Metallo-β-lactamase inhibitory activity of phthalic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.07.018

4-Butyl-3-methylphthalic acid was recognized as a metallo-β-lactamase inhibitor. The structure-activity relationship study of substituted phthalic acids afforded 3-phenylphthalic acid derivatives as potent IMP-1 inhibitors. On the other hand, 3-substituted with 4-hydroxyphenyl phthalic acid derivative displayed a potent combination effect with biapenem (BIPM) against Pseudomonas aeruginosa that produce IMP-1.

Full text of DOI:10.1016/j.bmcl.2009.07.018

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5162–5165
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Metallo-b-lactamase inhibitory activity of phthalic acid derivatives
*
Yukiko Hiraiwa , Akihiro Morinaka, Takayoshi Fukushima, Toshiaki Kudo
Pharmaceutical Research Center, Meiji Seika Kaisha, 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:
4-Butyl-3-methylphthalic acid was recognized as a metallo-b-lactamase inhibitor. The structure–activity
relationship study of substituted phthalic acids afforded 3-phenylphthalic acid derivatives as potent IMP-
1 inhibitors. On the other hand, 3-substituted with 4-hydroxyphenyl phthalic acid derivative displayed a
potent combination effect with biapenem (BIPM) against Pseudomonas aeruginosa that produce IMP-1.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 25 March 2009
Revised 29 June 2009
Accepted 4 July 2009
Available online 9 July 2009
Keywords:
Metallo-b-lactamase
Pseudomonas aeruginosa
Efflux pump
b-Lactam antibiotics are the most widely used as clinical agents
for the treatment of bacterial infection. Among the b-lactam anti-
biotics, carbapenems, such as imipenem (IPM), meropenem
(MEPM) and biapenem (BIPM), have potent broad-spectrum anti-
bacterial activities that are widely used to combat serious infec-
tions. It is well known that b-lactams are inactivated by b-
lactamases. Based on their amino acid sequence homology, b-lacta-
mases are divided into four classes A, B, C and D according to the
Amber classification.1 Among them, class B b-lactamases are
known as metallo-b-lactamases (MBLs) because their active sites
contain zinc.2 MBLs hydrolyze b-lactams, penicillins, cephalospo-
rins and carbapenems. Multi-drug resistant Pseudomonas aerugin-
osa (MDRP) are reported to produce MBL at high frequency,
which can result in significant problems in clinical practice.3 b-Lac-
tamase inhibitors that are effective against the class A serine b-lac-
tamase, clavulanic acid, sulbactam or tazobactam are used with
b-lactam antibiotics in the clinical field. Although some MBL inhib-
itors have been reported4 none of them are clinically approved.
Here, we aimed to develop MBL inhibitors that are effective against
Gram-negative bacteria, in particular P. aeruginosa. We focused on
the IMP-15 subclass of MBL because these enzymes are present at
high frequency in clinical isolates.6 Initially, we screened our com-
pound library for IMP-1 inhibitory activity using nitrocefin as sub-
strate and selected 230 candidate compounds. The 230 compounds
were then screened for a combination effect with MEPM or ceftaz-
idime (CAZ) against IMP-1 producing Escherichia coli and obtained
40 active compounds. Of these compounds, we selected the phtha-
lic acid derivative 1 as the lead compound (Fig. 1).
We reasoned that the two carboxyl groups of compound 1
might interact with zinc in the active site of IMP-1. Therefore, we
fixed the two carboxyl groups and investigated the effect of substi-
tution of the phenyl ring on MBL inhibitory activity. First, we syn-
thesized the 4-subsutituted phthalic acid derivatives (Scheme 1).
Esterification of commercially available 2 gave diester deriva-
tive 3, and then Pd catalyzed C–C bond formation afforded the 4-
substituted derivative 4. Hydrolysis under alkali conditions gave
compound 5a and 5c, respectively. Similarly, the hydrolysis of
commercially available 4-t-butylphthalic anhydride 6 gave com-
pound 5b. The IMP-1 inhibitory activities of 4-substituted phthalic
acid derivatives 5a–e are shown in Table 1.
IMP-1 inhibitory activity (IC50
) of lead compound 1 was
16.0 M. Because phthalic acid 5d displayed no IMP-1 inhibitory
l
activity, it was recognized that substitution of the phenyl ring
was necessary for IMP-1 inhibitory activity. Substitution at the
4-position with a methyl (5e), t-butyl (5b) or phenyl (5c) moiety
also resulted in a compound with no IMP-1 inhibitory activity.
By contrast, 4-n-butylphthalic acid 5a showed weak IMP-1 inhib-
itory activity (IC50 = 243 lM), but this activity was less than a
one-tenth that of the lead compound 1. From these results, we
* Corresponding author at present address: Kyoto Branch, Pharmaceutical
Marketing Division, Meiji Seika Kaisha, Ltd., 285 Fukuro-cho, 1-chome, Higashiy-
ama-ku, Kyoto 605-0911, Japan. Tel.: +81 75 541 2161; fax: +81 75 551 1452.
E-mail address: yukiko_hiraiwa@meiji.co.jp (Y. Hiraiwa).
Figure 1. Lead compound of MBL inhibitor.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.018

Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5162–5165
5163
Scheme 1. Synthesis of 4-substituted phthalic acid derivatives. Reagents and conditions: a) p-TsOH H2O, EtOH, 100 °C; b) Bop-reagent, iPr2NEt, EtOH, rt (79%); c) nBuB(OH)2,
K2CO3, Pd(PPh3)4, toluene, 80 °C (4a), PhB(OH)2, K2CO3, Pd(PPh3)4, toluene, 80 °C (93%, 4c); d) (1)NaOHaq, THF, MeOH, rt (2)HClaq (4.2%, 2steps, 5a), (88%, 5c); e) (1)NaOHaq,
THF, rt, (2)HClaq (55%).
Table 1
concluded that substitution of the phenyl ring, in particular at
the 3-position, is important for IMP-1 inhibitory activity. We
then synthesized a series of 3-substituted phthalic acid deriva-
tives (Scheme 2).11
IMP-1 inhibitory activity of 4-subsutituted phthalic acid derivatives12
O
OH
A two step esterification of commercially available 7 gave
diethylester derivative 8. Reduction of the nitro group then
afforded 9 in good yield. The 3-amino derivative 9 was subjected
to a Sandmeyer reaction to give the 3-bromo derivative 10. Pd
catalyzed C–C formation (and sequential reduction for 11b0,
11d0–f’ then) gave 3-substituted phthalic acid derivatives
11b–i.7 Hydrolysis of each diethylester 11b–i under alkali condi-
tions gave the corresponding compound 12b–i, respectively. By
contrast with the 4-substituted derivatives, hydrolysis of 3-
substituted derivatives was performed at elevated temperatures.
In the case of 12d, it was confirmed that the hydroxyl group on
OH
R4
R3
O
Compd
R3
R4
IMP-1 inhibitory activity IC50 (lM)
1
Me
H
H
H
H
n-Bu
n-Bu
t-Bu
Ph
H
Me
16.0
243
>300
>300
>100
>300
5a
5b
5c
5da
5ea
H
a
Compounds 5d and 5e were commercially available.
Scheme 2. Synthesis of 3-substituted phthalic acid derivatives. Reagents and conditions: a) H2SO4aq, EtOH, 80 °C; b) EtI, K2CO3, DMF, (90%, 2steps); c) H2/Pd-C, EtOH, rt,
(100%); d) NaNO2, HBr then CuBr, HBr, 70 °C (73%); e) allyl alchol, Pd(tBu3P)2, N,N-dicyclohexylmethylamine , K2CO3, Pd(PPh3)4, toluene, rt, (63%, 11b0), phenylboronic acid,
K2CO3, Pd(PPh3)4, toluene, 80 °C, (54%, 11c), 2-(2-benzyloxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolan, K2CO3, Pd(PPh3)4, toluene, 80 °C, (23%, 11d0), 2-(3-benzyloxy-
phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolan, K2CO3, Pd(PPh3)4, toluene, 80 °C, (28%, 11e0), 2-(4-benzyloxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, K2CO3,
Pd(PPh3)4, toluene, 80 °C, (52%, 11f0), ethyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate, K2CO3, Pd(PPh3)4, toluene, 80 °C, (11%, 11g), methyl 3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate, K2CO3, Pd(PPh3)4, toluene, 80 °C, (23%, 11h), methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate, K2CO3,
Pd(PPh3)4, toluene, 80 °C, (75%, 11i); f) NaBH4, EtOH, rt, (80%); g) H2, Pd-C, rt; h) (1)NaOH aq, 1,4-dioxane, 80 °C (2)HClaq (58%, 12b; 88%, 12c; 53%, 2steps, 12d; 59%,
2steps, 12e; 17%, 2steps, 12f; 60%, 12g; 32%, 12h; 27%).

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Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5162–5165
Table 2
Table 3
IMP-1 inhibitory activity of 3-subsutituted phthalic acid derivatives
Combination effect of 3-substituted phthalic acid derivatives with BIPM
O
Compd
Combination effect (50
lg/mL) with BIPM
MIC of BIPM (
l
g/mL)
OH
OH
P. aeruginosa KG50029/
P. aeruginosa PAO1/
pMS36310
(
D
mexAB)
pMS36310
R3
O
12a
4
16
12b
12c
12d
1
50.5
2
4
2
8
Compd
R3
IMP-1 inhibitory activity IC50
160
(lM)
12a
–Me
12e
12f
12g
0.5
50.25
64
4
1
64
12b
12c
13.2
0.968
2.44
OH
12h
12i
BIPM only
32
0.5
64–128
64
2
64–128
ONa
12da
by their substitution position. Although the ortho-substituted
derivative 12g displayed only weak IMP-1 inhibitory activity,
para-substituted derivative 12i increased the inhibitory activity
about 100-fold.
In this study, we found that 3-substituted phthalic acid deriva-
tives had potent IMP-1 inhibitory activity. Next, we investigated
the combination effect of 3-substituted phthalic acid derivatives
with the carbapenem antibiotic, Biapenem against P. aeruginosa.
The combination effect of 3-substituted phthalic acid derivatives
12a–12i with BIPM is shown in Table 3.13
OH
12e
12f
1.75
1.55
207
OH
COOH
The phenyl derivative 12c, m-hydroxyphenyl derivative 12e, p-
hydroxyphenyl derivative 12f and p-carboxyphenyl derivative 12i
showed potent combination effects with BIPM against P. aeruginosa
12g
12h
KG5002/pMS363(DmexAB).
Some interesting approaches to the development of MBLs inhib-
itors have previously been reported.4 However, to our knowledge,
there are very few examples of MBL inhibitors displaying a combi-
nation effect with carbapenem antibiotics against P. aeruginosa. In
this study, we discovered that the 3-substituted phthalic acid
showed potent MBL inhibitory activity. Moreover these com-
pounds displayed a combination effect with BIPM against P. aeru-
ginosa that produces IMP-1.
The efflux systems of P. aeruginosa make an important contri-
bution to antibiotic resistance. The resistance nodulation division
(RND) efflux system of MexAB-OprM is such examples. It has
been reported that carbapenems are effluxed by MexAB-OprM
of the RND efflux system. Therefore, we tested the combination
effect with BIPM against P. aeruginosa expressing efflux system
MexAB-OprM.8
COOH
17.9
2.25
12i
COOH
a
Bis-sodiumbenzoate.
the phenyl moiety at the 3-position and the carboxyl group at
the 2-position lactonized under acidic conditions. As a result,
12d was treated as the sodium salt. By contrast, 3-methylphtha-
lic acid 12a was obtained from the hydrolysis of 3-methylphtha-
lic anhydride under the same conditions used for the conversion
of 6–5b. The IMP-1 inhibitory activities of 3-substituted phthalic
acid derivatives 12a–i are shown in Table 2.
3-Methylphthalic acid 12a showed weak IMP-1 inhibitory
activity whereas compound 12b, with a 3-hydroxypropyl group
at the 3-position, displayed a 10-fold increase in IMP-1 inhibi-
tory activity. These results suggested that a bulky group at
the 3-position of the phenyl ring increases IMP-1 inhibitory
activity. Therefore, we decided to introduce the phenyl group,
as a more bulky substitution, at the 3-position. Substitution
with the phenyl ring at the 3-position 12c increased IMP-1
inhibitory activity 100-fold compared with the methyl deriva-
tive 12a. Next we investigated the influence of substitutions
of the phenyl ring at the 3-position of phthalic acid. Hydroxyl
derivatives 12d–12f all showed strong IMP-1 inhibitory activity.
Unlike the hydroxyl group derivatives, IMP-1 inhibitory activity
of carboxyl group derivatives 12g–12i are strongly influenced
The p-hydroxyphenyl derivative 12f showed the most potent
combination effect. It is interesting that the MIC of P. aeruginosa
KG5002/pMS363(DMexAB) is smaller than that of P. aeruginosa
PAO1/pMS363. Thus, the influence of the efflux system of Mex-
AB-OprM on the MIC is about fourfold. In conclusion, we found that
the 3-substituted phthalic acid showed potent MBL inhibitory
activity. Moreover these compounds displayed a combination ef-
fect with BIPM against P. aeruginosa that produces IMP-1. This
combination effect was also shown against P. aeruginosa with ef-
flux system MexAB-OprM. Based on these findings, further struc-
ture–activity relationship studies of this class of compound are
currently in progress.
Acknowledgements
We thank Professor Naomasa Gotoh from Kyoto Pharmaceutical
University for kindly providing us with the P. aeruginosa
PAO1
providing us with the IMP-1 producing plasmid. We thank Mizuyo
DmexAB-oprM strain. We thank Dr. Shizuko Iyobe for kindly

Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5162–5165
5165
10. Iyobe, S.; Tsunoda, M.; Mitsuhashi, S. FEMS Microbiol. Lett. 1994, 121, 175.
Ida and Erumi Murase for helpful discussion and technical assis-
tance. We also thank Shigeko Miki for NMR spectroscopic and mass
spectrometric analyses, respectively.
11. Selected spectral data. Compound 12b: 1H NMR (DMSO-d6, 400 MHz) d 1.69
(2H, m), 2.64 (2H, t, J = 8.0 Hz), 3.39 (2H, t, J = 6.4 Hz), 7.42 (1H, dd, J = 7.6,
7.6 Hz), 7.49 (1H, d, J = 7.6 Hz), 7.72 (1H, d, J = 7.6 Hz), FABMS: m/z 225 [M+H]+.
Compound 12c: 1H NMR (CDCl3, 400 MHz) d 7.40 (5H, m), 7.54–7.61 (2H, m),
8.05 (1H, dd, J = 1.5, 7.6 Hz), FABMS: m/z 243 [M+H]+. Compound 12f: 1H NMR
(CDCl3, 400 MHz) d 6.81 (2H, d, J = 8.3 Hz), 7.17 (2H, d, J = 8.3 Hz), 7.47 (2H, m),
7.87 (1H, dd, J = 2.7, 6.3 Hz), FABMS: m/z 259 [M+H]+. Compound 12i: 1H NMR
(DMSO-d6, 400 MHz) d 7.50 (2H, d, J = 8.2 Hz), 7.59 (2H, m), 7.92 (1H, dd,
J = 2.5, 6.6 Hz), 7.98 (2H, d, J = 8.2 Hz), ESIMS: m/z 287 [M+H]+.
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nitrocefin (Oxoid, Basingstoke, England) as the substrate. IMP-1 was PCR
amplified from plasmid DNA prepared from
aeruginosa MSC15369. The PCR product was cloned into pTrcHis2 TOPO
vector (Invitrogen, Carlsbad, CA) and expressed in E. coli DH5 (Toyobo,
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isopropyl-b-D(ꢀ)-
a carbapenems-resistant P.
a
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plate reader (Perkin Elmer, Waltham, MA) employing nitrocefin as the
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13. The in vitro activities were determined by the microbroth dilution 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 combination with the inhibitor at
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