Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 1. Discovery of a class C BLI for combination with imipinem

Article abstract of DOI:10.1016/j.bmcl.2009.12.069

Bridged monobactam β-lactamase inhibitors were prepared and evaluated as potential partners for combination with imipenem to overcome class C β-lactamase mediated resistance. The (S)-azepine analog 2 was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.

Full text of DOI:10.1016/j.bmcl.2009.12.069

Bioorganic & Medicinal Chemistry Letters 20 (2010) 918–921
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Side chain SAR of bicyclic b-lactamase inhibitors (BLIs). 1. Discovery
of a class C BLI for combination with imipinem
a
a
a
b
b
Timothy A. Blizzard a, , Helen Chen , Seongkon Kim , Jane Wu , Katherine Young , Young-Whan Park ,
Amy Ogawa b, Susan Raghoobar b, Ronald E. Painter b, Nichelle Hairston b, Sang Ho Lee b, Andrew Misura b,
Tom Felcetto b, Paula Fitzgerald a, Nandini Sharma a, Jun Lu a, Sookhee Ha a, Emily Hickey b, Jeff Hermes b,
Milton L. Hammond a,b
*
a Department 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:
Bridged monobactam b-lactamase inhibitors were prepared and evaluated as potential partners for com-
bination with imipenem to overcome class C b-lactamase mediated resistance. The (S)-azepine analog 2
was found to be effective in both in vitro and in vivo assays and was selected for preclinical development.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 October 2009
Revised 15 December 2009
Accepted 17 December 2009
Available online 23 December 2009
Keywords:
b-Lactamase
Inhibitor
Carbapenem
Imipenem
O
Carbapenem antibiotics (e.g., imipenem) have played an impor-
tant role in the treatment of nosocomial bacterial infections for
over twenty years.1 However, some Pseudomonas strains have
developed resistance to carbapenems via porin loss and 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 Class A BLs are a major
cause of penicillin resistance in Gram-positive bacteria, while carb-
apenem resistance in Pseudomonas is mediated by class C BLs such
as AmpC.4 Several b-lactamase inhibitors (BLIs) have been devel-
oped that effectively restore antibacterial activity against class A
BL-expressing strains when combined with a penicillin (e.g., cla-
vulanate with amoxicillin).5 Similarly, addition of an effective class
C BLI to a carbapenem antibiotic should restore antibacterial effi-
cacy against class C BL-producing strains. To date, no class C BLI
has been brought to market although several have been reported.6
Several years ago, we initiated a program to discover a class C
BLI for combination with imipenem/cilastatin (IPM/CIL). Our initial
focus was a collaboration with Methylgene based on their phos-
phonate BLIs.7 We subsequently expanded our effort to include
analogs of the bridged monobactam BLIs (e.g., Ro48-1256, 1) previ-
ously reported by Roche.6b, c
HN
N
N
H
H
H
N
O
SO3H
Ro48-1256 (1)
Molecular modeling studies using a crystal structure of 1 bound in
the active site of AmpC as a starting point indicated that there was
room for a larger side chain than the piperidine present in 1. To test
this hypothesis, we prepared the seven-membered (azepine) side
chain analogs 2 and 3 (Scheme 1). The requisite side chain was pre-
pared from the commercial N-benzylazepinone 15.8 Reductive ami-
nation of 15 afforded the racemic amine 16 in 52% yield. Reaction of
16 with N,N0-disuccinimidyl carbonate afforded the activated carba-
mate 17. Coupling of 17 and the chiral bridged monobactam core
186b,9 followed by debenzylation and HPLC separation of the diaste-
reomers afforded 2 and 3. The stereochemistry of the two diastereo-
mers was determined by X-ray crystal structures of the complexes
of 2 and 3 bound (separately) in AmpC (Fig. 1).10
We were pleased to find that the (S)-isomer 2 was more active
than 1 in our enzyme inhibition assay11a,b and in vitro synergy as-
say (Table 1).11a,c In addition to allowing assignment of side-chain
stereochemistry, the X-ray crystal structures also provided a possi-
ble explanation for the differential activity of 2 and 3. As expected,
* Corresponding author. Tel.: +1 732 594 6212; fax: +1 732 594 9556.
E-mail address: tim_blizzard@merck.com (T.A. Blizzard).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.069

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 20 (2010) 918–921
919
Once the stereochemistry of the active diastereomer was estab-
lished, a chiral synthesis of the side chain amine (S)-23 was devel-
oped (Scheme 2). Ring expansion of CBZ-piperidone 19, followed
by decarboxylation of 20, afforded the protected azepinone 21.
Reductive amination of 21 using (R)-t-butyl sulfinamide according
to the general method of Ellman et al.12 afforded a mixture of dia-
stereomeric sulfinamides 22 that were separated by chiral HPLC.
Hydrolysis of the slower eluting diastereomer afforded the desired
(S)-23.
Bn
Bn
N
N
i
ii
O
NH2
15
16
HN
O
Bn
O
N
N
H
H
iii-v
+
O
N
O
N
O
SO3H
H
17
18
H
H
Additional analogs (4–9) were prepared to explore the effect of
altering the heteroatom and its location (Scheme 3). DIBAL reduc-
tion of the seven-membered lactams 24a–c13 afforded the amines
25a–c. Acylation of the amines followed by condensation of the
activated intermediates with the bicyclic core amine 18 afforded
the penultimate intermediates, which were deprotected to afford
final products 4–6. Although the overall yields of this sequence
were generally low, sufficient material was obtained for evaluation
of all three analogs in our in vitro screens. The diastereomeric ana-
log 7 was obtained by the same route starting with the enantiomer
of 24c. Lactam 8 was prepared in 54% overall yield by direct con-
densation of amide 24a with N,N0-disuccinimidyl carbonate fol-
lowed by reaction with 18. Carbacycle 9 was prepared from
aminocycloheptane.
The eight-membered and nine-membered side chains 29, 30a,b,
and 31a,b were prepared by ring expansion of azepinone 21
(Scheme 4).
Side chains 29, 30a,b, and 31a,b were then converted to the
eight- and nine-membered analogs 10–14 via the standard activa-
tion, condensation, and deprotection sequence (Table 2). Note that
compounds 11–14 are single diastereomers of undetermined
stereochemistry.
As previously noted, the (S)-azepine analog 2 was a more effi-
cient inhibitor of the pseudomonal class C b-lactamase AmpC than
the piperidine 1. This improvement in enzyme inhibition was re-
flected in the increased in vitro synergy with IPM observed for 2.
The (R)-azepine analog 3 was less active against the enzyme and
exhibited reduced in vitro synergy. Interestingly, the closely re-
lated (S)-azepine analog 4, in which the ring nitrogen has been
shifted one atom closer to the exocyclic nitrogen, exhibited en-
zyme inhibition comparable to 2 but much reduced synergy with
IPM, perhaps due to reduced cell penetration or increased efflux.
Addition of a second nitrogen atom (e.g., 5) resulted in a substan-
tial reduction in both enzyme inhibition and synergy. Addition of
an oxygen atom at the same position (e.g., 6 and 7), led to a slight
N
O
N
O
S
R
N
N
N
N
+
H
H
H
H
H
H
N
N
O
SO3H
O
SO3H
2
3
Scheme 1. Reagents and conditions: (i) Ti(OiPr4), NH3, EtOH, then NaBH4, 52%; (ii)
N,N0-disuccinimidyl carbonate, MeCN; (iii) NaHCO3, MeCN, H2O, 51%; (iv) Pd(OH)2,
MeOH, AcOH, H2O, 40 psi H2, 20%; (v) HPLC on phenominex synergi polar RP80
column eluted with MeOH/H2O.
the bicyclic cores of 2 and 3 occupy essentially the same position
relative to the enzyme since the lactam carbonyl in this part of
the molecule forms a covalent bond to the active site serine. Sur-
prisingly, however, the side chain nitrogen atoms of 2 and 3 also
occupy the same position relative to the enzyme due to an interac-
tion (via a water molecule) with the oxygen of Y249 below. Be-
cause of the fixed locations of the core and the azepine nitrogen,
the (S)-azepine 2 has three carbon atoms to the left of the binding
pocket and two carbon atoms to the right, while the reverse is true
of the (R) isomer 3 (i.e., the azepine ring is flipped). This results in
an increased interaction of the azepine carbons of 3 with Q146 and
less freedom of motion for the side chain of this amino acid. In fact,
the side chain of Q146 is actually observed in two conformations
(ꢀ60:40 ratio) in the crystal structure of AmpC with bound 2 while
in the crystal structure with 3 bound this side chain is primarily in
one conformation. It is possible that the tighter interaction of the
hydrophobic azepine carbon atoms of 3 with the polar oxygen of
Q146 may account for the increased enzyme IC50 observed for this
compound. However, it should be noted that the azepine shift is
small and it is possible that the observed difference in activity
may be due to some other cause. Interestingly, before the X-ray
data was available, molecular modeling had qualitatively predicted
that 2 would be more active.
Figure 1. X-ray crystal structures of 2 (a) and 3 (b) in the active site of Amp C.

920
T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 20 (2010) 918–921
Table 1
CBZ
CBZ
ii
N
N
Enzyme inhibition and in vitro synergy
i
iii, iv
CBZ
CBZ
N
O
O
O
O
CO2Et
N
R
N
19
20
21
CBZ
H
H
H
N
CBZ
O
O
O
SO3H
v
N
N
N
S
S
tBu
tBu
+
N
N
NH2
(S)-23
#
R
P.a. AmpC
IC50
Conc. (lM) to restore
H
H
(lM)
IPM susc. (CL5701)b
a
(R)-22
(S)-22
H
N
N
Scheme 2. Reagents and conditions: (i) ethyl diazoacetate, BF3ꢁOEt2, Et2O, 100%; (ii)
K2CO3, H2O, reflux, 72%; (iii) (R)-tBuSONH2, Ti(OEt)4, NaBH4, THF; (iv) chiral HPLC
on OJ column eluted with 15% EtOH/heptane, 27% yield of (S)-22 (slower eluting)
and 23% yield of (R)-22 (faster eluting); (v) 4 M HCl/dioxane, MeOH.
1
6.8
21
H
H
2
3
1.0
4.0
9.4
25
N
H
N
O
R
R
N
N
H
O
N
ii - iv
NH2
i
N
X
N
H
4
5
1.9
17
50
H
H
NH2
X
X
N
O
SO3H
24
25
H
a X = CH2; R = Bn a X = CH2; R = Bn
b X = NR; R = Bn b X = NR; R = Bn
c X = O; R = PMB c X = O; R = PMB
4 X = CH2
5 X = NH
6 X = O
N
100
N
Scheme 3. Reagents and conditions: (i) DIBAL 100% (24a), 74% (24b), 40% (24c); (ii)
N,N0-disuccinimidyl carbonate, MeCN; (iii) 18, NaHCO3, MeCN, H2O; (iv) Pd(OH)2,
MeOH, AcOH, H2O, 40 psi H2, 14% (4), 23% (5), 23% (6).
H
H
N
6
0.6
25
O
CBZ
O
CBZ
CBZ
CBZ
CBZ
O
N
N
N
N
N
i-ii
O
+
7
8
1.1
0.8
50
O
O
N
21
H
i-ii
26
29
27
H
N
iii-v
iii-v
O
CBZ
N
>100
28
NH2
iii-v
9
0.4
3.4
>100
25
NH2
30a,b
H
H
CBZ
N
N
10
NH2
31a,b
Scheme 4. Reagents and conditions: (i) diazomethane, BF3ꢁOEt2; (ii)K2CO3, THF/
H20, 100 °C; (iii) (R)-tBuSONH2, Ti(OEt)4, NaBH4, THF; (iv) chiral HPLC; (v) 4 M HCl/
dioxane, MeOH.
N
(A)
11
12
1.3
25
12.5
H
N
iments with these and other analogs in efflux-deleted mutants
strongly suggest that a basic nitrogen in the side chain is required
to prevent efflux of the BLI.14 The azocine analogs 10 and 11
showed improved enzyme IC50 relative to 1. Isomer 11 also had
better synergy with IPM. Interestingly, 12, the other side chain dia-
stereomer of 11, showed reduced enzyme inhibition and no syn-
ergy, illustrating the importance of side chain stereochemistry.
The two nine-membered diastereomers, 13 and 14, while reason-
ably active against the enzyme, had no in vitro synergy with IPM.
Azepine 2 was evaluated in several in vivo infection models15
and found to be superior to 1. For example, in a mouse spleen
infection model with IPM-resistant Pseudomonas aeruginosa, (CL
(B)
>100
(A)
(B)
H
H
N
N
13
14
4
5
>100
>100
a
IC50 for inhibition of the hydrolysis of nitrocefin; see Ref. 11 for details.
Concentration of BLI (lM) required to reduce the imipenem (IPM) MIC for P.
aeruginosa strain CL5701 from 32 lg/mL to the susceptibility breakpoint of 4 lg/
mL; see Ref. 11 for details.
b
5701; MIC 16–32 lg/mL) treatment with 2.5 mpk of IPM/CIL and
increase in both enzyme activity and synergy. Surprisingly, 8, the
lactam analog of 4, exhibited improved enzyme inhibition but no
synergy at all.
Similarly, analog 9, which also lacks a basic nitrogen in the side
chain, showed improved enzyme inhibition but no synergy. Exper-
2.35 mpk of 2 (1.25 h infusion QID for 1 day) resulted in a 4.6 log
reduction (relative to IPM/CIL alone) of bacterial CFU (colony form-
ing units) in the spleen. Treatment with 2.5 mpk of IPM/CIL and
4.7 mpk of 1 (1.25 h infusion QID for 1 day) resulted in only a 1.0
log reduction of bacterial CFU.

T. A. Blizzard et al. / Bioorg. Med. Chem. Lett. 20 (2010) 918–921
2. Livermore, D. M. Antimicrob. Agents. Chemother. 1992, 36, 2046.
921
Table 2
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 Discovery 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.
Synthesis of analogs 10–14
Amine
29; 30a,b; 31a,b
i - iii
Product
10 - 14
Amine
Product
Yielda
6. (a) Silver, L. L. Expert Opin. Ther. Patents 2007, 17, 1175; (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.
7. (a) Besterman, J.M.; Delorme, D.l; Rahil, J. WO 2001002411; (b) DiNinno, F.;
Hammond, M.L.; Dykstra, K.; Kim, S.; Tan, Q.; Young, K.; Hermes, J.D.; Raeppel,
S.; Mannion, M.; Zhou, N.Z.; Gaudette, F.; Vaisburg, A.; Rahil, J.;
Georgopapadakou, N. WO 2007139729; (c) Martell, L. A.; Rahil, G.; Vaisburg,
A.; Young, K.; Hickey, E.; Hermes, J.; DiNinno, F.; Besterman, J. M. Abstract of
Papers, 49th ICAAC, San Francisco, CA; American Society for Microbiology:
Washington, DC, 2009; Abstract C1-1373.
CBZ
O
O
O
N
HN
N
NH2
NH2
N
H
8
H
H
29
N
O
SO3H
10
CBZ
CBZ
N
N
HN
N
N
H
24
11
10
5
H
H
H
30a
N
(isomer A)
O
SO3H
11
NH2
HN
HN
N
N
H
H
30b
31a
N
(isomer B)
O
SO3H
12
8. Azepinone 15 was purchased from Tyger Scientific (www.tygersci.com).
9. An improved synthesis of 18 was developed by Merck Process Research and
will be published separately.
10. X-ray coordinates for 2 (PDB code 2wzx) and 3 (2wzz) have been deposited in
the RCSB Protein Data Bank (PDB) database.
O
CBZ
CBZ
N
N
N
NH2
N
H
H
N
H
11. (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 P.
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.
(isomer A)
O
SO3H
13
O
HN
N
NH2
N
H
31b
H
H
N
(isomer B)
O
SO3H
14
Conditions: (i) N,N0-disuccinimidyl carbonate, MeCN; (ii) 18, NaHCO3, MeCN, H2O;
(iii) Pd(OH)2, MeOH, AcOH, H2O, 40 psi H2.
a
Overall yield (%) for three-step conversion of amine to final product.
Based on its superior in vitro and in vivo activity and acceptable
pharmacokinetics (matching imipenem, data not shown), 2 was se-
lected for further development in combination with IPM/CIL for
treatment of IPM-resistant Pseudomonas infections. Unfortunately,
development of 2 has since been terminated due to an inadequate
therapeutic margin in subsequent safety studies. Future reports
from this laboratory will describe our continuing efforts to identify
an optimal BLI for combination with imipenem.
12. Tanuwidjaja, J.; Peltier, H. M.; Ellman, J. A. J. Org. Chem. 2007, 72, 626.
13. Lactam 24a is commercially available (Matrix). Lactams 24b and 24c were
prepared by condensation of N,N0-bis-benzyl ethylene diamine and N-benzyl-
N-BOC-ethanolamine, respectively, with the methyl ester of N-CBZ-2-aziridine
carboxylic acid followed by cyclization of the intermediate amino ester to
afford the desired lactam.
References and notes
1. (a) Baughman, R. P. J. Intens. 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.
14. Young, K. et al., in preparation.
15. Lee, S. et al., in preparation.