Design, synthesis, and biological evaluation of tetrazole analogs of Cl-amidine as protein arginine deiminase inhibitors

Abstract

Protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs.

Figure 1

Protein arginine deiminases (PADs) convert arginine residues into citrulline.

Scheme 1. C-Terminal Bioisosteric Modification of Cl-amidine

Scheme 2. Synthesis of Tetrazole Analogs of Cl-amidine and F-amidine

Scheme 3. Synthesis of Biphenyl Tetrazole Analogs of Cl-amidine and F-amidine

Scheme 4. Synthesis of o-Carboxylate Containing Tetrazole Analogs of Cl-amidine and F-amidine

Figure 2

Selectivity of tetrazole haloacetamidines: (A) selectivity of tetrazole analogs of Cl-amidine; (B) selectivity of tetrazole analogs of F-amidine.

Figure 3

Cell efficacy studies. (A) Cell viability studies with the tetrazole analogs of Cl-amidine. U2OS cells were treated with compounds at final concentration of 20 μM, and cell viability was measured by the colorimetric XTT assay. Biphenyl tetrazole tert-butyl Cl-amidine (13) completely abolishes the growth of U2OS cells at 20 μM. (B) Cell viability studies with the tetrazole analogs of F-amidine. (C) EC₅₀ of biphenyl tetrazole tert-butyl compounds. Compounds 13 and 14 have lower EC₅₀ values compared to Cl-amidine in U2OS cells. (D) Stability of tetrazole analogs of Cl-amidine in mouse hepatic microsomes compared to Cl-amidine.

Figure 4

Biphenyl tetrazole tert-butyl Cl-amidine (13) and o-carboxyl tetrazole tert-butyl Cl-amidine (21) inhibit NET formation. The DNA/neutrophil elastase overlap assay suggests that compound 13 significantly reduces NET formation compared to Cl-amidine. Compound 21 also inhibits NET formation at higher doses: (∗) p < 0.05 and (∗∗) p < 0.01.