Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila

The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.


Column chromatography
The separation and purification of the crude products was achieved with a column chromatographic SP1 systems form Biotage (Sweden) and prepacked columns. The used solvent mixtures of n-hexane and EtOAc, CHCl 3 or DCM and MeOH were tested previously by TLC. Different flow rates of 15 mL/min, 30 mL/min, and 30 mL/min, where used for different columns of SNAP KP-Sil ® 10 g, 25 g and 50 g, respectively. After separation, collected fractions were combined according to the curve of the UV-chromatogram, and after retention factors from the TLC, respectively.

Minimum inhibitory concentration (MIC) determination
Minimal inhibitory concentrations (MICs) of nematophin and its derivatives were medium supplemented with the different compounds at various concentrations ranging from 128 to 0.06 µg/mL were added to each well of a 96-well microtiter plate. 5 µL of a bacterial suspension were added to each well to yield a final concentration of about 5 × 10 5 cells/mL. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited growth of the organism in a microdilution well as detected by the unaided eye. For quality control and to monitor for accuracy the gentamicin MIC was verified to fall within the acceptable limits. .

General procedure for synthesis of nematophin and related derivatives
To a mixture of -keto carboxylic acid (1.5 equiv), EDC·HCl (1.5 equiv), HOBt (1.5 equiv) and DIPEA (2.0 equiv) in DMF (c = 0.1 M) was added the appropriate amine (1.0 equiv) and reaction was conducted under microwave irradiation at 75 °C, and 25 W for 20 min (Discover system, CEM Corporation/ USA). Afterwards saturated aqueous NaHCO 3 was added and extracted three times with EtOAc. The combined organic layers were dried over MgSO 4 , concentrated and purified by flash chromatography (Biotage/ Sweden, SNAP KP-Sil ® , 10 g, EtOAc in n-hexane or MeOH in DCM as eluent).

7-azatryptamine (19 and 26)
First two steps of the synthesis were performed slightly modified as described previously. 1

chloro-1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone (21)
Under an atmosphere of nitrogen, a suspension of azaindole (1.0 equiv) and AlCl 3 (5 equiv) was stirred in dry DCM (c = 0.2 M) for 1 h. Afterwards a solution of chloroacetyl chloride (5 eq.) in dry DCM (c = 2.5 M) was added slowly and the mixture was stirred at rt overnight. The solvent was removed and the residue was dissolved in water and basified carefully with saturated Na 2 CO 3 solution until pH 9 and extracted three times with EtOAc. The combined organic layers were dried over MgSO 4 and the solvent was removed under reduced pressure to give title compounds as slightly yellow solids.
iii 10 %  100 % EtOAc in n-hexane) to give title compounds as slightly yellow solids.