Toyobo, Osaka, Japan) after
-(ꢀ)- thiogalactopyranoside
D
FABMS: m/z 292 (MꢀH)ꢀ.
HEPES, pH7.5) in the presence of 100
lM ZnSO4 and 20 lg/ml
BSA (Sigma–Aldrich, St. Louis, MO). Using initial velocity as a
measure of activity, inhibition was monitored spectrophotometri-
cally at 490 nm in a Wallac ARVOsx 96-well plate reader (Perkin
Elmer, Waltham, MA) employing nitrocefin as the reporter sub-
4.19. 3-(40-Hydroxy-40-phenylpiperidine-1-yl)phthalic acid (16h)
To a DMSO (2.0 mL) solution of 12 (120 mg, 0.500 mmol), 4-hy-
strate at 100 lM.
droxy-4-phenylpiperidine (440 mg, 2.48 mmol) was added. The

5850
Y. Hiraiwa et al. / Bioorg. Med. Chem. 21 (2013) 5841–5850
4.22. Mic
providing us with the IMP-1 producing plasmid. We thank Mizuyo
Ida and Erumi Murase for helpful discussion and technical assis-
tance. We also thank Shigeko Miki for NMR spectroscopic and mass
spectrometric analyses, respectively.
The in vitro activities were determined by the microbroth dilu-
tion method in accordance with CLSI. Mueller–Hinton II broth
(Becton, Dickinson and Company, Sparks, MD) was used for testing
procedure. MICs were determined for BIPM alone and in combina-
References and notes
tion with the inhibitor at a constant 50 lg/ml. The bacterium inoc-
ulum size was approximately 5 ꢁ 104 CFU/well.
1. Ambler, R. P. Philos. Trans. R. Soc. Landon, Biol. Sci. 1980, 289, 321.
2. Daiyasu, H.; Osaka, K.; Ishino, Y.; Toh, H. FEBS Lett. 2001, 503, 1.
3. Ida, T. Antibiot. Chemother. 2007, 23, 227.
Acknowledgements
4. Grossi, P.; Gasperina, D. D. Expert Rev. Anti Infect. Ther. 2006, 4, 639.
5. Hiraiwa, Y.; Morinaka, A.; Fukushima, T.; Kudo, T. Bioorg. Med. Chem. Lett. 2009,
19, 2162.
6. Okamoto, K.; Gotoh, N.; Nishino, T. J. Infect. Chemother. 2002, 8, 371.
7. Iyobe, S.; Tsunoda, M.; Mitsuhashi, S. FEMS Microbiol. Lett. 1994, 121, 175.
We thank Professor Naomasa Gotoh from Kyoto Pharmaceutical
University for kindly providing us with the P. aeruginosa
PAO1DmexAB-oprM strain. We thank Dr. Shizuko Iyobe for kindly