The Design, Synthesis, and Evaluation of Mechanism- Based -Lactamase Inhibitors CWRU 2009.
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Transcript of The Design, Synthesis, and Evaluation of Mechanism- Based -Lactamase Inhibitors CWRU 2009.
The Design, Synthesis, and Evaluation of Mechanism-
Based b-Lactamase Inhibitors
CWRU 2009
N
S
O
N
HR
OCH3
CH3
CO2Na
Penicillins
NO
N
HR1
O
Cephalosporins
S
R2
CO2Na
NO
CO2Na
Carbapenems
Me (H)OH
R
N
CH3
O
N
HR
O
Monobactams
SO3Na
N
S
OCO2Na
Penems
OH
R
H (OMe)
NO
N
HR1
O
Oxacephems
O
R2
CO2Na
OMe
Major Classes of b-Lactam Antibiotics
Potent, broad-spectrum antibiotics Usually well tolerated Structural similarities include a negatively charged carboxylate, (usually fused bicylic) b-lactam, and C6 appendage
N
S
O
N
HR
OCH3
CH3
CO2Na
Enz-OH
(PBP)HN
S
O
N
HR
OCH3
CH3
CO2NaOEnz
The b-lactam antibiotics interfere with one or more members of a crucial set of bacterial enzymes, known as the penicillin-binding-proteins (PBPs), that are responsible for cross-linking glycan strands through a protruding peptide side chain.
•The b-lactam antibiotics are believed to resemble the D-Ala-D-Ala terminus of the pentapeptide side chain (Strominger Hypothesis)•Bacterial transpeptidases cleave between the two D-Ala residues, to form an intermediate acyl-enzyme, which is then reacted with a free amino moiety (e.g. the w amino group of diaminopimelic acid) to form the cross link.
OH
PeptideChain
Gly
Blocked H2OBlocked
HCR
O
CO2H
NH
OMe
Me
N
S
S
HN
O
O
Me
Me
NH
CO2H
C
O
R H S
HN
O
O
Me
Me
NH
CO2H
C
O
R H
Link
Why are b-lactam antibiotics such good drugs?
• b-Lactam antbiotics still comprise approximately half the commercial antibiotic market.
• Formation of a covalent bond to the target(s) may be an effective strategy for avoiding resistance due to point mutations which lower affinity
• Targeting the bacterial cell wall avoids the necessity to accumulate in cytoplasm, thus avoiding efflux pumps.
• b-lactams do not penetrate most mammalian cell types, resulting in low toxicity (disadvantage when treating atypicals)
• Most commonly observed resistance is due to production of b-lactamase(s)
Resistance to b-Lactam Antibiotics1) Production of one or more enzymes (b-lactamases) that
hydrolytically destroy b-lactam antibiotics 2) Produce PBPs that do not recognize penicillin3) In the case of Gram-negative strains delete outer
membrane porins, which are responsible for the allowing the b-lactams to reach the periplasm and hence the cell wall
4) In the case of Gram-negative strains, upregulate efflux pumps, which are responsible for pumping out foreign substances (including b-lactams).
N
S
O
N
HR
OCH3
CH3
CO2Na
Enz-OH
(-lactamase)HN
S
O
N
HR
OCH3
CH3
CO2NaOEnz
HN
S
O
N
HR
OCH3
CH3
CO2NaOH
H2O
Enz-OH
+
Action of Serine b-Lactamases
The b-Lactamases
• More than 600 different b-lactamases, grouped into four classes A-D• Classes A, C, and D are serine enzymes• Class B are zinc metalloenzymes• Historically, the class A (serine) enzymes were the most prominent• Can be produced in large quantity (hyperexpressed)• Produced in the periplasm of Gram-negative organisms, or extracellularly in Gram-positive strains.
N
S
ON+
CO2-
HN
N O
CO2H
S
N
H2N ON
O
HN
O
S
CO2Na
OMe
MeO
Methicillin Ceftazidime
•One early strategy for countering b-lactamase mediated resistance was to design b-lactam antibiotics which would also be poor b-lactamase substrates.•This was achieved by incorporating sterically large substituents at C6 (penicillin) or C7 (cephalosporin).
Bulkygroup
Enzyme
C
R
HN
O
CO2H
Me
MeS
N
H H
O
•Unfortunately, this gave rise to new forms of resistance, such as the appearance of a penicillin binding protein with reduced affinity for all b-lactam antibiotics (PBP2a in MRSA) and also the appearance of b-lactamases with enlarged active sites (extended spectrum b-lactamases or ESBLs) that could accommodate the larger antibiotics.
Methicillin-resistant Staphylococcus aureusMRSA
Recent Trends in b-Lactamase-mediated Resistance
• Broad spectrum b-lactamases, known as extended spectrum b-lactamases (ESBLs) capable of hydrolyzing third generation cephalosporins, are disseminated widely (e.g. class A, CTX-M)
• Class C b-lactamases (AmpC) are more widely disseminated, now including many plasmid-mediated AmpCs (e.g. FOX and CMY)
• Classes A and D enzymes have evolved the ability to hydrolyze the carbapenem class of antibiotics. These serine carbapenemases are increasingly widespread (e.g. KPC).
• Class B metallo-b-lactamases are disseminating widely. These enzymes were originally seen in Asia and in Europe, but cases of resistance due to class B b-lactamases are now appearing in the US (e.g. IMP and VIM).
NO
O
CO2Na
OH
NO
O2S
CO2Na
NO
O2S
CO2Na
NN
N
Clavulanate Sulbactam Tazobactam
Current Commercial b-Lactamase Inhibitors
•A second approach was to develop inhibitors of b-lactamase•Unfortunately, current commercial inhibitors target only class A enzymes
Since the inhibitors have no independent antibacterial activity (i.e. ability to bind PBPs), they must be coadministered with b-lactam antibiotics
NO
O
CO2Na
OH
Clavulanate
NO
S
CO2Na
Amoxicillin
HN
O
NH2
HO
= Augmentin(GSK)
+
NO
O2S
CO2Na
Sulbactam
NO
S
CO2Na
Ampicillin
HN
O
NH2
= Unasyn(Pfizer)+
+NO
O2S
CO2Na
NN
N
Tazobactam
NO
S
CO2Na
Piperacillin
HN
O
HNN
O
NEt
O O
= Zosyn(Wyeth)
How do these commercial inhibitors work?
•Placing sulfur at the sulfone oxidation state predisposes the thiazolidine ring to fragment, producing the iminium ion shown above.•The iminium ion can then tautomerize to the b-aminoacrylate, or be captured by a second active site serine, producing in both cases, a stabilized acyl-enzyme.
N
O2S
OCO2Na
Enz-OHHN
O2S
O
CO2NaO-Ser70
HN+
SO2-
O
CO2NaO-Ser70
HN
SO2-
O
CO2NaOEnz
HN
SO2-
OCO2Na
OEnz
-Aminoacrylate (Stabilized Acyl-Enzyme)
HN
SO2-
O
CO2NaO-Ser70
OSer130
O
O-Ser70
OSer130
Doubly covalently-boundAcyl-enzyme
How can we build a better mousetrap?
E + I E I E-IMichaelisComplex
InitialAcyl-Enzyme
E-I'StabilizedAcyl-Enzyme
H2OE I'
Complex ofHydrolyzed Inhibitor
E + I'
Irreversible inhibitors offer numerous opportunities for improving the inhibitory efficiency.
enzymatic mechanism
active site dimensions and
binding characteristics
synthetic feasibility
generate a library of prospective inhibitors
Assay againstall relevant enzymes
The Inhibitor Design Process
Initially we focused on designing inhibitors which held the potential to quickly form very stable acyl-enzymes.
N
O2S
OCO2Na
R1
R2
NO
CR1
R2
O2S
OAc
CO2Na
E + I E I E-IMichaelisComplex
InitialAcyl-Enzyme
E-I'StabilizedAcyl-Enzyme
H2OE I'
Complex ofHydrolyzed Inhibitor
E + I'
Focus Here
NO
S
CO2CHPh2
CH2OAc
H2N
NO
S
CO2CHPh2
CH2OAc
O
1) Tf2O, Et3N
2) aq HCl
MgBr1)
2) H3O+ NO
S
CO2CHPh2
CH2OAc
OH
1) Tf2O, pyr
2) (t-Bu)2CuCNLi2, -100 oCN
O
S
CO2CHPh2
CH2OAc
CH
t-Bu
1) xs mCPBA
2) TFA, anisole3) NaHCO3
NO
O2S
CO2Na
CH2OAc
CH
t-Bu
N
O
O
OH
CO2Na
N
O2S
CO2NaO
N NN
NO
O2S
CO2Na
CH2OAc
CH
t-Bu
> 2000 M 51.9 M 11.8 M
IC50 Values against the class C b-lactamase derived from Enterobacter cloacae, strain P99
NO
O2S
CO2Na
CH2OAc
CD
t-Bu
HNO
O2S
CO2-
CH2OAc
CD
t-Bu
OEnz
HNO
SO2-
CO2-
CH2OAc
t-Bu
OEnz
EnzOH
Further mechanistic investigations uncovered an isotope effect on the rate of inactivation. A mechanism consistent with this observation is shown below.
StabilizedAcyl-Enzyme
N
S
OCO2CHPh2
H2Ni-PrONO
cat. TFAN
S
OCO2CHPh2
N2
3 mol % Rh2OAc4
xs propylene oxideN
S
OCO2CHPh2
O
quantitative yield
New chemical methodology facilitated the preparation of new inhibitors.
N
S
OCO2CHPh2
O
RCH=PPh3N
S
OCO2CHPh2
R
xs mCPBAN
O2S
OCO2CHPh2
R
1) TFA, anisole
2) NaHCO3N
O2S
OCO2Na
R
The availability of 6-oxopenicillanate simplifies the synthesis of 6-alkylidene penams, as shown.
N
S
OCO2CHPh2
R1
N
S
OCO2CHPh2
R1O
H
N
SOH
OCO2CHPh2
R1
N
S
OCO2CHPh2
R1 S N
S
1 eq mCPBA
S
N
HS
AgOAc
R2CO2HN
S
OCO2CHPh2
R1
O
O
R2
a) R1 = CO2-t-Bu, R2 = CH2O2CCH3
b) R1 = CO2-t-Bu, R2 = CH2O2CCH2Cl
c) R1 = CO2-t-Bu, R2 = CH2O2CH
d) R1 = CO2-t-Bu, R2 = CH2O2CCH2Ph
e) R1 = CO2Me, R2= CH2O2CCH3
f) R1 = CO2Me, R2 = CH2O2CCH2Cl
N
S
OCO2CHPh2
R1
S S
N
N
S
OCO2CHPh2
R1
O
O
R2
+Ag
N
S+
OCO2CHPh2
R1
-O2CR
A
BN
O
R1
S
CO2CHPh2
CH3
O2CR
A B
+
N
S
OCO2CHPh2
H2N1) alloc-Cl, Et3N
2) 1 eq mCPBA N
S
OCO2CHPh2
allocNHO
S
N
HS
tol. N
S
O
allocNHSBt
CO2CHPh2
N
S
OCO2CHPh2
allocNHAgOAc
RCO2H
O
O
R a) R = CH3b) R = CH2Ph
c) R = CH2
O-t-Bu
O-t-Bu
N
S
OCO2CHPh2
allocNH
O
O
R1(n-Bu)3SnH, AcOH
cat. Pd(PPh3)4N
S
OCO2CHPh2
H2N
O
O
R1
N
S
OCO2CHPh2
O
O
R1O1) i-PrONO, cat. TFA
2) cat. Rh2OAc4, propylene oxide
N
Ph3P
R2
N
S
OCO2CHPh2
O
O
R1
N
R2
N
O2S
OCO2Na
O
O
R1
N
R2
1) xs mCPBA2) TFA, anisole
3) NaHCO3
Table 1. Inhibitory activity on Three Representative Serine b-Lactamases IC50 (mM)R1 R2 P99 TEM-1 PC1
None (Tazo) CH2C2H2N3 51.9 0.297 2.57
CO2Na CH3 4.50 1.8 108
CO2Na CH2O2CCH3 0.708 0.180 76.53
CO2Na CH2O2CCH2Cl NT 0.196 7.2
CO2Na CH2O2CH 0.592 1.84 173
CO2Na CH2O2CCH2Ph 0.54 0.0154 579.0
CO2Na CH2O2CCH23’,4’-C6H3(OH) 2 0.37 0.105 116
CO2Me CH2O2CCH3 9.51 2.72 NT
CO2NH2 CH2O2CH3 8.48 0.31 2.21
CO2Na CH2Cl 527.0 120.5 2100
CO2Me CH2Cl 13.91 44.51 432
CO2Na CH=CHCN 6.76 21.67 504
CO2Na CH2O2CCH2-S-tet 0.64 0.233 NT
CO2Me CH2O2CCH2-S-tet 13.2 2.37 939.7
a’-pyr CH2O2CCH3 0.062 0.004 0.66
a’-pyr CH2O2CCH2Ph 0.001 0.04 0.39
a’-pyr CH2O2CCH2-3’,4’-C6H3 (OH) 2 0.026 0.060.7
N
O2S
O
R2
CO2Na
R1
Buynak, J. D. et. al. BMCL 1999, 9, 1997-2002.
Piperacillin PIP:TAZ PIP:JDB/LN-1-255
P. aeruginosa Ps505A1 (AmpC derepressed) >64 16 2
A. sobria ((Asb A, OXA-12, AsbM) 64 64 1
S. marcescens GC 4132 (Amp C, in vivo) 64 32 4
E. coli C600N (no b-lactamase) 2 2 1
E. coli C600N +(TEM-1) >64 4 2
E. coli C600N + (IRT – 2) >64 8 2
E. coli C600N + (SHV – 4) >64 2 2
E. coli C600N + (PSE – 1) 32 1 2
E. coli C600N + (OXA-10) {PSE-2} >64 2 2
E. coli C600N + (MIR-1) 64 8 8
E. coli C600N + (Imi-1) >64 16 8
E. coli 300 + (TEM-1) >64 4 1
E. coli 300 + (ampRampC) 16 4 2
K. Pneumoniae KC 2 (TEM-10) >64 2 4
E. coli GC6265 (TEM-1, in vivo) >64 4 4
N
O2S
OCO2Na
N
O
O
OH
OH
JDB/LN-1-255
N
O2S
OCO2Na
O
NO
NH
N
O2S
OCO2Na
O
NO
OH
OH
N
S
NH2N
O2S
OCO2
-
O
NO
NH
O
HN
NH3+
N
O2S
OCO2Na
O
NO
NH2
O
NH2
N
O2S
OCO2Na
O
NO
NH2
NH3+
N
O2S
OCO2
-
N
NH3+
N
O2S
OCO2
-
O
NO
NH2
HNNH2
NH2+
N
O2S
OCO2
-
O
NO
NH2
NH3+
JDB/SA-3-18JDB/SA-4-11 JDB/SA-4-17 JDB/SA-4-141
JDB/SA-4-157 JDB/SA-4-196 JDB/SA-4-198JDB/LN-1-255
NH2
Inhibition of Representative -lactamases (IC50, M)
Inhibitor TEM-1E. Coli
AmpCP. aeruginosa
AmpCA. baumannii
OXA-40A. baumannii
In serumAmpC P. aeruginosa
JDB/SA-3-18 0.0004 0.008 0.017 0.0060 0.012
JDB/SA-4-11 0.00010 0.185 0.191 0.191 0.028
JDB/SA-4-17 0.00003 0.012 0.020 0.007 0.014
JDB/SA-4-141 0.0002 0.065 0.071 0.583 0.029
JDB/SA-4-157 0.0006 0.201 0.515 0.888 0.080
JDB/SA-4-196 0.0001 0.006 0.015 0.046 0.003
JDB/SA-4-198 0.0001 0.039 0.052 0.079 0.015
JDB/LN-1-255 0.00003 0.006 0.004 0.011 0.082
N
O2S
OCO2Na
O
NO
NH
N
O2S
OCO2Na
O
NO
OH
OH
N
S
NH2N
O2S
OCO2
-
O
NO
NH
O
HN
NH3+
N
O2S
OCO2Na
O
NO
NH2
O
NH2
N
O2S
OCO2Na
O
NO
NH2
NH3+
N
O2S
OCO2
-
N
NH3+
N
O2S
OCO2
-
O
NO
NH2
HNNH2
NH2+
N
O2S
OCO2
-
O
NO
NH2
NH3+
JDB/SA-3-18JDB/SA-4-11 JDB/SA-4-17 JDB/SA-4-141
JDB/SA-4-157 JDB/SA-4-196 JDB/SA-4-198JDB/LN-1-255
NH2
Synergy of Inhibitors with Imipenem Against Resistant P. aeruginosaImipenem
(mg/L)JDB/SA-
3-18(mg/L)
JDB/SA-4-11
(mg/L)
JDB/SA-4-17
(mg/L)
JDB/SA-4-141(mg/L)
JDB/SA-4-157(mg/L)
JDB/SA-4-196(mg/L)
JDB/SA-4-198(mg/L)
JDB/LN-1-255(mg/L)
MIC 20 0 0 0 0 0 0 0 0
0.5 MIC 10 12.5 25 25 12.5 3.125 6.25 12.5 6.25
0.25 MIC 5 100 50 50 25 12.5 12.5 25 25
0.125 MIC
2.5 100 100 100 25 25 12.5 50 50
0.0625 MIC
1.25 >100 >100 >100 50 50 25 100 100
0.0313 MIC
0.625 >100 >100 >100 >100 >100 >100 >100 >100
Inhibition of b-Lactamase (IC50 mM)
R Escherichia coli W3310(Class A)
Enterobacter cloacae P99 (Class C)
t-butylmethylidene (allene) >2000 11.8
a-pyridyl 44 1.30
CO2But 0.28 429
tazobactam 1.37 51.9
clavulanic acid 3.3 >2000
NO
N
O2S
CO2Na
OAcN
O
CO2S
CO2Na
OAc
H
t-Bu
NO
CO2-t-Bu
O2S
CO2Na
OAc
Initial attempts to improve the cephalosporin series of b-lactamase inhibitors relied on analogy with the cephalosporin antibiotics themselves.
N
S
ON+
CO2-
HN
N O
CO2H
S
N
H2N O
Ceftazidime
But these efforts resulted in an abysmal failure!
N
O2S
ON+
CO2-
Ceftazidime-like analog
N
N
O2S
OOAc
CO2-
N
IC50 values against class C P99 -lactamase
1.3 M >2000 M
HN
O2S
XO
OEnz
N
CO2-
N
O2S
O
OEnz
N
CO2-
Pathway 1 H2O
N
O2S
O
OH
N
CO2-
EnzOH+
HN
O2S
XO
OEnz
N
CO2-
N
SO2-
XO
OEnz
N
CO2-
Pathway 2
Stabilized Acyl-enzyme
?
•Since the charge neutral pyridine moiety is a better leaving group than the negatively charged acetate, it is more likely to follow pathway 1 above.•Yet all the inhibitory mechanisms we have proposed follow pathway 2.
Type R1 R2 TEM-1 PC1 P99 GC1
Tazo 0.32 2.8 49.8 3.4
I 2’-py E-CH=CH-CN 0.014 0.72 0.01 0.012
I 2’-py E-CH=CHCO2Me 0.02 0.30 0.20 0.30
I 2’-py E-CH=CHCONH2 0.09 0.10 0.026 0.01
I 2’-py Z-CH=CClCO2Me 0.07 1.4 0.90 0.18
I 2’-py E-CH=CH-CH=CH2 68 75 24 NT
I 2’-py E-CH=CHCO2But NT 240 1.48 NT
I 2’-py E-CH=CHCO2Na 2.5 31 0.31 NT
I 2’-py E-CH=CHNO2 0.07 0.20 0.02 0.10
I 2’-py E-CH=CH-2’-py 0.20 4.3 0.18 NT
I 2’-py E-CH=CH-2”py-N-ox 0.006 8.6 0.60 0.10
I 2’-py CN 2.34 280 0.029 NT
I 2’-thzl E-CH=CHCONH2 0.90 154 0.29 NT
II 2’py E-CH=CHCO2Me 2.9 6.0 0.03 0.06
II 2’-py E-CH=CH-CO2But NT NT 440 150
II 2’-py E-CH=CHCO2Na 2.5 NT 6.60 NT
NO
R1
O2S
CO2Na
R2N
O
O2S
CO2Na
R2
CH2
I II
R1
R TEM-1 InhibitionIC50, M
P99 InhibitionIC50, M
Tazobactam 0.25 101.6
CH=CH-CONH2 0.2615 0.022
CH=CH-CONHCH2CF3 0.078 1.18
CH=CH-CONHCH2CH2OH 0.0701 0.212
CH=CH-CONHCH(CH2) 2 0.240 0.824
CH=CH-CONH-CH2CH2 (CN3H4) 0.0083 0.0055
CH=CH-CONHOH 7.69 0.128
CH=CH-CONHC6F5 4.28 0.127
CH=CH-CON(CH2CH2) 2NMe 0.053 6.34
CH=CH-CONHCH 2Ph 1.4 0.11
CH=CH-CONHNH 2 0.39 1.1
CH=CH-CO-NHC 6H4OH 0.11 0.035
CH=CH-CONHCH 2CO 2Na 4.2 0.31
CH=CH-CONH(CH 2) 3NH 2 1.59 4.2
N
O2S
O
N
CO2Na
R
How do my inhibitors work?
N
O2S
OCO2Na
RHN+
SO2-
O
CO2Na
R
O
Ser70
NN
HN
SO2-
O
CO2Na
R
O
Ser70
N+
HN
SO2-
O
CO2Na
R
O
Ser70
N
Stabilized Acyl-Enzyme(several crystal structures now in PDB)
H
- H+
• Intramolecular capture of intermediate imine is more efficient than intermolecular capture (and/or tautomerization)• Inhibitors tend to be more general to all (serine) b-lactamases, since inhibitory mechanism does not depend on enzyme active site groups
Next goal: Prepare penicillin-derived inhibitors of metallo-b-lactamases
Problem: Metallo-b-lactamases are still a small portion of total number of b-lactamase producing strains
Solution: Prepare a single molecule that can function as dual inhibitor of both metallo- and serine-b-lactamases.
Problem: Metallo and serine b-lactamases have profoundly different mechanisms of action.
NO
S
CO2-
R2
R1
Zn1 Zn2
HO
H84
H86
H160
D88
H89
H225OH2
N
O
S
C
R2R1
Zn1 Zn2H84
H86
H160
D88
H89
H225OH2
O
OO
HN
O S
C
R2
R1
Zn1 Zn2H84
H86
H160
D88
H89
H225OH2
O
OH
O
Proposed series of events involved in the hydrolysis of a cephalosporin substrate by the L1 metallo-b-lactamase.
Inhibiting metallo-b-lactamases
Like most metalloenzymes, metallo-b-lactamases are inactivated by good zinc chelators.
Potential problem is that zinc chelating agents would likely be nonspecific, thus resulting in toxicity.
Solution: Generate a zinc chelating moiety that relies on the action of the enzyme itself to achieve optimal inhibitory activity (i.e. generate a mechanism-based metalloenzyme inhibitor).
Proposed Mechanism-based Inhibitors of the Zinc Metallooenzymes
N
O2S
O
HS
CO2Na
H2O
-lactamase HN
SO2-
O
S
CO2NaO
Zn+2
N
OnS
O
HX
CO2Na
Two epimers
Targeted Sulfone and Sulfide oxidation statesBoth alcoholand thiol
N
S
O
H3N+
CO2-
N
S
O
Br
CO2CHPh2
1) NaNO2, H2SO4, Br2
2) PhC=N2
Br
1) t-BuMgBr, -78 oC
2) CH2O, -78 to rtN
S
O
Br
CO2CHPh2
HOCH2
N
S
OCO2CHPh2
HOCH2
(n-Bu)3SnH
cat AIBN,
1) MeSO2Cl, DMAP
2) CH3COSCs N
S
OCO2CHPh2
AcSCH2
NaOCH3
-78 to -40 oCN
S
OCO2CHPh2
HSCH2
N
S
OCO2Na
HSCH2
1) TFA, anisole
2) NaHCO3
N
S
OCO2CHPh2
HSCH2troc-Cl, DMAP
0 oC to rtN
S
OCO2CHPh2
TrocSCH2
KMnO4/HOAc
CH2Cl2, rt N
O2S
OCO2CHPh2
TrocSCH2
Zn/Cu, HOAc
THF/MeOH, rtN
O2S
OCO2CHPh2
HSCH2 1) m-cresol, 50 oC, 2.5 h
2) NaHCO3N
O2S
OCO2Na
HSCH2
N
S
OCO2CHPh2
HOCH2
2.2 eq mCPBA
CH2Cl2/pH 6.4 BufferN
O2S
OCO2CHPh2
HOCH2
1) m-cresol, 50 oC
2) NaHCO3
N
O2S
OCO2Na
HOCH2
N
S
OCO2CHPh2
HOCH21) m-cresol, 50 oC
2) NaHCO3
N
S
OCO2Na
HOCH2
N
S
OCO2CHPh2
HOCH2
Br Bu3P, anh MeOH
1 h, rtN
S
OCO2CHPh2
HOCH2
H
N
S
OCO2CHPh2
AcSCH2
H1) MsCl, DMAP
2) AcSCs, MeCN
NaOMe, THF/MeOH
-78 oC to -40 oCN
S
OCO2CHPh2
HSCH2
H1) m-cresol, 50 oC, 6h
2) NaHCO3N
S
OCO2Na
HSCH2
H
Compound TEM-1(class A)(Serine)
P99(class C)(Serine)
L1(class B)(Metallo)
BCII(Class B)(Metallo)
Tazobactam 0.122 53.2 >200 >200
752 409 >200 >200
275 96.2 >200 >200
0.65 3.9 72.3 >200
14.6 10.0 >200 >200
Inhibition of Serine and Metallo-b-lactamases IC50 (mM)
TEM-1(class A)(Serine)
P99(class C)(Serine)
L1(class B)(Metallo)
BC1(Class B)(Metallo)
Tazobactam 0.122 53.2 >200 >200
601 0.10 32.1 2.9
648 3.75 10.9 1.7
6.8 10.5 0.10 1.4
51.7 7.5 0.30 2.0
Inhibition of Serine and Metallo-b-lactamases IC50 (mM)
Van den Akker Strategy: Stabilize the E-b-aminoacrylate intermediate in the active site.
• Designed by analogy with acyl-enzyme of Tazobactam.• This should result in an acyl-enzyme with increased affinity for the site.• May retain occupancy of the site subsequent to hydrolysis of the covalent
ester linkage of the acyl-enzyme.
E + I E I E-IMichaelisComplex
InitialAcyl-Enzyme
E-I'StabilizedAcyl-Enzyme
H2OE I'
Complex ofHydrolyzed Inhibitor
E + I'
Focus Here Focus Here
N
O2S
OCO2Na
Enz-OHHN
O2S
O
CO2NaO-Ser70
HN+
SO2-
O
CO2NaO-Ser70
HN
SO2-
O
CO2NaOEnz
HN
SO2-
OCO2Na
OEnz
-Aminoacrylate (Stabilized Acyl-Enzyme)
HN
SO2-
O
CO2NaO-Ser70
OSer130
O
O-Ser70
OSer130
Doubly covalently-boundAcyl-enzyme
R
Tightly inBinding Pocket
Design a 2’-substituent that stabilized the E-form of the b-aminoacrylate
N
S
O
BrBr
CO2CHPh2
1) 1 eq mCPBA
2) mercaptobenzothiazole, tol. N
S
O
BrBr
CO2CHPh2
S
N
S
1) AgOAc
2) HO2C(CH2)3CO2CH2CCl3N
S
O
BrBr
CO2CHPh2
O
O
OCH2CCl3
O
Zn
CH3CN N
O2S
OCO2CHPh2
O
O
OCH2CCl3
O
Zn
HOAc
xs mCPBA
N
O2S
O
BrBr
CO2CHPh2
O
O
OCH2CCl3
O
N
O2S
OCO2CHPh2
O
O
OH
O
1) TFA, anisole
2) NaHCO3 N
O2S
OCO2Na
O
O
ONa
O
N
O2S
OCO2Na
O
O
ONa
O HN
SO2-
O
NaO2C
O
O
-O2CO
Enz
NH3+
K234
EnzOH
Thanks to my collaborators:
Robert BonomoPaul CareyMarion HelfandFocco van den Akker
And my funding sources:
Robert A. Welch FoundationNational Institutes of Health