Propenylamide and propenylsulfonamide cephalosporins as a novel class of anti-MRSA β-lactams

Article abstract of DOI:10.1016/j.bmcl.2010.05.110

Novel C(3) propenylamide and propenylsulfonamide cephalosporins have been synthesized and tested for their ability to inhibit the penicillin-binding protein 2′ (PBP2′) from Staphylococcus epidermidis and the growth of a panel of clinically relevant bacter

Full text of DOI:10.1016/j.bmcl.2010.05.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4635–4638
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Propenylamide and propenylsulfonamide cephalosporins as a novel class of
anti-MRSA b-lactams
b
b
a
a
Jens Pohlmann a, , Natalya I. Vasilevich , Andrei I. Glushkov , Laurenz Kellenberger , Stuart Shapiro ,
*
Patrick Caspers a, Malcolm G. P. Page a, Franck Danel a
a Basilea Pharmaceutica International Ltd, Grenzacherstrasse 487, CH-4005 Basel, Switzerland
b Asinex Ltd, ul. Geroev Panfilovtzev 20, Bldg 1, RU-125480 Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel C(3) propenylamide and propenylsulfonamide cephalosporins have been synthesized and tested
for their ability to inhibit the penicillin-binding protein 20 (PBP20) from Staphylococcus epidermidis and
the growth of a panel of clinically relevant bacterial species, including methicillin-resistant Staphylococ-
cus aureus (MRSA). The most potent compounds inhibited the growth of MRSA strains with minimum
Received 3 May 2010
Revised 27 May 2010
Accepted 29 May 2010
Available online 8 June 2010
inhibitory concentrations (MIC) as low as 1
lg/mL. The structure–activity relationship revealed the
potential for further optimization of this new cephalosporin class.
Keywords:
Cephalosporins
MRSA
Ó 2010 Elsevier Ltd. All rights reserved.
Cephalosporins have a history of success in the treatment of
bacterial infections. Their favorable safety profile and generally
bactericidal mode of action have made them one of the most fre-
quently used classes of antibacterial drugs.1 However, the emer-
gence of resistance has created the need for new antibacterial
compounds.2,3 Therefore, an extensive search for cephalosporins
with efficacy also against resistant bacteria, especially methicil-
lin-resistant Staphylococcus aureus (MRSA), has been made in re-
tent anti-MRSA activity that is not attributable to a leaving group
in these novel side-chains.
Cephalosporin building block 6 served as key intermediate for
all propenylamide and propenylsulfonamide cephalosporins in this
study. Orthogonally protected 6 was prepared from 3-hydroxy
cephalosporin 4 by formation of the corresponding triflate 5 and
subsequent palladium acetate catalyzed Stille coupling with the
E-vinylstannane 3 to introduce the N-Boc-protected aminoprope-
nyl side-chain (Scheme 1).15 Vinylstannane 3 was obtained stere-
oselectively by hydrostannylation of the corresponding
propargylamine 2.16
cent years.4 These efforts yielded
a number of anti-MRSA
cephalosporins, with the most advanced representative, ceftobi-
prole, recently being approved in first countries.5,6
A common feature of most anti-MRSA cephalosporins is the
presence of a basic group, or even a permanent positive charge in
the C(3) side-chain.7,8 This positive charge increases the apparent
binding affinity of the drug towards PBP20, the enzyme that is
the main cause for the resistance of staphylococci towards older
generations of penicillinase-stable b-lactams.9,10 A special sub-
group among these charged cephalosporins comprises derivatives
with a quaternary ammonium moiety that is linked to the cepha-
losporin core via a propenyl group.11–14 The favorable activity of
these compounds against MRSA can be attributed to the leaving
group properties of the ammonium moiety upon reaction with
penicillin-binding proteins (Fig. 1). However, the zwitterionic nat-
ure of these drugs can lead to an unfavorable physico-chemical
profile that poses challenges for further development.
Treatment of 6 with trifluoroacetic acid allowed simultaneous
deprotection of the amino and carboxylic acid moieties, thus pro-
viding cephalosporin 7. Different approaches were evaluated for
the acylation of the terminal amino group of 7. Reaction of 7 with
aromatic acylchlorides proceeded rapidly, but with poor chemose-
lectivity for the amino versus the carboxylic acid group. Coupling
by activating the acid as a mixed-anhydride with methyl- or i-bu-
tyl-chloroformate worked well for aliphatic acids, but led to mix-
tures of the desired product and the corresponding carbamate
when aromatic acids were applied. However, employing CDI or
TBTU as coupling reagent gave good yields for both aliphatic and
aromatic acids.
The preparation of sulfonamides was achieved by treatment of
7 with sulfonyl chlorides. The choice of base greatly affected the
outcome of these reactions: DIPEA, for example, led to complex
product mixtures, whereas the use of BSA allowed the reproducible
preparation of the desired products in good yield.
Here, we report for the first time non-basic, uncharged C(3)
propenylamide and propenylsulfonamide cephalosporins with po-
To obtain cephalosporins with variations in the 7-aminoacyl
* Corresponding author. Tel.: +41 61 606 1385; fax: +41 61 606 1112.
E-mail address: jens.pohlmann@basilea.com (J. Pohlmann).
moiety, the phenylacetyl group of
6 was removed by the
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.110

4636
J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4635–4638
Figure 1. Reaction of propenylammonium cephalosporins with penicillin-binding proteins.
Scheme 1. Reagents and conditions: (a) Boc2O, DIPEA, CH2Cl2, rt; (b) n-Bu3SnH, cat. AIBN, benzene, 80 °C; (c) Tf2O, DIPEA, CH2Cl2, ꢀ78 °C; (d) 3, cat. Pd(OAc)2, NMP, rt; (e)
TFA, CH2Cl2, rt; (f) RCO2H, CDI, NMP, rt; (g) RSO2Cl, BSA, CH2Cl2, reflux.
standard imino-chloride method.17 Careful evaluation of the
subsequent acylation conditions led to the selection of TBTU
or PyBOP as preferred coupling reagents that precluded isomer-
described above for the 7-phenylacetamide cephalosporins
(Schemes 2–4).
All new propenylamide and propenylsulfonamide cephalospo-
rins were tested for their ability to inhibit purified recombinant
PBP20 from Staphylococcus epidermidis and for their activity against
a panel of bacterial strains, comprised of S. aureus, Streptococcus
pneumoniae and Escherichia coli.18,19
ization of the cephalosporin D2–D3 double bond. Aminothiazol-
yl-oximino acetyl and aromatic thioacetyl side-chains were
introduced by this method. Subsequent deprotection and deriv-
atization of the propenylamino group were carried out as
Scheme 2. Reagents and conditions: (a) (1) PCl5/Py, CH2Cl2, ꢀ10 °C; (2) MeOH, ꢀ20 to 10 °C; (b) (2-amino-thiazol-4-yl)-methoxyimino-thioacetic acid S-benzothiazol-2-yl
ester, NMP, rt; (c) TFA, CH2Cl2, rt; (d) RCO2H, CDI, NMP, rt; (e) RSO2Cl, BSA, CH2Cl2, reflux.

J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4635–4638
4637
Scheme 3. Reagents and conditions: (a) TBTU, DIPEA, NMP, rt; (b) TFA, CH2Cl2, rt; (c) RSO2Cl, BSA, CH2Cl2, reflux.
Scheme 4. Reagents and conditions: (a) (2-Naphthylthio)acetic acid, TBTU, DIPEA, NMP, rt; (b) TFA, CH2Cl2; (c) RCO2H, CDI, NMP, rt; (d) RSO2Cl, BSA, CH2Cl2, reflux.
Table 1 shows that cephalosporins with a simple C(7) phenyl-
acetamide side-chain exhibited good antibacterial activity against
sensitive S. aureus and S. pneumoniae, but that there was generally
no significant activity against MRSA (S. aureus COL BLAꢀ) and
E. coli. Replacement of the C(7) phenylacetamide side-chain by
an aminothiazolyl-methoxyimino acetamide group gave some
activity against E. coli (compounds 11a–11c in Table 2), but re-
sulted in poor inhibition of PBP20 and decreased activity against
sensitive S. aureus strains. However, this is a known effect of the
methyl-substituted oximino group.21,22 Therefore, the correspond-
Table 1
MIC values of C(7) phenylacetamide cephalosporins
Compound
MIC (lg/mL)
S. aureus
S. pneumoniae
E. coli
ATCC 25923
887a
COL BLAꢀ20
COL BLA+a
R6
UB1005
8a
8b
8c
8d
0.25
60.06
0.5
0.13
0.25
0.25
8
>32
>32
>32
32
32
>32
>32
>32
>32
>32
>32
>32
60.06
60.06
0.13
60.06
60.06
0.125
>32
>32
>32
>32
8
n.d.b
16
2
Amoxicillin
Cephalothin
32
0.5
8
a
b-Lactamase producer.
Not done.
b
Table 2
IC50 and MIC values of C(7) aminothiazolyl-oximino acetamide cephalosporins
Compound
IC50
(l
M)
MIC (
lg/mL)
PBP20
S. aureus
COL BLAꢀ
S. pneumoniae
E. coli
ATCC 25923
887
COL BLA+
R6
UB1005
11a
11b
11c
13a
13b
>500
>500
235 110
2
4
1
0.5
2
8
8
2
1
4
>32
>32
>32
8
>32
>32
>32
16
60.06
60.06
60.06
0.25
1
0.25
1
>32
16
6
12
0.4
2
16
32
60.06

4638
J. Pohlmann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4635–4638
Table 3
IC50 and MIC values of C(7) thioacetamide cephalosporins
Compound
IC50
(lM)
MIC (lg/mL)
PBP20
S. aureus
S. pneumoniae
E. coli
ATCC 25923
887
COL BLAꢀ
COL BLA+
COL BLA+
(+4 g/mL
R6
UB1005
l
sulbactam)
15a
15b
15c
15d
15e
15f
129
31 16
50
69 16
20 1.5
22
2
8
0.13
0.13
1
1
1
0.5
2
4
4
4
1
1
8
1
>32
>32
32
16
32
1
1
4
n.d.
n.d.
n.d.
1
0.13
60.06
60.06
60.06
60.06
60.06
60.06
>32
>32
>32
>32
>32
>32
>32
6
60.06
60.06
60.06
60.06
60.06
8
0.5
2
0.5
>32
16
15g
ing chloro-aminothiazolyl-hydroxyimino acetamide derivatives
13a and 13b were evaluated, and, indeed, these compounds exhib-
ited significantly increased affinities towards PBP20, which trans-
the physico-chemical profile, thus prompting further evaluation
to discover the full potential of this novel cephalosporin class.
lated into MICs as low as 8
l
g/mL against MRSA. Compounds 13a
Acknowledgments
and 13b were apparently penicillinase-stable as the MICs were
not significantly affected by the presence of the staphylococcal
PC-1 b-lactamase. Further improvement of anti-MRSA activity
was achieved by introducing an aromatic thioacetamide side-chain
at C(7) (Table 3). The growth of S. aureus COL BLAꢀ was inhibited at
The authors would like to thank Elena I. Mayboroda and Inna
Yankovich for their excellent support in the synthesis of the com-
pounds and Laure Thenoz for the determination of the MIC values.
concentrations as low as 1
l
g/mL with still an excellent coverage
References and notes
of sensitive Gram-positive bacteria. In general, it was interesting
to note that a relatively broad structure–activity relationship
(SAR) was observed for the propenyl substituent. Aromatic and
non-aromatic amides and sulfonamides showed similar antibacte-
rial activity that was apparently independent of the presence of a
basic moiety in the side-chain. A disadvantage of the thioaceta-
mide side-chain is that it confers lability towards b-lactamases,
as seen by the increased MIC values for PC-1 b-lactamase-produc-
ing strain S. aureus COL BLA+. Combination with a b-lactamase
inhibitor would be a feasible option to counter this problem. In-
deed, addition of sulbactam rendered the b-lactamase-producing
strain of S. aureus sensitive towards the propenylamide and prope-
nylsulfonamide cephalosporins.
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amides are not good leaving groups and it was therefore question-
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evaluate this, 15b was treated with purified IMP1 b-lactamase23
and the resulting mixture was checked for 3-pyridylcarboxamide,
which should have been formed if the reaction had occurred.24 In
these experiments, no free 3-pyridylcarboxamide was found de-
spite the successful hydrolysis of the cephalosporin by IMP1 b-lac-
tamase, indicating that the enhanced anti-MRSA activity of the
novel propenylamide and, by inference, propenylsulfonamide
cephalosporins is not due to leaving group characteristics of the
C(3) side-chain substituent.
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In summary, we have identified propenylamide and propenyl-
sulfonamide cephalosporins as members of a new subclass of anti-
bacterial b-lactams. Depending on the nature of the C(7) side-
chain, there is the potential to optimize either for activity against
MRSA or enterobacteriaceae. The broad SAR observed for the pro-
penyl substituent allows optimization of potency as well as of
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24. Compound 15b was mixed in water with IMP1 b-lactamase. After 5 min of
incubation, the reaction was stopped by addition of acetonitrile and the
solution was analyzed by LC–UV–MS. In this analysis, 15b and 3-
pyridylcarboxamide were used as reference.