Spirocyclopropyl β-lactams as mechanism-based inhibitors of serine β-lactamases. Synthesis by rhodium-catalyzed cyclopropanation of 6-diazopenicillanate sulfone

Article abstract of DOI:10.1021/jm034056q

Class A-class C mechanism-based β-lactamase inhibitors were designed on the basis of the intermediacy of an oxycarbenium species capable of cross-linking with amino acids residues in the active site. Penams 24 and 27 were very potent against AmpC in vitro. The MIC values of 24 in combination with piperacillin against class A and class C producing organisms showed improvement over clinically used tazobactam.

Full text of DOI:10.1021/jm034056q

J . Med. Chem. 2003, 46, 2569-2571
2569
Ch a r t 1. Structures of Clinically Approved
â-Lactamases Inhibitors
Sp ir ocyclop r op yl â-La cta m s a s
Mech a n ism -Ba sed In h ibitor s of Ser in e
â-La cta m a ses. Syn th esis by
Rh od iu m -Ca ta lyzed Cyclop r op a n a tion of
6-Dia zop en icilla n a te Su lfon e
Vincent P. Sandanayaka, Amar S. Prashad,
Youjun Yang, R. Thomas Williamson,
Yang I. Lin, and Tarek S. Mansour*
Ch a r t 2. Structure of Class A-Class C Inhibitors
Chemical and Screening Sciences and Infectious Diseases,
Wyeth Research, 401 North Middletown Road,
Pearl River, New York 10965
Received March 3, 2003
Abstr a ct: Class A-class C mechanism-based â-lactamase
inhibitors were designed on the basis of the intermediacy of
an oxycarbenium species capable of cross-linking with amino
acids residues in the active site. Penams 24 and 27 were very
potent against AmpC in vitro. The MIC values of 24 in
combination with piperacillin against class A and class C
producing organisms showed improvement over clinically used
tazobactam.
for further cross-linking with other active-site residues
of the enzyme as an alternative to fragmentation8 or
rearrangement transformations9,10 of the acyl-enzyme
intermediate reported to date (Scheme 1). This concept
of involving nucleophilic sites of the enzyme in addition
to active serine has been postulated in both class A8 and
class C â-lactamases.4 In this report, we communicate
our preliminary results dealing with cyclopropanation
reactions of 6-diazopenicillanate sulfone 10 and the
utility of the resultant spirocyclopropyl sulfones as a
novel class of â-lactamase inhibitors.
Resu lts a n d Discu ssion . Ch em istr y. Retrosyn-
thetically, we envisioned the use of Rh(II)-catalyzed
cyclopropanation reaction of 10 as the key step to
assemble the requisite tricyclic ring system based on
literature precedents in the preparation of 6-spirocy-
clopropyl penicillanate.11 Thus, the commercially avail-
able (+)-6-aminopenicillanic acid (7) was converted to
the corresponding (+)-6-aminopenicillanic acid sulfone
(8) in 78% yield by employing permanganate oxidation.
The carboxylic acid group of 8 was protected as the
diphenylmethyl ester, with concurrent formation of the
ammonium salt for easy purification, leading to com-
pound 9. The salt was converted to the free amine,
which was treated with NaNO2/HClO4 to obtain the
novel diazosulfone 10 in 90% yield. The overall process
is very efficient in that it does not require any chroma-
tography and can be used to make multigram quantities
of 10 in excellent overall yield (Scheme 2).
We next examined the insertion reactions between 10
and various monosubstituted olefins in the presence of
rhodium(II) catalyst.12 No attempts were made to
improve the stereoselectivity in these reactions at this
stage, since lack of stereochemical differentiation would
be advantageous to gain more insights into the struc-
tural and mechanistic aspects of enzyme interactions.
Some of the vinyl ether insertion partners were chosen
such that the R moiety of 6 was a hydroxyl-protected
group. If the hydroxycyclopropyl compound 6 (R ) H)
were stable enough to be prepared, it might give
additional mechanistic implications of â-lactamase in-
hibition.13
In tr od u ction . Bacterial â-lactamases are enzymes
that hydrolyze the â-lactam ring in all classes of
â-lactam antibiotics before these drugs can exert their
desired therapeutic advantage. These enzymes, which
are the leading cause of resistance to â-lactam antibiot-
ics,1 now comprise a class of nearly 340 discrete â-lac-
tamases and thus represent a significant challenge in
combating bacterial infections.2 In particular, serine
enzymes classified as class A and class C are clinically
relevant because of the emergence of extended spectrum
â-lactamase (ESBLs) resistance and AmpC â-lacta-
mases.1,3 A therapeutically successful strategy to over-
come the resistance problem relies on the administra-
tion of a â-lactamase inhibitor along with an antibiotic.
Thus, in combination with antibiotics, clavulanic acid
(1), sulbactam (2), and tazobactam (3) are â-lactamase
inhibitors commercially used against class A â-lacta-
mases but are not effective against the clinically rel-
evant class C enzymes (Chart 1).
Very few broad-spectrum class A-class C inhibitors
have been reported to date4-6 that include borates as
transition-state analogue inhibitors, acyl phosphonates,
or phosphates that act by phosphorylation of the nu-
cleophilic serine of the â-lactamase active site, cepha-
losporonic acid derivatives, and penem inhibitors such
as 4 and 5 (Chart 2).
During the course of our continuing studies on â-lac-
tamase inhibitors,7 we hypothesized that the incorpora-
tion of a cyclopropyloxy group at the C6 position of 2
leading to compounds 6 should enhance the enzyme-
inhibitor interactions within the active site and thus
may lead to an inhibitor with a broad-spectrum activity
against serine â-lactamases. Mechanistically, the cy-
clopropyloxy group can promote the subsequent chemi-
cal events after initial acylation of the enzyme to
unravel the aldehyde or the oxycarbenium functionality
Compound 10 was treated with 2 equiv of styrene in
the presence of a catalytic amount of Rh2(OAc)4 to obtain
* To whom correspondence should be addressed. Phone: 845-602-
3668. Fax: 845-602-5580. E-mail: mansout@wyeth.com.
10.1021/jm034056q CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/23/2003

2570 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Sch em e 1. Proposed Mechanism of Inactivation
Letters
F igu r e 1. Products of Rh(II)-catalyzed insertion reactions of
10 with alkenes.
Sch em e 2. Synthesis of Diazosulfone 10a
a
Conditions: (a) (i) KMnO4, H2SO4, -10 °C (78%); (ii) Ph2CN2,
0 °C, then anhydrous HCl in ether (84%); (b) aqueous NaNO2, 1
N HClO4, 0 °C (90%).
F igu r e 2. Structures of spirocyclopropylpenam sulfones.
Ta ble 1. In Vitro Data for Selected Compounds
Sch em e 3. Insertion Reactions of 10 with Alkenesa
a
IC50 (µM)
enzyme
24
0.65 12.0 17.0 0.51 >13
0.02 0.4 4.1 0.03
25
26
27
28
29
2
3
TEM-1
AmpC
1.7
1.4
0.1
>3.2 3.2 66.0 48.0
a
Determined graphically from six different concentrations of
the inhibitor.
bon, providing isomers 21, 22, and 23 in a 1:1:0.75 ratio.
In all cases, the diastereomers were readily separated
by column chromatography (Figure 1).
a
Conditions: (a) 2 equiv of alkene in CH2Cl2 (0.2-4.0 M) added
dropwise, Rh2(OAC)4 in CH2Cl2 (0.2 M) (46-60%); (b) flash
chromatography on silica gel.
Unmasking the protecting groups of 13 and 15 proved
to be problematic with typical deprotection agents.
Catalytic hydrogenolysis (H2, 10% Pd/C, EtOAc) condi-
tions were used to convert the benzhydryl esters to the
corresponding acids. In this regard, the acid derivatives
24-29 corresponding to 15-20 were obtained in good
yield (Figure 2). Despite the apparent instability of the
parent hydroxycyclopropyl sulfone penam, the acids 24-
29 seemed to be viable potential inhibitors based on the
mechanistic proposal involving the oxycarbenium cation
as outlined in Scheme 1.
11 and 12 in a ratio of 1.7:1 in 46% overall yield after
separation by chromatography on silica gel. The stere-
ochemical outcome of this transformation is controlled
by the sterically demanding exo face of the bicyclic
penam system. Importantly, diphenylmethyl vinyl ether
resulted in the formation of 13 and 14 in a combined
yield of 56% as a 1:1 mixture of diastereomers (Scheme
3).
Diazosulfone 10 was also treated with TBDMS vinyl
ether under the same reaction conditions to afford a
mixture of diastereomers 15, 16, and 17 in a total yield
of 52% and a ratio of 2:2:1. The stereochemistry of the
above isomers was assigned on the basis of 1H-13C
heteronuclear multiple-bond correlation (HMBC), 2D
nuclear Overhauser effect spectrometry (NOESY), and
1D NOE experiments. Correlation of chemical shift
assignments by 1H-13C HMBC experiment and with 1D
1H NMR spectra enabled the differentiation of the four
methine protons present in the molecule. H5 proved to
be the most important probe for the determination of
the stereochemistry of these compounds because of its
proximity to the cyclopropyl functionality in question.
Moreover, the absolute configuration of 15 was con-
firmed by X-ray crystallography as shown in Figure 3b.
Cyclohexyl vinyl ether under the above catalytic condi-
tions gave the corresponding spiro compounds 18, 19,
and 20 in 59% yield as a 3:3:2 mixture of diastereomers.
The insertion reaction with allyl vinyl ether occurred
chemoselectively at the more electron-rich olefinic car-
En zym e a n d Cellu la r Assa ys. Acids 24-29 were
evaluated for their inhibitory activities14 against two
clinically important â-lactamases, TEM-1 and AmpC,
representatives of class A and class C enzymes, respec-
tively. As expected, alkoxycyclopropyl-containing com-
pounds 24-29 showed good inhibitory activity against
the â-lactamases tested (Table 1). In comparison to
sulbactam, sulfone penams 24 and 27 exhibited excel-
lent activity against TEM-1 and AmpC, respectively,
with over a 2500-fold improvement against the class C
enzyme. Within the same series, 24 and 27 are ap-
preciably more potent than their corresponding diaster-
eomers particularly against the AmpC enzyme. These
penams have their H5 proton on the same side as the
methylene moiety of the spirocyclopropyl ring. In the
less active isomers, H5 is in proximity to the methine
proton of the spirocyclopropyl ring, resulting in a 100-
to 1000-fold reduction in potency. Such an outcome
results in a stereochemical preference of the alkoxy
substituent of the cyclopropyl ring that affects the

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2571
nism-based inhibitors and as an internal mechanistic
probe for the investigation of enzyme mechanisms.16
Ack n ow led gm en t. We thank Mr. Peter Petersen for
MIC for determinations, Dr. D. Ho (Princeton Univer-
sity) for X-ray studies, and Drs. Hollinger and A.
Agarwal for modeling studies.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures, spectral data for all relevant compounds, and NOE
studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
F igu r e 3. (a) Molecular modeling for 24. (b) X-ray ORTEP
diagram for 15.
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MIC c (µg/mL)
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E. coli GC6265
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binding affinities of these inhibitors. Within the silyloxy
derivatives, 24 is appreciably more potent than 25 or
26 against both enzymes. The difference in potency of
25 versus 26 of ca. 10-fold against AmpC appears to
reaffirm the stereochemical preference of the alkoxy
substituent of the cyclopropyl ring affecting the binding
affinity of these inhibitors.
Further support for the strong binding of 24 to AmpC
was deduced from molecular modeling studies based on
the reported crystal structure of class C enzymes. The
model revealed that silyloxy penam 24 was well accom-
modated in the AmpC active site for initial binding
compared to its isomers 25 and 26. The cyclopropyl
moiety benefits from hydrophobic interactions with
cyclopropyl-CH2-Tyr-221, tert-Bu-Leu-119, and dimeth-
ylsilyl-Val-211 in addition to lactam CO interaction with
Ser-64 and the salt bridge between COOH and Lys-315
(Figure 3a).
Further in vitro evaluations in a cell-based assay
(MIC) established the effectiveness of 24 as a potent
broad-spectrum â-lactamase inhibitor (Table 2). Silyloxy
sulfone 24, in combination with piperacillin at a ratio
of 1:1, was two dilutions better than tazobactam against
class C producing organisms, consistent with its more
potent AmpC enzyme activity. Moreover, the combina-
tion of 24 with cefotaxime increases the antimicrobial
activity of cefotaxime against Gram-negative and Gram-
positive organisms.15 The relatively higher MIC values
of 27 against class C producing organisms may be
attributable to piperacillin not being the best partner
with it or simply due to poor penetration into the cell.
(13) Crichlow, G. V.; Michiyoshi, N.; Doppalapudi, V. R.; Buynak, J .
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Kinetic Interactions of Tazobactam with â-Lactamases from All
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acid 4,4-dioxides. U.S. Patent 6,489,316, 2002.
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as beta-lactamase inhibitors. G.BB Patent 2202533, 1988.
6-Spiroepoxycephems have been reported to be weak inhibitors
of â-lactamases.
In conclusion, a new class of mechanism-based class
A and class C â-lactamase inhibitors has been synthe-
sized using a Rh(II)-catalyzed cyclopropanation reaction
of diazosulfone 10.
Sulfone penam 24 emerged with good MIC activity
against class A and class C producing microorganisms.
The cyclopropyloxy structural motif, especially with an
acceptor at the vicinal position on the cyclopropane ring,
should find applications in the design of other mecha-
J M034056Q