Thiophenyl oxime-derived phosphonates as nano-molar class C beta-lactamase inhibitors reducing MIC of imipenem against Pseudomonas aeruginosa and Acinetobacter baumannii

Article abstract of DOI:10.1016/j.bmcl.2011.04.122

The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.

Full text of DOI:10.1016/j.bmcl.2011.04.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4363–4365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiophenyl oxime-derived phosphonates as nano-molar class C
beta-lactamase inhibitors reducing MIC of imipenem against Pseudomonas
aeruginosa and Acinetobacter baumannii
Qiang Tan a, , Aimie M. Ogawa b, Susan L. Raghoobar b, Douglas Wisniewski b, Lawrence Colwell a,
⇑
Young-Whan Park b, Katherine Young b, Jeffrey D. Hermes b, Frank P. DiNinno a, Milton L. Hammond a
a Department of Medicinal Chemistry, Merck & Co., Inc., Rahway, NJ 07065, USA
b Department of Infectious Diseases, Merck & Co., Inc, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhib-
itors is described. A number of these analogs were potent and selective inhibitors of class C beta-lacta-
mases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of
imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title
compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter
baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a
highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice com-
pound 3b exhibited phamacokinetics similar to imipenem.
Received 6 March 2011
Revised 24 April 2011
Accepted 26 April 2011
Available online 13 May 2011
Keywords:
Thiophenyl
Oxime
Phosphonate
Class C
Ó 2011 Elsevier Ltd. All rights reserved.
Beta-lactamase inhibitors
Imipenem
Pseudomonas aeruginosa
Acinetobacter baumannii
MIC
OH
β-lactamase-serine-O
Beta-lactamases, enzymes that catalyze the chemical degrada-
tion of beta-lactam antibiotics, are a leading cause of the fading
efficacy of beta-lactam antibiotics, particularly in problematic
Gram-negative organisms.1 Co-administration of beta-lactam anti-
biotics with beta-lactamase inhibitors (BLI’s) has been a time hon-
ored strategy to restore the clinical efficacy. Beta-lactamases have
been categorized into four classes:2 classes A, C and D are serine-
based hydrolases; class B comprises zinc–metallo hydrolases.
Unfortunately, the currently marketed beta-lactamase inhibitors,
such as clavulanate, tazobactam and sulbactam, are only effective
on a subset of class A beta-lactamases. Thus there is an important
medical need to develop new beta-lactamase inhibitors to combat
the growing threat of antibiotic resistance.
O
P
HO
P
O
O
O
O
N
β-lactamase-serine-OH
N
N
H
O
N
H
S
S
H2N
H2N
NO2
N
N
O
A
HO
NO2
compound, it possesses a number of liabilities among which are
moderate potency, and a problematic nitro-phenol leaving group.
Moreover, it has not been reported whether compound A improves
the efficacy of beta-lactam antibiotics against resistant bacterial
strains.
We selected 1, a close analog of compound A, as an initial target
for our studies, and tested it against class C as well as class A and D
beta-lactamases. Similar to compound A, 1 carries a good phenolic
leaving group; but unlike compound A, 1 does not have a nitrophe-
nol as a leaving group. As Table 1 shows, 1 is a selective class C
Recently phosphonate A was reported as a class C beta-lacta-
mase inhibitor. Incubation of compound A with a susceptible
beta-lactamase results in an irreversible phosphorylation of the ac-
tive site serine.3 While compound A is a valuable proof-of-principle
beta-lactamase inhibtor, with a low lM IC50 for AmpC and P99. Ini-
tial SAR by capping the amino group as trichloroacetamide (2a &
2b) did not improve potency.
⇑
Corresponding author.
E-mail address: qiang_tan@merck.com (Q. Tan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.122

4364
Q. Tan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4363–4365
Table 1
IC50 (lM) against different classes (bold letter in parenthesis) of beta-lactamases
Compound
TEM-1 (A)
SHV-5 (A)
AmpC (C)
P99
OXA-40 (D)
(Pseudomonas aeruginosa)
(Klebsiella pneumoniae)
(Pseudomonas aeruginosa)
(C) (Enterobacte cloacae)
(Acinetobacter baumannii)
1
>50
>50
>50
>50
>50
28
>50
25
15
>50
>12.5
22
1.5
3.0
3.0
0.0096
0.046
0.0075
0.20
6.5
3.2
0.80
0.34
0.71
0.039
0.10
0.032
0.10
21
>50
>12.5
>12.5
>50
26.
2a
2b
3
3a
3b
4
5a
5b
6a
6b
7
17
21
23
27
>50
>50
17
>50
16
>50
>50
>50
>50
-
>50
>50
27
>50
31
3.0
0.021
0.087
0.0050
0.087
0.17
0.019
HO
P
O
HO
O
O
O
O
HO
O
HO
P
O
O
O
O
O
N
N
S
P
CN
CN
N
H
O
P
N
H
O
CN
N
O
CN
N
S
Cl3C
F
N
H
O
H2N
F
H
N
H
N
S
S
S
F
2a (C=N isomer A)
2b (C=N isomer B)
N
O
F
N
1
O
3
3a
HO
P
O
O
N
O
HO
P
O
O
A carries an amino-thiazole oxime moiety common to many 2nd
and 3rd generation Cephlosporins (Fig. 1), a design3 intended to re-
duce recognition by beta-lactamases. This perhaps explains its
moderate potency. Thus to improve potency we replaced the ami-
no-thiazole with a thiophene (3 ꢀ 7), a design inspired by the
following:
CN
N
H
CN
N
H
OH
O
F
S
F
N
O
3b
4
HO
HO
P
O
OH
O
OH
P
O
N
H
N
O
H
S
S
S
O
O
N
N
O
5b
5a
O
F
-O
P
H2N
NH
O
NH
O
Me
N
Me
S
O
O
N
N
OR
CN
N
H
O
N
OR
N
-
-
O
SO3
SO3
HO
S
O
O
F
N
P
aztreonam
(SQ 26,776)
B
CN
6a
6b
(C=N isomer A)
N
O
H
(C=N isomer B)
S
Cl
N
N+
O
7
Aztreonam, a narrow spectrum monobactam antibiotic, carries
a similar amino-thaizole oxime moiety. However, it has been re-
ported that certain thiophene analogs such as compound B were
significantly better than aztreonam in acting as a beta-lactamase
inhibitor and synergizing the antibacterial activity of ceftazidime.4
Syntheses of thiophene analogs (3 ꢀ 7, Fig. 2) are straightfor-
ward. The published general chemistry3 gave access to both oxime
isomers 14 (Scheme 1). A slight modification as shown in Scheme 2
Figure 2. Thiophenyl oxime-derived phosphonate as novel BLI’s.
provided isomer 3b stereoselectively, employing oxime C@N bond
isomerization to yield a single isomer 17 during TMSBr-induced es-
ter cleavage of isomer mixture 16.5 Conveniently, as shown next,
3b is the more potent isomer.
As shown in Table 1 replacement of the amino-thiazole with a
thiophene resulted in a significant improvement in potency against
the two class C b-lactamases tested. The Z oxime stereoisomer 3b
is more potent than the E isomer, 3a. The desmethyl analog 4 is
slightly less potent, as are 6a & 6b, compounds designed to modify
the overall molecular charge. Compounds 5a & 5b confirm the
presence of the phenolic leaving group is necessary for inhibitory
activity, consistent with the mechanism of action. The chloro thio-
phene 7 exhibited potency similar to 3b.
O
OH
N
N
H
H
N
S
N
N
S
N
H2N
H2N
O
N
O
O
S
O
N
O
S
O
O
O
OH
OH
All of these compounds are much less potent on class A and D
Cefdinir
O
Cefotaxime
enzymes, with double-digit
lM or more IC50. Noteworthy is that
the best class C compounds, 3b & 7, also possesses the best class
A and class D activities, suggesting that it might be possible to de-
sign an optimal compound with broad spectrum inhibitory
activity.
Compounds 3b and 7 were evaluated for their abilty to syner-
gize the antimicrobial activity of imipenem against an AmpC pro-
ducing strain of imipenem-resistant Pseudomonas aeruginosa. As
O
N
N+
N
H
H
N
S
N
S
N
N
H2N
H2N
O
O
N
O
N
O
N+
S
S
O
O
OH
OH
Cefepime
Cefpirome
shown in Table 2 co-administration with up-to-100 lM 3b or 7
Figure 1. Representative cephalosporins carrying amino-thiazole oxime moiety.

Q. Tan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4363–4365
4365
O
O
nisms including the high level expression of both class C and class
D beta-lactamases. As a proof of principle experiment we evalu-
ated the ability of compounds 3b and 7 to synergize the activity
of imipenem against the highly imipenem-resistant strain of A.
baumannii (CL6188).6 A. baumannii (CL6188) expresses multiple
beta-lactamases of class A, C and D. For this strain, compound 3b
was able to significantly lower the MIC of impenem from 128 to
O
a
b
OEt
OEt
OEt
S
S
S
N
N
O
OH
OR
8
9
10
O
O
O
c
d
OH
O
N
S
S
N
O
N
12
OR
OR
11
O
16
lg/mL. Interestingly, compound 7 was ineffective. No antibiotic
O
activity was observed with either inhibitor alone at 100
lM.
O
O
f
e
N
H
P
N
H
P
Taking all the data together we decided to take a further look at
3b. This compound in mice at 2.5 mg/kg i.v. dosing gave the follow-
ing pharmacokinetics:
OH
S
OH
S
N
O
N
OH
OR
OR
14
13
CN
Half life 0.54 h; AUC(Norm) 0.98 lM/h/kg/mg; Clearance
F
43 mL/min/kg; volume of distribution 1 L/kg.
Its half life matches or exceeds the imipenem mouse half life;7
together with the MIC reduction data, 3b warrants further study as
Scheme 1. Reagents and conditions: (a) hydroxyamine hydrochloride, pyridine,
EtOH; gave ca. 1:1 mixture of oxime isomers; (b) cesium carbonate, DMF, RBr or RI;
(c) KOH, MeOH, THF, water; (d) EDC, hydroxysuccinimide, dioxane, DMF; (e)
(aminomethyl)phosphonic acid, sodium bicarbonate, water, DMF, EtOH, THF; (f)
ArOH, trichloroacetonitrile, pyridine, 80 °C.
a
potential beta-lactamase inhibitor in combination with
imipenem.
In summary, we discovered a series of novel thiophenyl oxime-
derived phosphonates as potent and selective class C beta-lacta-
mase inhibitors, with weak class A and class D activities. The most
potent compounds displayed suitable in vitro and PK profile sug-
gesting further studies of their potential to enhance the efficacy
of beta-lactam antibiotics, in particular imipenem, against resis-
tant bacterial strains. These studies and further SAR exploration
will be reported in due course.
O
O
O
P
OEt
a
b
OH
N
H
S
c
OEt
S
N
N
N
15
OMe
OMe
16
O
O
P
OH
N
H
3b
S
OH
References and notes
MeO
5b
1. Matagne, A.; Dubus, A.; Galleni, M.; Frere, J.-M. Nat. Prod. Rep. 1999, 16, 1.
2. Hall, B. G.; Barlow, M. J. Antimicrob. Chemother. 2005, 55, 1050.
3. Nukaga, M.; Kumar, S.; Nukaga, K.; Pratt, R. F.; Knox, J. R. J. Biol. Chem. 2004, 279,
9344.
4. (a) Maiti, S. N.; Setti, E. L.; Phillips, O. A.; Reddy, A. V. N.; Micetich, R. G.; Singh,
R.; Higashitani, F.; Kunugita, C.; Nishida, K.; Uji, T. PCT Int. Appl. 1998, WO
9847895 A1.; (b) Setti, E. L.; Maiti, S. N.; Phillips, O. A.; Reddy, A. V. N.; Micetich,
R. G.; Higashitani, F.; Kunugita, C.; Nishida, K.; Uji, T. PCT Int. Appl. 1999, WO
9910324 A1.; (c) Nishida, K.; Kunugita, C.; Uji, T.; Higashitani, F.; Hyodo, A.;
Unemi, N.; Maiti, S. N.; Phillips, O. A.; Spevak, P.; Atchison, K. P.; Salama, S. M.;
Atwal, H.; Micetich, R. G. Antimicrob. Agents Chemother. 1999, 43, 1895; (d)
Danes, C.; Navia, M. M.; Ruiz, J.; Marco, F.; Jurado, A.; Jimenez de Anta, M. T.;
Vila, J. J. Antimicrob. Chemother. 2002, 50, 261.
Scheme 2. Reagents and conditions: (a) HATU, Hunig’s base, diethyl (amino-
methyl)phosphonate hydrochloride, DMF; (b) TMSBr, DCM; (c) ArOH, trichloroace-
tonitrile, pyridine, 80 °C.
Table 2
MIC of Imipenem against Pseudomonas aeruginosa (CL5701) in the presence of BLI
Imipenem MIC (
lg/mL)
With 3b (
l
M)
With 7 (lM)
20
10
5
0
25
50
>100
0
12.5
25
5. 1H NMR (CD3OD, 500 MHz) d (ppm): Compound 5b 7.91 (dd, J = 4.0, 1.1 Hz, 1H),
7.68 (dd, J = 5.1, 1.1 Hz, 1H), 7.11 (dd, J = 5.1, 4.0 Hz, 1H), 4.12 (s, 3H), 3.73 (d,
J = 12.6 Hz, 2H). NOE: irradiation of –OMe protons at 4.12 ppm gave NOE signal
at 7.91 ppm corresponding to proton at thiophene C-3. Compound 3b 7.95 (d,
J = 3.4 Hz, 1H), 7.70 (d, J = 4.3 Hz, 1H), 7.54 (t, J = 8.2 Hz, 1H), 7.14 (t, J = 4.5 Hz,
1H), 6.74 (dd, J = 8.5, 2.2 Hz, 1H), 6.70 (dd, J = 11.3, 2.2 Hz, 1H), 4.15 (s, 3H), 3.77
(d, J = 12.6 Hz, 2H).
2.5
100
6. (a) Montefour, K.; Frieden, J.; Hurst, S.; Helmich, C.; Headley, D.; Martin, M.;
Boyle, D. A. Crit. Care Nurse 2008, 28, 15; (b) Perez, F.; Endimiani, A.; Bonomo, R.
A. Exp. Rev. Anti-Infect. Ther. 2008, 6, 269; Bou, G.; Martinez-Beltran, J.
Antimicrob. Agents Chemother. 2000, 40, 428; 2006, 50, 2280.; (d) Bou, G.;
Oliver, A.; Martinez-Beltran, J. Antimicrob. Agents Chemother. 2000, 44, 1556.
7. An independent PK study reported imipenem half life in mice to be
approximately 17 min. (Fluckiger, U.; Segessenmann, C.; Gerber, A. U.
Antimicrob. Agents Chemother. 1991, 35, 1905.).
reduced the imipenem MIC against P. aeruginosa in a dose-depen-
dent fashion. In terms of both dose-response and maximum MIC
reduction, 7 is slightly more potent than 3b in this assay (Table
1). In a control experiment, neither compound 3b or 7 alone dis-
played antibiotic activity at maximum dose of 100 lM.
Highly resistant Acinetobacter baumannii strains are known to
resist the activity of beta-lactam antibiotics by a variety of mecha-