Targeting clinically-relevant metallo-β-lactamases: from high-throughput docking to broad-spectrum inhibitors

Figures & data

Figure 1. Structure of the HTD hit compound 1, of the optimized derivative 3i and general structure of inhibitors identified in this study.

Table

Time (min)	CH3CN (0.1% TFA)	Water (0.1% TFA)	Flow (mL/min)
0	20	80	1.5
10	80	20	1.5

Table 1. Reaction conditions explored for hydrazone bond reduction for conversion of compound 2a to 4b (Scheme 1 and Table 2).

Entry	Reducing agent	Solvent	Temp	Time	Yield
1	NaBH4 (1, 2, 4 or 10 eq)	MeOH	25–70 °C	1–12 h	SM^a
2	NaBH4 (9 eq)+TiCl3/LiCl/Ni°/InCl3 (3 eq)	MeOH	25 °C	1–18 h	SM
3	NaBH4 (9 eq)+NiCl2 (3 eq)	MeOH/THF 1:1	25 °C	1 h	SM
4	NaBH4 (9 eq)+Ni° (3 eq)	MeOH/4 N NaOH 1:1	60 °C	3–18 h	SM
5	NaBH4 (3 eq)+TiCl4 (1.1 eq)	1,2-dimethoxyethane	25 °C	3 h	Dec.^b
6	LiAlH4 (3 eq)	THF	0–25 °C	2–18 h	Dec.
7	H2, Pd/C 10%	MeOH	25 °C	18 h	Dec.
8	Zn (3 eq)	AcOH/H2O 1:1	60 °C	1–2 h	Dec.
9	NaBH4 (3 eq)	TFA	25 °C	1–18 h	Dec.
10	NaCNBH3 (1 eq)	TFA	25 °C	0.5 h	11%
11	Na(OAc)3BH (10 eq)	TFA or TFA/DCM 1:5	25 °C	0.5–18 h	4% max
12	Et3SiH (2 eq)	TFA	25 °C	1 h	SM
13	Et3SiH (2 eq)	TFA	25 °C	4 h	72%
14	Et3SiH (2 eq)	TFA	25 °C	18 h	Dec.
15	(iPr)3SiH (1.1 eq)	TFA	25 °C	4 h	32%
16	(Cl)3SiH (1.1 eq)	TFA	25 °C	4 h	17%
17	PhSiH3 (1.1 eq)	TFA	25 °C	4 h	24%

^aSM = only starting material recovered from reaction mixture.

^bDec.=decomposition of starting material observed.

Scheme 2. Synthetic procedure for the preparation of final compounds 6–8. Reagents and conditions: (a) TEA, EtOH, reflux, 76 h; (b) 2-aminoethanol, 150 °C, 15 min then 190 °C, 30 min (c) i. p-TsCl, pyridine, dry DCM, 0–25 °C, then 50 °C, 4 h; ii. 15 or 16, DIPEA, dry DMSO, 70 °C, 20 h.

Table 2. K_i values for VIM-2 and NDM-1 for compounds, 1, 2a–e, 3a–l, 4a–c and 5–8.

General formula for compounds 6–8 are reported in Scheme 2
						VIM-2	NDM-1
Cpd	R1	R2	R3	X	[I]max (μM)	Ki (μM)
1	6,7-OCH2O-	2-Me	NHSO2Me	–	80–200	10	10
2a	6,7-OCH2O-	2-Me	SMe	–	50–200	5	N.I.^a
2b	6,7-OCH2O-	2-Me	SO2Me	–	50–200	N.I.	N.I.
2c	6,7-OCH2O-	2-Me	SOMe	–	80–200	N.I.	N.I.
2d	6,7-OCH2O-	2-Me	CH(OEt)2	–	80–200	15	–
2e	6,7-OCH2O-	2-Me	CHO	–	200	10	10
3a	6,7-OCH2O	2-Me	SO2NH2	–	400	17	N.I.
3b	6,7-OCH2O	H	NHSO2Me	–	100	N.I.	9
3c	6,7-OCH2O	H	SMe	–	100	10	N.I.
3d	H	2,3-(CH2)4	NHSO2Me	–	500	24	N.I.
3e	6,7-OCH2O-	2-Me	CH(OMe)2	–	100	6	N.I.
3f	H	2,3-(CH2)4	SMe	–	100	2	∼45
3g	6-OMe	Me	NHSO2Me	–	100	5	11
3h	6-OMe	Me	SMe	–	100	9	6
3i	7-OMe	Me	NHSO2Me	–	100	8	9
3j	7-OMe	H	NHSO2Me	–	100	3	N.I.
3k	H	H	NHSO2Me	–	100	N.I.	N.I.
3l	H	H	SMe	–	100	N.I.	N.I.
4a	6,7-OCH2O	2-Me	NHSO2Me	CH2	100	N.I.	N.I.
4b	6,7-OCH2O	2-Me	SMe	CH2	100	36	13
4c	6,7-OCH2O	2-Me	SO2NH2	CH2	100	15%	N.I.
5	6,7-OCH2O	2-Me	NHSO2Me	CO	100-200	N.I.	6
6	–	–	–		100	N.I.	34
7	–	–	–		100	N.I.	N.I.
8	–	–	–		100	N.I.	N.I.

^aNI, no or poor inhibition. K_i values were not determined for inhibitors showing poor inhibition; in this case, the enzyme inhibition in the presence of the inhibitor at the highest tested concentration (indicated in column 6) was below 20% (residual activity in the presence of inhibitor, ≥80%).

Figure 2. Docked pose of 1 (sticks) into VIM-2 binding site (A), NDM-1 (B) and IMP-1 (C) as found by HTD protocol. Regarding IMP-1, two representative clusters were reported: in light sticks the docked pose of the first cluster, in dark sticks the docked pose of the second cluster. The contacts established by 1 into the active sites of the enzymes are represented by dotted lines. Metal ions were represented by spheres. The numbering of NDM-1 is referred to Uniprot KB protein sequence F6IAY7, while the numbering of IMP-1 is referred to Uniprot KB protein sequence P52699. The contacts established by 1 into the active sites of the enzymes are represented by dotted lines. Metal ions were represented by spheres. The numbering of NDM-1 is referred to deposited Uniprot KB protein sequence F6IAY7. Nonpolar hydrogens were omitted for the sake of clarity. The pictures were generated by PyMOL (The PyMOL Molecular Graphics System, v1.6-alpha; Schrodinger LLC, New York, 2013).

Figure 3. Docked pose of 3b into VIM-2 binding site (A) and NDM-1 (B) as found by docking protocol. The contacts established by the mentioned compounds into the active sites of the enzymes are represented by dotted lines. Metal ions were represented by spheres. Nonpolar hydrogens were omitted for the sake of clarity. For 3b in panel A, the clusters are as follows: cluster 1, ChemScore 38.86 and ΔG_bind −61.74 kcal/mol; cluster 2, ChemScore 31.79 and ΔG_bind −53.29 kcal/mol; cluster 3, ChemScore 27.11 and ΔG_bind −40.73 kcal/mol). The pictures were generated using the PyMOL software.

Figure 4. Docked pose of 3i (sticks) into VIM-2 binding site (A) and NDM-1 (B) as found by HTD protocol. The contacts established by 3i into the active sites of the enzymes are represented by dotted lines. Metal ions were represented by spheres. The numbering of NDM-1 is referred to deposited protein sequence (Uniprot KB code F6IAY7). Nonpolar hydrogens were omitted for the sake of clarity. The pictures were generated by PyMOL (The PyMOL Molecular Graphics System, v1.6-alpha; Schrodinger LLC, New York, 2013).

Scheme 1. Synthetic procedure for the preparation of final compounds 3a–l, 4a–c, and 5. Reagents and conditions: (a) EtOH, reflux, 2–16 h; (b) HBF₄, MeOH, 25 °C, 2 min; (c) Et₃SiH, TFA, 25 °C, 4–6 h; (d) DCC, HOBt, dry DMF, 25 °C, 12 h.

Table 3. Synergistic antibacterial activity (combo test agar disk diffusion assay) of selected compounds with cefoxitin on VIM-2-producing E. coli AS19 strain.

Compound	Diameter of growth inhibition (mm)
–	19
100% DMSO	19
2a	20
3e	23
3f	19
3g	21
3i	24
3j	23