RhII‐Catalyzed De‐symmetrization of Ethane‐1,2‐dithiol and Propane‐1,3‐dithiol Yields Metallo‐β‐lactamase Inhibitors

Abstract Diversity‐oriented synthesis (DOS) is a rich source for novel lead structures in Medicinal Chemistry. In this study, we present a DOS‐compatible method for synthesis of compounds bearing a free thiol moiety. The procedure relies on Rh(II)‐catalyzed coupling of dithiols to diazo building blocks. The synthetized library was probed against metallo‐β‐lactamases (MBLs) NDM‐1 and VIM‐1. Biochemical and biological evaluation led to identification of novel potent MBL inhibitors with antibiotic adjuvant activity.


Introduction
Multiresistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens are a major global threat for human health. Among other resistance factors, β-lactamases are most prevalent and effectively protect the bacteria against the different kind of β-lactam antibiotics, including last resort penems and cephalosporins. [1] The βlactamase-mediated mechanism of β-lactam hydrolysis relies either on nucleophilic serine residue (in serine β-lactamases, SBLs) or metal ions (in metallo-β-lactamases, MBLs) in the active site of the enzyme. Although in general a β-lactamase inhibitor does not exhibit antimicrobial activity itself, it prevents the rapid degradation of β-lactam antibiotics and thereby acts as antibiotic adjuvant. [2] While SBL inhibitors are widely established, agents inhibiting MBLs or both type of βlactamases are still under clinical evaluation. [3] Fast evolution of β-lactamases, caused by high selection pressure make the search for new inhibitors highly urgent. One of the possible MBL inhibition mechanism involves inhibitors possessing a thiol moiety. Thiols bind tightly to the Zn 2 + ions in the MBL active site. [4] Although a great variety of thiol-based inhibitors have been developed which reached very significant binding potency in vitro, [5] none of them reached clinical evaluation yet. Previously we showed that approved drugs exhibiting free thiol moieties potently inhibit different MBLs in vitro. [6] Our efforts to follow the SOSA (selective optimization of side activities) approach [7] to optimize the approved drugs thiorphan and captopril towards efficient MBL inhibitors revealed numerous challenges in the development of thiol-based MBL inhibitors. [8] Recently, we discovered an efficient Rh(II)-catalyzed SÀ H insertion reaction of α-diazo-γ-butyrolactams with a variety of aromatic and aliphatic thiols. [9] Interestingly, the same reaction with ethane-1,2-dithiol led to the formation of the monoinsertion product in good chemical yield. To the best of our knowledge, the latter reaction represents the first example to a general approach to de-symmetrization of symmetrical aliphatic dithiols 2 using chemistry of diazo compounds 1 (Figure 1). In this article, we describe an application of this novel synthetic approach towards the linking of a thiol moiety to a range of chemically diverse aliphatic scaffolds which led to identification of potent MBL inhibitors among resulting alkylthio-substituted thiols 3 ( Figure 1).

Synthesis of alkylthio-substituted aliphatic thiols 3
The arsenal of diazo compounds 1 a-i selected for this study has been reported previously as prepared via the recently developed 'sulfonyl-azide-free' (SAFE) protocol (vide infra). [10][11][12] α-Diazo-γ-butyrolactams 1 j-n were prepared via the Danheiser diazo transfer protocol using 4-nitrobenzene sulfonyl azide as diazo transfer reagent. [13,14] It should be noted that all aforementioned diazo compounds are stable to storage except for 1 j which undergoes a rapid dimerization [14] and had, therefore, been used immediately [13] in the subsequent Rh(II)-catalyzed SÀ H insertion reaction without isolation ( Figure 2).
In addition to known α-diazocarbonyl compounds 1 a-n, we synthesized a small set of α-diazo acetamides 1 o-r. Amines 4 reacted with 2,2,6-trimethyl-4H-1,3-dioxin-4-one (5) in refluxing xylene which led to ring opening towards α-acetyl acetamides 6. The latter, after change of the solvent to acetonitrile, were subjected to the SAFE diazo transfer protocol. [12] After the diazo transfer was complete, brief reaction workup and removal of the acetyl group by treatment with KOH solution in aqueous acetonitrile led to the formation of α-diazo acetamides 1 o-r. The latter were isolated by chromatography in modest yields as calculated over 3 chemical steps (Scheme 1).
With the arsenal of 18 structurally diverse α-diazocarbonyl compounds 1 a-r in hand, we proceeded with coupling the respective Rh(II) carbenes to either ethane-1,2-dithiol or propane-1,3-dithiol, or both, which led to the expected desymmetrization of the latter and the formation of alkylthiosubstituted thiols 3 a-x in modest to good yields (Scheme 2).

Biochemical evaluation
In order to investigate the structure-activity relationships of the dithiol library, we measured the inhibitory activity of 3 a-x in a fluorescence-based enzyme activity assay (Table 1). Two relevant β-lactamase isoforms, NDM-1 and VIM-1 were selected for in vitro assays. The fluorogenic substrate fluorocillin [15] was prepared according to literature and its conversion was monitored as described previously. [6] In general, a linear relationship between the potency of 3 a-x towards both enzymes could be observed, however, inhibitory potency towards VIM-1 was almost tenfold higher ( Figure 3). A very clear preference for the propane-1,3-dithiol over ethane-1,2-dithiol compounds could be observed from matched molecular pairs. The most potent compound with a balanced inhibitory activity towards both enzymes was the N-phenyl-γ-lactam derivative  In order to rationalize the structure-activity relationships of the prepared library, molecular docking experiments with the most potent derivative 3 o and the most simple analogue 3 a were conducted. Therefore, structures of both possible enantiomers of 3 o and 3 a were docked into the X-ray structure of NDM-1 (PDB code 4EXS [16] ) in complex with a thiol-containing inhibitor L-captoptril. The obtained docking mode of 3 a revealed that the free thiol group, which was assumed to be negatively charged, is located between the Zn 2 + ions in the catalytic center. It thereby displaces the polarized water responsible for β-lactam hydrolysis. Furthermore, the thioether moiety forms a directed hydrogen bond towards backbone NH of Asn220. The carbonyl oxygen of the cyclopentanone moiety forms a hydrogen bond towards the side chain of Lys211. Both interactions towards Asn220 and Lys211 are described to be important for recognition of the carboxylate moiety of βlactam. [17] The binding mode explains the preference of the propane-1,3-dithiol over ethane-1,2-dithiol derivatives, due to the optimal distance of three carbons between the thiol and the thioether groups. (Figure 4) The docking of the most potent derivative 3 o reveals the same preferred interactions as the simplified analogue 3 a.
( Figure 3B) The N-phenyl ring reaches out to a flat subpocket which is only partially lipophilic and open to solvent. Due to its open nature, a certain variability can be assumed in this area. This hypothesis fits to the observation that various moieties fit this subpocket without significant loss in activity e. g. N-phenyl . Notably, only (S)-enantiomers of 3 a and 3 o were able to display these favourable binding modes while (R)-enantiomers were unable to form all directed interactions in a low-energy conformation. This observation paves the way for future investigations of the enantioselective synthesis route and subsequent biochemical evaluation of the enantiomers.   For further biological evaluation we concentrated on the compounds with the balanced potency towards both, NDM-1 and VIM-1, 3 o and 3 s. Furthermore, the minimalistic derivative 3 a was used for comparison to ensure that the aromatic derivative does not change the mode of action of the compound. Some classes of MBL inhibitors do not directly bind to the active site but act as zinc chelators and withdraw catalytically essential zinc ions. [18] We verified the direct inhibitory mode of action by adding 100 μM ZnCl 2 to the recombinant MBL in vitro assay. As Figure 5 shows, addition of zinc ions does not impair the inhibitory activity of 3 a, 3 o, and 3 s, suggesting direct inhibition and binding to the active site.
The next step was the investigation of the binding thermodynamics of 3 a, 3 o, and 3 s. For these experiments we used a closely related enzyme VIM-2 which is an isoform of VIM-1 and can be recombinantly expressed in very high concentrations, suitable tor isothermal titration calorimetry (ITC) experiments ( Figure 6). ITC titration of 250 μM of 3 a, 3 o, or 3 s, respectively, into 50 μM of VIM-2 revealed potent entropydriven binding of all three compounds, with 3a being the weakest (K d = 0.88 μM). Notably, binding of 3 o displays almost double enthalpy ΔH = À 49 kJ/mol compared to 3 a and 3 s (ΔH = À 22 kJ/mol and À 27 kJ/mol).
The considerable inhibition of purified NDM-1 and VIM-1 in vitro suggested that compound 3 a, 3 o, or 3 s, which themselves exhibit no intrinsic antimicrobial activity (Table 2), can potentially restore the activity of imipenem against bacterial isolates producing MBL.
To investigate this, the MIC of imipenem or combined with various concentrations of compound 3a, 3o, or 3 s was determined against E. coli transformants producing NDM-1 or VIM-1 (Table 3 and 4). Especially compound 3o and 3 s substantially reduced the MIC of imipenem against NDM-1 or VIM-1 producing bacteria up to 8-fold and 32-fold, respectively.

Conclusion
In this study we could show that Rh(II)-catalyzed introduction of dithiols is a highly useful method for diversity-oriented synthesis of chemical libraries which are intended to contain free sulfhydryl groups. We prepared a diverse library of thiol-based inhibitors of MBLs and evaluated them in vitro. Biochemical and biological evaluation of the prepared library showed that potent MBL inhibitors with antibiotic adjuvant activity could be generated.

Experimental Section
Chemical synthesis

General methods
Known diazocarbonyl compounds 1 a-n were prepared according to literature procedures, [10][11][12][13][14] other reagents were obtained from commercial sources and used without any additional purification. Solvents were distilled over suitable drying agents. Mass spectra were recorded with a Bruker Maxis HRMS-ESI-qTOF spectrometer (electrospray ionization mode). NMR spectroscopic data were recorded with Bruker Avance 400 spectrometer (400.13 MHz for 1 H and 100.61 MHz for 13 C) in CDCl 3 and were referenced to residual solvent proton peaks (δ H = 7.28) and solvent carbon peaks (δ C = 77.0). Melting points were determined with a Stuart SMP50 instrument in open capillary tubes.

Preparation of α-diazo acetamides 1 o-r
A mixture of appropriate amine 4 (1 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (5, 1 mmol) in o-xylene (6 mL) was heated at reflux for 1 h and the solvent was removed under reduced pressure. The residue was dissolved in acetonitrile (8 mL) and a mixture of 3-(chlorosulfonyl)benzoic acid (292 mg, 1.34 mmol), sodium azide (98 mg, 1.5 mmol) and potassium carbonate (276 mg, 2 mmol) in water (8 mL), pre-stirred over 30 min, was added. The resulting emulsion was stirred for 2 h at room temperature whereupon the diazo transfer was complete. The reaction mixture was extracted with chloroform (2 × 10 mL). The chloroform solution was separated, dried over anhydrous Na 2 SO 4 , filtered and concentrated to dryness. The residue was dissolved in acetonitrile (20 mL) and was treated with a solution of KOH (140 mg, 5 mmol) in water (4 mL). The resulting mixture was stirred for 3 h at room temperature and extracted with chloroform (2 × 10 mL). The organic phase was dried over anhydrous Na 2 SO 4 , filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography using ethyl acetate-n-hexane 1 : 4 as eluent.

General procedure for the preparation of compounds 3 a-x
To a vigorously stirred solution of diazo compound 1 (0.5 mmol) and symmetrical dithiol (5 mmol) in dichloromethane (10 mL) an appropriate rhodium(II) catalyst (0.005 mmol of Rh 2 (OAc) 4 for lactams 3 m-t and 0.0025 mmol of Rh 2 (esp) 2 in all other cases) was added. The reaction mixture was stirred at room temperature over 18 h. The volatiles were removed using a rotary evaporator and the desired product was isolated by a column chromatography on silica gel using ethyl acetate-n-hexane 1 : 4 as eluent.

3-((2-Mercaptoethyl)thio)-1-(3-(trifluoromethyl)phenyl) pyrrolidin-2-one (3 p)
concentration of 250 μM containing a final DMSO concentration of 1 %. The VIM-2 protein was diluted to a final concentration of 50 μM, supplemented with 1 % DMSO using the dialysis buffer and pure DMSO. For the control measurements dialysis buffer was supplemented with pure DMSO to a final concentration of 1 %. The measurements were performed using an "Affinity ITC" (TA-Instruments) in reversed mode, with a stir rate of 75 rpm and a temperature of 25°C. The VIM-2 was placed in the cell and the respective compound in the syringe. For blank measurements either the protein or compound dilution were exchanged with buffer containing 1 % DMSO. For the measurements of 3 a and 3 s one time 0.5 μl was injected followed by 50 injections of 2 μl with a spacing of 240 s. 3 o was measured with one injection of 0.5 μl followed by 35 injections of 2 μl with a spacing of 240 s. The data were analyzed using the "NanoAnalyze Data Analysis" software (version 3.10.0; TA-Instruments).

Minimal inhibitory concentration determination
Minimal inhibitory concentrations (MICs) of imipenem monohydrate (Sigma-Aldrich) � compound 3 a, 3 o, or 3 s against transformed E. coli strains producing NDM-1 or VIM-1 were determined according to microdilution method established by Clinical and Laboratory Standards Institute (CLSI). 20 The checkerboard assay was performed to test for synergy in vitro. The microtiter-plates were set up with serial doubling dilutions of compound 3 a, 3 o, or 3 s (2-128 mg/L) and imipenem (0.125-128 mg/L).

Molecular modeling
Docking was performed using MOE2019.0102 (Chemical Computing Group, Montreal, Canada). X-ray structure of NDM-1 (PDB code 4EXS 16 ) was downloaded from PDB and prepared using QuickPrep routine. Co-crystallized ligand captopril was selected to define the binding site. Induced-fit docking was employed to dock both enantiomers of 3 a and 3 o, respectively. For initial placement, template "CHCH 2 S À " was used, while rescoring of the obtained conformations was performed by GBVI/WSA dG scoring function to generate 5 low-energy docking poses, which were inspected manually. Poses with the highest score were used for generation of Figure 4.