Design, synthesis, and crystal structures of 6-alkylidene-2′- substituted penicillanic acid sulfones as potent inhibitors of acinetobacter baumannii OXA-24 carbapenemase

Article abstract of DOI:10.1021/ja104092z

Class D β-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial β-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel β-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2′-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important β-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 β-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC₅₀ values against OXA-24 and two OXA-24 β-lactamase variants ranged from 10 ± 1 (4 vs WT) to 338 ± 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K_i (500 ± 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k_inact/ K_i = 0.21 ± 0.02 μM^-1 s^-1). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 A) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2′-substituted penicillin sulfones are effective mechanism-based inactivators of class D β-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D β-lactamases is proposed.

Full text of DOI:10.1021/ja104092z

Table 1. Kinetic Parameters
kcat (s-1)
Km (µM)
kcat/Km (µM-1s-1)
NCF
OXA-24 WT
538 ± 40
571 ± 55
394 ± 40
28 ± 3
19.2 ± 2.6
2.4 ± 0.3
2.8 ± 0.4
OXA-24Y112A
238 ± 20
143 ± 13
OXA-24Y112A M223A
Imipenem
OXA-24 WT
3.8 ± 0.4
4.8 ± 0.5
5.3 ± 0.2
2.3 ± 0.1
12.0 ± 0.4
22 ± 4
1.7 ± 0.2
0.40 ± 0.04
0.24 ± 0.04
OXA-24 Y112A
OXA-24Y112A M223A
SI Tables p1

Table 2a. IC50
CENTA
1
3
4
5
(nM)
(nM)
(nM)
(nM)
OXA-24 WT
127 ± 42
384 ± 13
509 ± 100
187 ± 17
975 ± 81
500 ± 37
192 ± 56
309 ± 55
594 ± 64
237 ± 7
648 ± 240
898 ± 93
OXA-24 Y112A
OXA-24 Y112A M223A
Table 2b. IC50
NCF
1
2
3
4
5
(nM)
(nM)
27 ± 1
57 ± 5
180 ± 9
(nM)
(nM)
(nM)
OXA-24 WT
17 ± 1
22 ± 1
10 ± 1
55 ± 5
297 ± 15
338± 20
OXA-24 Y112A
OXA-24 Y112A M223A
142 ± 10
250 ± 44
105 ± 16
187 ± 8
68 ± 7
185 ± 12
SI Tables p2

Table 3. Kinetic Parameters of Inhibition
Ki (µM)
2.22 ± 0.88
12.0 ± 0.7
kinact (s-1)
kinact/Ki (µM-1s-1)
0.040 ± 0.008
0.008 ± 0.002
OXA-24 Y112A
0.08 ± 0.01
0.09 ± 0.01
1
2
OXA-24Y112A M223A
OXA-24 Y112A
8.8 ± 0.3
0.03 ± 0.01
0.10 ± 0.01
0.003 ± 0.001
OXA-24 Y112A M223A
22.6 ± 0.2
0.0040 ± 0.0004
OXA-24Y112A
8.0 ± 0.9
0.22 ± 0.02
0.15 ± 0.01
0.030 ± 0.005
0.013 ± 0.001
3
4
5
OXA-24 Y112A M223A
12.0 ± 1.5
OXA-24 Y112A
5.0 ± 0.7
3.0 ± 0.2
0.05 ± 0.01
0.05 ± 0.01
0.010 ± 0.001
0.020 ± 0.003
OXA-24Y112A M223A
OXA-24 Y112A
11 ± 2
19 ± 2
0.04 ± 0.01
0.07 ± 0.01
0.004 ± 0.001
0.004 ± 0.001
OXA-24 Y112A M223A
Tazobactam Ki was determed by Drawz, et al and was 271 ± 37 M (ref).
SI Tables p3

Table 4. ESI-MS Analysis
Expected Mass
(amu)
Actual Mass (amu)
± 3 amu
29071
OXA-24WT
29073
28980
28920
29560
29467
29407
29495
29402
29342
29565
29472
29412
29497
29404
29344
OXA-24Y112A
28980
OXA-24Y112A M223A
OXA-24WT /1
28919
29560
OXA-24 Y112A /1
OXA-24 Y112A M223A /1
OXA-24 WT/3
29467
29410
29496
OXA-24 Y112A /3
OXA-24 Y112A M223A/3
OXA-24 WT/4
29404
29344
29565
OXA-24 Y112A/4
OXA-24 Y112A M223A/4
OXA-24 WT/5
29473
29412
29496
OXA-24 Y112A/5
OXA-24 Y112A M223A/5
29404
29343
SI Tables p4

Supplementary Information FIGURE CAPTIONS
Schemes 1, 2, and 3. See text
Figure 1. Mass spectrometry. Intact mass spectrometry was performed to determine the
covalent intermediates in the inactivation pathway. Here we show the deconvoluted mass
spectra of OXA-24 and variants Y112A and Y112A, M223A) before and after 15-min incubation
with the inhibitors (1, 3, 4, 5). Spectra were obtained on a Q-STAR XL quadrupole-time-of-
flight mass spectrometer equipped with a nanospray source. A single mass shift was identified.
OXA-24 and variants with 2 was not performed.
Figure 2. (A) Detail of the region at the lower border of the active binding cleft of the 1
complex. The catechol moiety runs almost parallel to the right side of the active site. (B), (C)
Close-up view of the surface potential at the active binding site of OXA-24 in complex with 5
drawing in two different orientations. The molecular surface of the enzyme is depicted as a
transparent solid surface coloured according the electrostatic potential. The 5 molecule is
represented as sticks.
SI, Figures (text p1)

Scheme 1. Synthesis of compounds 4 and 5
O
SBt
S
S
N
O
allocNH
O
H3N+
allocNH
O
S
allocNH
O
allocNH
O
1.0 eq
SH
S
S
S
1) Ph2CN2, CH2Cl2/MeOH, rt, 15 h, 40%
CH2Cl
20 eq ClCH2CO2H, 2 eq AgOAc
CH2Cl2, rt, 3 h, 81%
O
CH3
+
N
N
N
N
N
2) ClCO2allyl, py, CH2Cl2, -10 oC, 30 min, 91%
3) 1 eq MeCO3H, -10 oC, 86%
O2CCH2Cl
O
tol. reflux, 3 h, 82%
-
CO2CHPh2
CO2CHPh2
CO2
CO2CHPh2
CO2CHPh2
6-APA
6
7 (Bt = benzothiazole)
8 54%
9 27%
O
O
H2
N
O
O
S
S
H2N
O
O
1) xs i-prONO, 0.05 eq TFA, EtOAc, rt, 30 min
S
S
CH2Cl
CH2Cl
1.1 eq (n-Bu)3SnH, 2.5 eq AcOH
O
O
CH3
CH3
O2CCH2Cl
CO2CHPh2
+
+
N
N
2) 0.0025 eq Rh2(OOct)4, propylene oxide, benzene
rt, 30 min, 90% overall
N
N
O2CCH2Cl
CO2CHPh2
0.01 eq Pd(PPh3)4, CH2Cl2, rt, 30 min, 92%
O
O
CO2CHPh2
CO2CHPh2
10
11
12
13
N
N
N
N
O
O
N
O
O
O2
S
PPh3
O2
S
S
1.5 eq mCPBA, CH2Cl2, rt
30 min, 33%
N
S
S
CH2Cl
CH2Cl
CH2Cl
O
O
O
CH3
CH3
O2CCH2Cl
CO2CHPh2
+
+
+
N
N
CH2Cl2/THF, -78 oC, 30 min, 33%
N
N
N
O2CCH2Cl
O
O
O
O
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
14
15
16 52%
17 5%
18 13%
(separable by column chromatography)
N
O
O
N
N
N
O
O
O2
S
O2
S
O2
S
5 eq thiourea
EtOH, 60 oC, 20 min, 65%
2 eq p-ClCO2C6H4NO2, 0.5 eq DMAP
1.2 eq mCPBA, rt
CH2Cl2, 12 h, 85%
S
CH2Cl
O
CH2Cl
OPNP
O
OH
O
py, rt, 5 h, 72%
N
N
N
N
O
O
O
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
16
18
19
20 (PNP = p-nitrophenyl)
N
N
O
N
N
O
O
H2N
O2
S
NH2
N
N
O2
S
O2
S
2 eq
1) m-cresol, 50 oC, 3 h
NH2
NH2
OPNP
O
Compound 4
N
S
N
H
O
H
O
S
S
N
N
N
2) aq NaHCO3
(51% overall)
O
py, rt, 10 min, 70%
O
O
CO2CHPh2
CO2Na
CO2CHPh2
20
21
22
Ph
N
O
H
N
N
N
O2
S
O
O
Ph
O
H2N
2 eq
O2
OPNP
O
O2
S
1) TFA - anisole
S
H
N
+
NH3
Compound 5
O
O
O
N
NH
O
NH
2) aq NaHCO3
N
O
N
(37% overall)
CO2CHPh2
O
py, rt, 10 min, 66%
O
O
-
CO2
CO2CHPh2
20
23
24
SI Figures p1

Scheme 2. Synthesis of compound 2
CO2allyl
CH3
CO2H
CO2allyl
CO2allyl
CO2allyl
1) 2 eq KMnO4, H2O,
80 oC, 12 h
1) SOCl2, cat. DMF, 80 oC, 2 h
1.13 eq POCl3, 1.13 eq Et3N
1) 2 eq PPh3, reflux
1.4 eq mCPBA, CH2Cl2
rt, 2 h, 93%
CH2Cl2, rt to 40 oC, 2 h
2) KO-t-Bu, THF, rt, 2 h
PPh3
N
O
CH3
2) allyl-OH, Et3N, CH2Cl2,
-78 oC to rt, 2 h, 71%
2) aq HCl, 30% overall
N
CH3
N
CH3
N
CH3
N
CH2Cl
N
25
26
27
28
29
allylO2C
allylO2C
HO2C
CO2allyl
N
N
N
O2
S
O2
S
H2N
O
O
1) 1.1 eq p-TolSO2Na, 0.1 eq Pd(PPh3)4
THF/MeOH, 1 h, rt
1) i-pr-ONO, cat. TFA
2.5 eq mCPBA
CH2Cl2, rt, 3 h, 65%
PPh3
S
S
S
N
2) propylene oxide,
cat Rh2(OOct)4
N
N
N
N
N
2) aq HCl, EtOAc, 70%
-78 oC, CH2Cl2, THF, 37%
O
O
O
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
30
31
32
33
34
MeO
H
N
O
MeO
H
N
N3
O
O
N
N
N
1) TFA - anisole
MeOH, 90 oC,
1 h, 9% overall from 34
O2
S
O2
S
1 eq DPPA, 1 eq Et3N,
DMF, rt, 2 h
Compound 2
O2
S
2) aq NaHCO3
(25% overall)
N
N
N
O
O
O
CO2Na
CO2CHPh2
CO2CHPh2
35
36
SI Figures p2

Scheme 3. Synthesis of compound 3
allylO2
C
allylO2C
allyO2C
allylO2C
allylO2C
CO2allyl
N
N
N
N
N
O
O
O
O
O2
S
O
O
O
O
O
S
1.5 eq mCPBA,
CH2Cl2, rt
O2
S
S
S
S
CH2Cl
+
S
CH2Cl
CH2Cl
O
CH2Cl
PPh3
CH3
O
O
CH3
O2CCH2Cl
CO2CHPh2
O
CH3
+
N
N
N
N
N
N
N
30 min, 33%
N
O2CCH2Cl
+
+
O2CCH2Cl
O
O
O
O
-78 oC, CH2Cl2, THF, 32%
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
12
13
37
38
39 54%
40 7%
41 16%
(separable by column chromatography)
HO2C
allylO2C
allylO2C
allylO2C
N
N
N
N
O
O
O
1) 1.3 eq mCPBA
CH2Cl2, 12 h, rt
O2
S
O2
S
1) 1.1 eq p-TolSO2Na, 0.1 eq Pd(PPh3)4
THF/MeOH, 1 h, rt
O2
S
S
5 eq thiourea
EtOH, 60 oC, 20 min, 65%
CH2Cl
CH2Cl
OH
O
O
OH
2) aq NaHSO3
(79% yield)
N
N
N
2) aq HCl, EtOAc, 84%
N
O
O
O
O
CO2CHPh2
CO2CHPh2
CO2CHPh2
CO2CHPh2
43
41
39
42
O
O
O
PNPO
H2N
H2N
N
N
O
N
O
O
1) TFA - anisole
O2
S
O2
S
3.0 eq NH3, dioxane
py, 30 min, rt 96%
O2
S
3.0 eq p-ClCO2C6H4NO2
0.5 eq DMAP, py, rt, 2 h, 63%
OPNP
NH2
Compound 3
O
2) aq NaHCO3
(26% overall)
NH2
O
O
N
N
N
O
O
O
CO2CHPh2
CO2Na
CO2CHPh2
44
45
46
SI Figures p3

OXA-24 Double mutant (40uM)
OXA-24
(Y112A M223A)
28919 +/- 3
OXA-24Tyr112 (40uM)
OXA-24WT (40uM)
OXA-24 (Y112A)
OXA-24 WT
29071 +/- 3
28980 +/- 3
Mass (amu)
Mass (amu)
Mass (amu)
OXA-24WT (40uM)
OXA-24Tyr112 (40uM)
OXA-24
(Y112A M223A) +
JDB/LN-1-255
29410 +/- 3
LN-1-255 (200 uM)
LN-1-255 (200uM)
OXA-24 (Y112A) +
15min
OXA-24 WT +
15min
JDB/LN-1-255
29467 +/- 3
JDB/LN-1-255
29560 +/- 3
Mass (amu)
Mass (amu)
Mass (amu)
OXA-24 Double mutant:SA-4-17
OXA-24WT:SA-4-17
OXA-24Tyr112:SA-4-17
1:5 15min
OXA-24 (Y112A) +
OXA-24
1:5 15min
1:5 15min
OXA-24 WT +
JDB/SA-4-17
29496 +/- 3
JDB/SA-4-17
29404 +/- 3
(Y112A M223A) +
JDB/SA-4-17
29344 +/- 3
Mass (amu)
Mass (amu)
Mass (amu)
OXA-24WT
SA-3-18
OXA-24Tyr112 (40uM)
OXA-24 Double mutant (40uM)
SA-3-18 (200 uM)
SA-3-18 (200uM)
OXA-24
(Y112A M223A) +
15min
15min
OXA-24 WT +
JDB/SA-3-18
29565 +/- 3
15min
OXA-24 (Y112A) +
JDB/SA-3-18
29473 +/- 3
JDB/SA-3-18
29412 +/- 3
Mass (amu)
Mass (amu)
Mass (amu)
OXA-24Tyr112 (40uM)
OXA-25 Double mutant (40uM)
OXA-24WT (40uM)
OXA-24 (Y112A) +
SA-3-53 (200uM)
OXA-24 WT +
OXA-24
SA-3-53 (200uM)
SA-3-53 (200uM)
15min
JDB/SA-3-53
15min
JDB/SA-3-53
(Y112A M223A) +
15min
29404 +/- 3
29496 +/- 3
JDB/SA-3-18
29343 +/- 3
Mass (amu)
SI Figures p4
Mass (amu)
Mass (amu)
Fig. 1

SI Figures p5
Fig. 2A

B
Y112
M223
C
SI Figures p6
Fig. 2 B, C