Green and Facile Assembly of Diverse Fused N-Heterocycles Using Gold-Catalyzed Cascade Reactions in Water

The present study describes an AuPPh3Cl/AgSbF6-catalyzed cascade reaction between amine nucleophiles and alkynoic acids in water. This process proceeds in high step economy with water as the sole coproduct, and leads to the generation of two rings, together with the formation of three new bonds in a single operation. This green cascade process exhibits valuable features such as low catalyst loading, good to excellent yields, high efficiency in bond formation, excellent selectivity, great tolerance of functional groups, and extraordinarily broad substrate scope. In addition, this is the first example of the generation of an indole/thiophene/pyrrole/pyridine/naphthalene/benzene-fused N-heterocycle library through gold catalysis in water from readily available materials. Notably, the discovery of antibacterial molecules from this library demonstrates its high quality and potential for the identification of active pharmaceutical ingredients.

Figure S2 13 C NMR spectrum of SF5a in methanol-d 4 .

Antibacterial bioassay
Bacterial strains, culture and growth conditions, and sample preparation Staphylococcus aureus (S. aureus) was used in this study and cultured at 37 o C in Mueller-Hinton broth (MH broth). 5 mg compounds were dissolved in 100 μl DMSO, and the resulting solution was used as the sample stock. All the experiments were repeated at least three times.

Preliminary screening of antibacterial activities
The preliminary antibacterial activities against S. aureus strain were investigated in 96-well plates, and DMSO was used as the blank control. Briefly, S. aureus strain was seeded into 200 μl MH broth per well to make a density of 1×10 5 CFU/ml. Subsequently, an aliquot of the sample stock was added to make a final compound concentration of 100 μg/ml. After that, the optical density (OD) of the mixture in each well at 600 nm wavelength was immediately measured by a spectrometer, and recorded as OD 0 . Then the plate was incubated at 37 o C for 24 h, after that, the OD of the mixture in each well was immediately measured again and recorded as OD 24 . ∆OD (∆OD = OD 24 − OD 0 ) was calculated and used to evaluate the antibacterial potency of the compounds. Finally, compounds with ∆OD lower than 0.1 were selected out for further study.

Minimal inhibitory concentration (MIC) study
The determination of minimal inhibitory concentration (MIC) of tested compounds was carried out in 96-well plates with DMSO as the blank control. Briefly, S. aureus strain was seeded into 200 μl MH broth per well to make a density of 1×10 5 CFU/ml. Subsequently, an aliquot of the sample stock was added to make 5 final compound concentrations (5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml). After that, the OD of the mixture in each well at 600 nm wavelength was immediately measured by a spectrometer, and recorded as OD 0 . Then the plates were incubated at 37 o C for 24 h, after that, the OD of the mixture in each well was immediately measured again and recorded as OD 24 . ∆OD (∆OD = OD 24 − OD 0 ) was calculated and used to determine the MIC 90 . MIC 90 was determined as the lowest concentration that inhibited 90% bacteria growth as compared with DMSO control group.

Time-Kill Assays
Time-kill assays were performed in 96-well plates, and DMSO was used as the blank control. Briefly, S. aureus strain was seeded into 200 μl MH broth per well to make a density of 1×10 5 CFU/ml. Subsequently, an aliquot of the sample stock was added to make 5 final compound concentrations (5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml). After that, the OD of the mixture in each well at 600 nm wavelength was instantly measured by a spectrometer, and recorded as OD 0 . Then the plate was incubated at 37 o C for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h, after that, the OD of the mixture in each well was immediately measured again and recorded as OD t . ∆OD (∆OD = OD t − OD 0 ) was calculated and used to draw the time-kill curves.

Colony-forming units (CFU) study
At the end of the time-kill assays, the plates were used for CFU study. Concisely, parallel wells were randomly selected and diluted by 10 5 times with MH broth. Then 100 μl diluent was taken and spread on Mueller-Hinton agar. The agar plates were incubated at 37 o C for 24 h. After that, the agar plates were recorded.
Statistical analysis: Statistical calculations were processed with Origin Pro 7.5 and Excel 2016.

Antibacterial results and discussion
Preliminary screening results Preliminary screening disclosed that 21 compounds from the library showed antibacterial activities against the growth of S. aureus strain at the concentration of 100 μg/ml ( Figure S13), and five of them (compounds SF9d, SF29b, SF33, SF36 and SF41) showed good antibacterial activities, which were selected for further study.

Figure S13
Preliminary screening of antibacterial activities of compounds at 100 μg/ml.

Time-kill assays and colony-forming unit (CFU) studies of compounds SF9d, SF29b, SF33, SF36 and SF41
As shown in Figure S14-S24, time-kill assays and colony-forming units (CFU) studies were also conducted with compounds SF9d, SF29b, SF33, SF36 and SF41. Among them, compound SF36 displayed the most potent antibacterial activity against S. aureus strain. Time-kill assay showed that SF36 was bactericidal within 2-24 h at the concentration of 25 μg/ml, preventing bacterial growth of S. aureus strain completely ( Figure 17). Colony-forming units (CFU) study of SF36 was also carried out ( Figure 23). The results showed that the number of clones on the agar plate decreased significantly in a dose-dependent manner, and only few clone was observed at the concentration of 50 μg/ml, indicating the antibacterial potency of this compound intuitively.