Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria

Abstract

Nα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide [MMV688845, Pathogen Box; Medicines for Malaria Venture; IUPAC: (2R)-N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide)] is a hit compound, which shows activity against Mycobacterium abscessus (MIC₉₀ 6.25-12.5 μM) and other mycobacteria. This work describes derivatization of MMV688845 by introducing a thiomorpholine moiety and the preparation of the corresponding sulfones and sulfoxides. The molecular structures of three analogs are confirmed by X-ray crystallography. Conservation of the essential R configuration during synthesis is proven by chiral HPLC for an exemplary compound. All analogs were characterized in a MIC assay against M. abscessus, Mycobacterium intracellulare, Mycobacterium smegmatis, and Mycobacterium tuberculosis. The sulfone derivatives exhibit lower MIC₉₀ values (M. abscessus: 0.78 μM), and the sulfoxides show higher aqueous solubility than the hit compound. The most potent derivatives possess bactericidal activity (99% inactivation of M. abscessus at 12.5 μM), while they are not cytotoxic against mammalian cell lines.

Figure 1.

Docking poses of MMV688845 (A), compound 20 (B), compound 14 (C) and the β’ subunit of RNAP_Mtb, and compound 39 after exchange of phenylalanine for tyrosine (D). Hydrogen bonds are displayed as yellow dashed lines. 2D visualizations of the shown interactions can be found in the Supporting Information (Figure S2). Visualization generated with Maestro graphical interface (Schrödinger Release 2022-3: Maestro, Schrödinger, LLC, New York, NY, 2021).

Figure 2.

Molecular structures of 6 (A), 20 (B) and 14 (C), as determined by X-ray crystallography. Displacement ellipsoids are drawn at the 50 % probability level. Hydrogen atoms on nitrogen and the chirality center, are represented by small spheres of arbitrary radius otherwise omitted for clarity. Dashed lines present N─H⋯O hydrogen bonds. Minor positional disorder of thiophene rings (ca. 6 %) in 6 (A) and 20 (B) is also not shown for the sake of clarity. Solvent water in the crystal structure of 14 (C)· 1.5 H₂O is not shown. The structure of 14 (C) was refined with aspherical atomic scattering factors using NoSpherA2^,.

Figure 2.

Molecular structures of 6 (A), 20 (B) and 14 (C), as determined by X-ray crystallography. Displacement ellipsoids are drawn at the 50 % probability level. Hydrogen atoms on nitrogen and the chirality center, are represented by small spheres of arbitrary radius otherwise omitted for clarity. Dashed lines present N─H⋯O hydrogen bonds. Minor positional disorder of thiophene rings (ca. 6 %) in 6 (A) and 20 (B) is also not shown for the sake of clarity. Solvent water in the crystal structure of 14 (C)· 1.5 H₂O is not shown. The structure of 14 (C) was refined with aspherical atomic scattering factors using NoSpherA2^,.

Figure 3.

Comparison of MIC₉₀ values of compounds that only differ in R¹. Compounds that only differ in R¹ are shown in the same color. Displayed values were generated by RFP measurement. Only Mtb MIC₉₀ values were generated by OD measurement. Compounds that only differ in R¹are displayed in the same color; the symbols represent the respective R¹ substituent.

Figure 4.

Comparison of MIC₉₀ values of compounds that only differ in R². Compounds that only differ in R² are shown in the same color. Displayed values were generated by RFP measurement. Only Mtb MIC₉₀ values were generated by OD measurement. Compounds that only differ in R²are displayed in the same color; the symbols represent the respective R² substituent.

Figure 5.

Comparison of MIC₉₀ values of compounds that only differ in R³. Compounds that only differ in R³ are shown in the same color. Displayed values were generated by RFP measurement. Only Mtb values were generated by OD measurement. Compounds that only differ in R³are displayed in the same color; the symbols represent the respective R³ substituent. Compounds 32 and 34 were excluded in this figure.

Figure 6.

Comparison of MIC₉₀ values of compounds that only differ in R⁴. Compounds that only differ in R⁴ are shown in the same color. Displayed values were generated by RFP measurement. Only Mtb values were generated by OD measurement. Compounds that only differ in R⁴ are displayed in the same color; the symbols represent the respective R⁴ substituent.

Figure 7.

MBC determination against Mabs ATCC19977 of a selection of AAP derivatives. Bacteria reduction levels are displayed as dashed, horizontal lines. Substances were analysed in triplicates as indicated by the white dots.

Figure 8.

Plasma stability of MMV688845 and compound 24 in human and murine plasma over five different time points. Substances were analysed in duplicates. Error bars display standard deviations.

Figure 9.

A: Relationship of cLogP values and their corresponding kinetic solubilities of all the synthesized AAP analogs. B: Relationship of cLogP values and their corresponding MIC₉₀ values against Mabs of all the synthesized AAP analogs. C: Relationship of cLogP values and their corresponding MIC₉₀ values against Mabs within the macrophage infection model of all the synthesized AAP analogs. The cLogP values were calculated with ChemDraw (Perkin Elmer Informatics Inc.).

Scheme 1.

Structures of the hit compound MMV688845 and D-AAP1

Scheme 2.

Synthetic pathway for derivatization of MMV688845 analogs (T3P: propanephosphonic acid anhydride, TFA: trifluoroacetic acid, DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one, DIPEA: N,N-diisopropylethylamine)

Scheme 3.

Preparation of the aniline building block (BINAP: 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; TFA: trifluoroacetic acid)

Scheme 4.

Synthesis of thiomorpholine sulfoxides and sulfones (m-CPBA: meta-chloroperoxybenzoic acid)

Scheme 5.

AAP base scaffold and derivatized residues

Scheme 6.

Overview of the influence of the different substituents on the antimycobacterial activity of AAPs. Red arrows indicate a loss in activity, green arrows indicate an increased activity caused by the respective residues.