Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712

Article abstract of DOI:10.1016/j.bmcl.2011.05.065

The bridged monobactam β-lactamase inhibitor MK-8712 (1) effectively inhibits class C β-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C β-lactamase mediated resi

Full text of DOI:10.1016/j.bmcl.2011.05.065

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4267–4270
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Side chain SAR of bicyclic b-lactamase inhibitors (BLIs). 2. N-Alkylated
and open chain analogs of MK-8712
Helen Chen a, , Timothy A. Blizzard a, Seongkon Kim a, Jane Wu a, Katherine Young b, Young-Whan Park b,
⇑
Aimie M. Ogawa b, Susan Raghoobar b, Ronald E. Painter b, Doug Wisniewski b, Nichelle Hairston b,
Paula Fitzgerald a, Nandini Sharma a, Giovanna Scapin a, Jun Lu a, Jeff Hermes b, Milton L. Hammond a,b
a Departments of Medicinal Chemistry, Merck Research Labs, Rahway, NJ 07065, USA
b Department of Infectious Diseases, Merck Research Labs, 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 bridged monobactam b-lactamase inhibitor MK-8712 (1) effectively inhibits class C b-lactamases.
Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with
imipenem to overcome class C b-lactamase mediated resistance. Although some analogs were more
potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 15 April 2011
Revised 16 May 2011
Accepted 18 May 2011
Available online 27 May 2011
Keywords:
b-Lactamase
Inhibitor
Carbapenem
Imipenem
Carbapenem antibiotics (e.g., imipenem) are an important ther-
apeutic option for the treatment of hospital-acquired bacterial
infections.1 Recently, some Pseudomonas strains have developed
resistance to carbapenems through either porin loss or expression
of b-lactamase enzymes (BLs) that can hydrolyze and inactivate
carbapenems.2 The hundreds of known BLs are divided into four
classes (A–D) based on their structures.3 While Class A BLs are a
major cause of penicillin resistance in bacteria, carbapenem resis-
tance in Pseudomonas is primarily mediated by class C BLs such as
AmpC.4 Over the years, several b-lactamase inhibitors (BLIs) have
been developed that effectively restore antibacterial activity
against class A BL-expressing strains when combined with a peni-
cillin (e.g., clavulanate with amoxicillin).5 Likewise, addition of an
effective class C BLI to a carbapenem antibiotic should restore anti-
bacterial efficacy against class C BL-producing strains. To date, no
class C BLI has been brought to market although several have been
reported.6
albeit in low yield (Scheme 1). The hydroxyl-alkyl analogs 4 and 5
were readily synthesized from the chiral side chain amine 166a as
outlined in Scheme 2. BOC protection of the free amine followed by
CBZ removal afforded the protected diamine 17 in 82% overall
yield. Alkylation with unprotected bromoethanol was messy but
reaction with the TIPS-protected alcohol proceeded smoothly with
moderate yields. Simultaneous TFA removal of the TIPS and BOC
protecting groups afforded the requisite hydroxyalkyl amine inter-
mediates 18 and 19. Activation of the amine as the hydroxy-
succinimide mixed carbonate followed by coupling with the core
amine 86b,7 provided 4 and 5.
The amino-alkyl analogs 6 and 7 were similarly prepared as
outlined in Scheme 3. Alkylation of 17 with dimethylamino ethyl
tosylate or N-Cbz-iodoethyl amine followed by deprotection affor-
H
N+
R'
N+
We recently reported the discovery of MK-8712 (1),6a a class C
BLI for combination with imipenem/cilastatin (IPM/CIL). MK-8712
is an analog of Ro48-1256.6b,6c
H
R
O
O
S
S
N
N
N
N
i or ii, iii
H
H
H
H
H
H
In the search for a backup for 1, we synthesized and report here-
in numerous N-substituted and open chain analogs. The N-methyl
(2) and N,N-dimethyl (3) analogs were prepared from MK-8712 by
reductive amination (for 2) or alkylation with methyl iodide (for 3),
N
N
-
-
O
SO3
O
SO3
2 R = Me, R' = H
3 R = R' = Me
MK-8712 (1)
Scheme 1. Reagents and conditions: (i) CH2O, NaCNBH3, AcOH, ACCN, 23%; (ii) MeI,
Et3N, DMAP, DMF, 29%; (iii) HPLC on Phenominex Synergi Polar RP80 column eluted
with MeOH/H2O.
⇑
Corresponding author. Tel.: +1 732 594 5323.
E-mail address: helen_chen@merck.com (H. Chen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.065

4268
H. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4267–4270
Open chain analogs 11–15 were prepared by coupling of the BLI
core with the appropriate hydroxysuccinimide carbamate
(Table 2).
Cbz
HO
H
N
N
i, ii
iii, iv
v, vi
NH2
NHBOC
In general, N-substitution on 1 was discovered to be detrimen-
tal to in vitro synergy with IPM (Table 1). As seen in the simple N-
methyl analog 2, both enzymatic inhibition against pseudomonal
class C b-lactamase AmpC, and synergy were decreased about 3-
fold. Although the N,N-dimethyl analog 3 restored enzymatic activ-
ity, a continued decrease in synergy with IPM was observed. By
tethering a longer hydrophilic side chain to the azepine ring, com-
pounds (4, 5, and 6) with comparable or improved enzymatic inhi-
bition over 1 could be obtained; however, all of these analogs
suffered substantial loss in synergy. This loss in synergy was even
more pronounced in the amino-alkyl analog 7.
Investigation into replacing the azepine side chain was also pur-
sued. Truncating the side chain to the unsubstituted urea 9 par-
tially restored enzymatic inhibition relative to the inactive
bridged monobactam core 8. By incorporating an isopropyl group,
a compound (10) with comparable enzymatic inhibition to 1 was
obtained, albeit with a complete loss of synergy. Experiments with
10 in efflux-deleted mutants strongly suggest that the loss of
in vitro synergy with IPM is due primarily to increased efflux.9
Open chain analogs 11–13 demonstrated that extending a tertiary
amine, up to 5-carbons, was also well tolerated by the enzyme, but
remained less effective in the synergy assay with IPM. Surprisingly,
the corresponding secondary amines (14 and 15) offered no
improvement in synergy as hoped, and proved to be inferior in
the enzymatic inhibition against class C BLs.
16
( )n
17
HN
-
N
TFA
H
N
H
+
+
NH3
O
SO3H
18
n = 1
8
19 n = 2
R
N
O
S
N
N
4 R = CH2CH2OH
5 R = CH2CH2CH2OH
H
H
H
N
O
SO3H
Scheme 2. Reagents and conditions: (i) Boc2O, Hunig’s base, DMAP, THF, 82%; (ii)
20% Pd(OH)2/C, 40 psi H2, MeOH; (iii) Br(CH2)nCH2OTIPS, Hunig’s base, DMF, rt,
48 h, 53% (n = 1), 48% (n = 2); (iv) TFA, CH2Cl2; (v) 18 or 19, N,N0-disuccinimidyl
carbonate, MeCN; Et3N; (vi) 8, NaHCO3, MeCN, H2O, 31%; (vii) HPLC on Phenominex
Synergi Polar RP80 column eluted with MeOH/H2O.
R'
N
H
R
-
N
N
TFA
i, ii
iii, iv
+
NHBOC
NH3
17
20
The structures of 6 and 15 bound to AmpC were solved to 1.4
and 1.6 Ang resolution, respectively.10 Figure 1 shows a compari-
son of the binding of MK-8712 (1) (panel A), compound 6 (panel
R = H, R' =Cbz
21 R = R' = Me
H2N
R'
N
R
N
O
N
O
v
Table 2
Synthesis of Analogs 11–15
N
N
N
N
H
H
H
H
H
H
O
N
N
O
O
SO3H
O
SO3H
N
R
O
N
H
i
ii, or
ii, iii
6
22
H
R = H, R' = Cbz
R'NH2
24a-e
R'
N
H
N
H
O
7 R = R' = Me
N
O
O
SO3H
25a-e
11-13
Scheme 3. Reagents and conditions: (i) ICH2CH2NHCO2Bn, Hunig’s base, DMF, rt,
24 h, 70–96%, or TsOCH2CH2NMe2, Hunig’s base, NaI, DMF, rt, 24 h, 73%; (ii) TFA,
CH2Cl2; (iii) N,N0-disuccinimidyl carbonate, MeCN, Et3N, 10% (R = R0 = Me), 49–54%
(R = H, R0 = Cbz); (iv) 8, NaHCO3, MeCN, H2O, 43–49%; (v) 22, 20% Pd black, H2,
MeOH, AcOH, 68%.
Conditions: (i) N,N0-Disuccinimidyl carbonate, MeCN; (ii) 8, NaHCO3, MeCN,
H2O; (iii) TsOH, AcOH, H3PO4
R0NH2
R
Yields (i, ii,
iii)a
NH2
24a
N
N
H
N
N
11
ded intermediates 20 or 21 in good to excellent yield. Coupling to
the core 8 provided 7 directly and the advanced intermediate 22,
which was further deprotected to afford amino-ethyl analog 6.
Reaction of the bridged monobactam core 8 and isopropyl iso-
cyanate or p-methoxybenzyl isocyanate afforded the isopropyl
amide 10 directly and the protected amide 23 (Scheme 4). Depro-
tection of PMB amide 23 afforded unsubstituted amide 9 in good
yield.
(35, 11, n/a)
(95, 18, n/a)
24a
11
N
NH2
24b
N
12
H
24b
12
NH2
24c
N
N
N
N
(NIb, 41, n/a)
(NIb, 23, 20)
13
H
24c
13
NH2
24d
O
O
N
N
H
H
14
BOC
24d
BOC
HN
H
N
N
R
N
H2N
i
ii
14
H
H
H
H
H
H
H
N
N
N
N
O
SO3H
O
SO3H
O
SO3H
N
H
N
(NIb, 35, 29)
24e
NH2
15
8
10 R = iPr
23 R = PMB
9
24e
15
a
Yields (%) for steps (i), (ii), and (iii) (if applicable).
NI = not isolated, crude used in next step without further purification.
Scheme 4. Reagents and conditions: (i) RNCO, H2O, THF, 19% (R = iPr) or 54%
(R = PMB); (ii) 23, CAN, H2O, MeCN, 66%.
b

H. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4267–4270
4269
Table 1
Enzyme inhibition and in vitro synergy
R
N
H
H
N
-
O
SO3
a
b
#
R
P.a. AmpC IC50
1.0
(l
M)
conc. (
lM) to restore IPM susc. CL5701
+H2N
O
1
9.4
N
H
HN+
O
2
3
4
5
6
7
2.6
1.5
0.9
1.1
0.6
0.9
25
50
25
50
25
100
N
H
N+
O
N
H
HN+
O
HO
N
H
HN+
O
HO
N
H
HN+
O
H2N
N
H
HN+
O
N
N
H
8
9
H
>250
16
>100
>100
O
H2N
O
10
11
12
1.8
2.1
1.6
>100
25
N
H
O
N
H
N
N
O
25
N
H
O
13
N
1.9
100
H
N
O
14
15
5.3
3.5
50
25
N
H
N
H
O
H
N
N
H
a
IC50 for inhibition of the hydrolysis of nitrocefin; see Ref. 8 for details.
Concentration of BLI (lM) required to reduce the imipenem (IPM) MIC for Pseudomonas aeruginosa strain CL5701 from 32 lg/mL to the susceptibility breakpoint of 4 lg/
b
mL; see Ref. 8 for details.

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H. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4267–4270
Figure 1. X-ray crystal structures of (a) MK-8712 (1), (b) compound 6, and (c) compound 15 bound to AmpC.
Fitzgerald, P.; Sharma, N.; Lu, J.; Ha, S.; Hickey, E.; Hermes, J.; Hammond, M. L.
Bioorg. Med. Chem Lett. 2009, 20, 918; (b) Heinze-Krauss, I.; Angehrn, P.;
Charnas, R. L.; Gubernator, K.; Gutknecht, E.-M.; Hubschwerlen, C.; Kania, M.;
Oefner, C.; Page, M. G. P.; Sogabe, S.; Specklin, J.-L.; Winkler, F. J. Med. Chem.
1998, 41, 3961; (c) Hubschwerlen, C.; Angehrn, P.; Gubernator, K.; Page, M. G.
P.; Specklin, J.-L. J. Med. Chem. 1998, 41, 3972; (d) Bonnefoy, A.; Dupuis-
Hamelin, C.; Steier, V.; Delachaume, C.; Seys, C.; Stachyra, T.; Fairley, M.;
Guitton, M.; Lampilas, M. J. Antimicrob. Chemother. 2004, 54, 410; (e) Morandi,
F.; Caselli, E.; Morandi, S.; Focia, P. J.; Blasquez, J.; Shoichet, B. K. J. Am. Chem.
Soc. 2003, 125, 685; (f) Morandi, S.; Morandi, F.; Caselli, E.; Shoichet, B. K.; Prati,
F. Bioorg. Med. Chem. 2008, 16, 1195; (g) Weiss, W. J.; Petersen, P. J.; Murphy, T.
M.; Tardio, L.; Yang, Y.; Bradford, P. A.; Venkatesan, A. M.; Abe, T.; Isoda, T.;
Mihira, A.; Ushirogochi, H.; Takasake, T.; Projan, S.; O’Connell, J.; Mansour, T. S.
Antimicrob. Agents Chemother. 2004, 54, 410; (h) Paukner, S.; Hesse, L.; Prezelj,
A.; Solmajer, T.; Urleb, U. Antimicrob. Agents Chemother. 2009, 53, 505; (i) Silver,
L. L. Expert Opin. Ther. Patents 2007, 17, 1175.
B) and compound 15 (panel C). The three compounds are cova-
lently bound to Ser 90, and extend along a narrow groove on the
surface of the protein. All three compounds are anchored at the
bottom of the groove by several hydrogen bonds to protein atoms.
The s-azepine of MK-8712 and 6 are stacked against the side chain
of Tyr249. In addition, the amino alkyl chain of 6 hydrogen bonds
to the main chain oxygen of Gly 240, the side chain oxygen of Tyr
249 and a water molecule. These interactions are not present in
MK-8712, and may account for the slightly higher enzymatic activ-
ity observed for 6. Conversely, the slight loss in potency observed
for 15 can be explained by the fact that the open alkyl chain does
not optimally interact with Tyr249; the reduced hydrophobic
interactions may be partially compensated by the hydrogen bond
made by the amino group.
7. An improved synthesis of 8 was developed by Merck Process Research and will
be published separately.
8. (a) Blizzard, T.A.; Chen, H.Y.; Wu, J.Y.; Kim, S.; Ha, S.; Mortko, C.; Variankaval,
N.; Chiu, A. WO 2008039420. Detailed experimental procedures for the enzyme
inhibition assay and the in vitro synergy assay are provided in Example 32 of
this patent. For convenience, brief summaries of the protocols for these assays
are provided below. (b) Enzyme inhibition assay: Hydrolysis of the
commercially available substrate nitrocefin by AmpC in the presence of the
BLI was measured in a spectrophotometric assay. The enzyme AmpC (from
Pseudomonas aeruginosa) and the substrate were dissolved in 100 mM KH2PO4
buffer (pH 7) containing 0.005% BSA. The BLI was dissolved in DMSO and
serially diluted in a 96-well microplate. The BLI and AmpC were incubated for
40 min at room temperature then the substrate solution was added and the
incubation continued for another 40 min. The spectrophotometric reaction was
quenched by the addition of 2.5 N acetic acid and the absorbance at 492 nm
was measured. The IC50 was determined from semi-logarithmic plots of
enzyme inhibition versus inhibitor concentration. (c) In vitro synergy Assay: The
assay determines the concentration of BLI required to reduce the MIC of
imipenem by one-half, one-quarter, one-eighth, one-sixteenth and one-thirty-
second against resistant bacteria. The BLI was titrated in a serial dilution across
a microtiter plate while at the same time imipenem was titrated in a serial
dilution down the microtiter plate. The plate was inoculated with the bacterial
strain in question then incubated overnight and evaluated for bacterial growth.
Each well in the microplate checkerboard contains a different combination of
concentrations of the inhibitor and the antibiotic thus allowing determination
of synergy between the two.
In conclusion, our efforts to identify a back-up to 1, demon-
strated that the azepine side chain is optimal both for enzymatic
inhibition against pseudomonal class C b-lactamase AmpC, and
synergy with IPM. Although several N-alkylated azepine and open
chain analogs have been discovered with slightly improved or sim-
ilar enzymatic inhibition against class C BLs, none showed im-
proved in vitro synergy with IPM. Further reports from this
laboratory will describe our efforts to clarify the factors contribut-
ing to this loss in synergy and to identify an optimal b-lactamase
inhibitor for combination with imipenem.
References and notes
1. (a) Baughman, R. P. J. Intensive Care Med. 2009, 24, 230; (b) Bradley, J. S.; Garau,
J.; Lode, H.; Rolston, K. V. I.; Wilson, S. E.; Quinn, J. P. Int. J. Antimicrob. Agents
1999, 11, 93.
2. Livermore, D. M. Antimicrob. Agents. Chemother. 1992, 36, 2046.
3. Jacoby, G. A.; Munoz-Price, L. S. N. Eng. J. Med. 2005, 352, 380.
4. Livermore, D. M. Clin. Microbiol. Rev. 1995, 8, 557.
5. (a) Coleman, K. Drug Disc. Today: Ther. Strat. 2006, 3, 183; (b) Buynak, J. D.
Biochem. Pharmacol. 2006, 71, 930; (c) Perez-Llarena, F. J.; Bou, G. Curr. Med.
Chem. 2009, 16, 3740; (d) Shahid, M.; Sobia, F.; Singh, A.; Malik, A.; Khan, H. M.;
Jonas, D.; Hawkey, P. M. Crit. Rev. Microbiol. 2009, 35, 81.
6. (a) Blizzard, T. A.; Chen, H.; Kim, S.; Wu, J.; Young, K.; Park, Y. W.; Ogawa, A.;
Raghoobar, S.; Painter, R. E.; Hairston, N.; Lee, S. H.; Misura, A.; Felcetto, T.;
9. Young, K. et al. manuscript in preparation.
10. Coordinates for the two complexes described in the paper have been deposited
with the Protein Data Bank (PDB), accession codes 3S1Y for compound 6, and
3S22 for compound 15.