Structure-activity relationships in nucleotide oligomerization domain 1 (Nod1) agonistic γ-glutamyldiaminopimelic acid derivatives

Abstract

N-acyl-γ-glutamyldiaminopimelic acid is a prototype ligand for Nod1. We report a detailed SAR of C(12)-γ-D-Glu-DAP. Analogues with glutaric or γ-aminobutyric acid replacing the glutamic acid show greatly attenuated Nod1-agonistic activity. Substitution of the meso-diaminopimelic (DAP) acid component with monoaminopimelic acid, L- or D-lysine, or cadaverine also results in reduced activity. The free amine on DAP is crucial. However, the N-acyl group on the D-glutamyl residue can be substituted with N-alkyl groups with full preservation of activity. The free carboxylates on the DAP and Glu components can also be esterified, resulting in more lipophilic but active analogues. Transcriptomal profiling showed a dominant up-regulation of IL-19, IL-20, IL-22, and IL-24, which may explain the pronounced Th2-polarizing activity of these compounds and also implicate cell signaling mediated by TREM-1. These results may explain the hitherto unknown mechanism of synergy between Nod1 and TLR agonists and are likely to be useful in designing vaccine adjuvants.

Figure 1

A. NF-κB induction activity in stable transfectants of HEK-293 cells expressing human Nod1. B. Rank-order plot of potencies (EC₅₀ values) of iE-DAP analogues.

Figure 2

Dose-responses of p38 MAP kinase (A) and CD11b (B) induction in the granulocytic population in whole human blood by 16 and 27 determined by flow cytometry.

Figure 3

Pathway analysis of transcriptomal activation patterns in human PBMCs exposed to 20 μg/mL of 16 and 27. The dashed line corresponds to the threshold of −log (base 10) value for P = 0.05 (after Benjamini-Hochberg multiple testing correction).

Scheme 1

Reagents: i. ROH, HBTU, TEA, DMAP, DMF; ii. (a) CF₃COOH (b) FmocCl, Na₂CO₃, Dioxane, H₂O; iii. (a) (Boc)₂O, TEA, H₂O (b) R′OH, HBTU, DMAP, TEA, DMF; iv. (a) HCl-dioxane (b) (Boc)₂O (1eq.), TEA, MeOH; v. PS-Carbodiimide, PS-DMAP, CH₂Cl₂.

Scheme 2

Reagents: i. 30% Piperidine, CH₂Cl₂; ii. C₁₁H₂₃COCl, pyridine, DMAP; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. (a) CF₃COOH (b) C₁₁H₂₃COCl, TEA, CH₂Cl₂; v. H₂, Pd(OH)₂/C, AcOH, MeOH, 60 psi; vi. CF₃COOH; vii. (a) C₁₁H₂₃COCl, pyridine, CH₂Cl₂ (b) H₂, Pd(OH)₂/C, MeOH, 60 psi; viii. C₁₁H₂₃CHO (1eq.), MP-CNBH₃, CH₂Cl₂, MeOH, AcOH; ix. RCHO(excess), MP-CNBH₃, CH₂Cl₂, MeOH, AcOH; x. 1H-Pyrazole-1-carboxamidine.HCl, pyridine, MW irradiation, 60°C; xi. H₂, Pd(OH)₂/C, MeOH, 60 psi; xii. N,N′-Di-Boc-1HPyrazole- 1-carboxamidine•HCl, pyridine, THF, 50°C.

Scheme 3

Reagents: i. (a) 30% Piperidine, CH₂Cl₂ (b) (Boc)₂O, TEA, MeOH; ii. (a) Chlorotris (triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. (a) Chlorotris(triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) C₁₁H₂₃NH₂, HBTU, TEA, DMAP, CH₂Cl₂; v. (a) Chlorotris (triphenylphosphine) rhodium(I), EtOH, H₂O, reflux (b) ROH, HBTU, DMAP, DMF; vi. (a) 30% Piperidine, CH₂Cl₂ (b) C₁₁H₂₃COCl, pyridine, DMAP; vii. (a) 30% Piperidine, CH₂Cl₂ (b) 1H-Pyrazole-1- carboxamidine•HCl, pyridine, MW irradiation, 60°C.

Scheme 4

Reagents: i. 30% Piperidine, CH₂Cl₂; ii. (a) (Boc)₂O, TEA, MeOH (b) H₂, Pd(OH)₂/C, MeOH, 60 psi (c) C₁₁H₂₃SH, HBTU, DMAP, TEA, DMF; iii. CF₃COOH; iv. C₁₁H₂₃COCl, pyridine; v. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH.

Scheme 5

Reagents: i. Fmoc-GABA-OH, PS-Carbodiimide, PS-DMAP, CH₂Cl₂; ii. (a) 30% Piperidine, CH₂Cl₂ (b) C₁₁H₂₃COCl, pyridine; iii. (a) H₂, Pd(OH)₂/C, MeOH, 60 psi (b) CF₃COOH; iv. Glutaric anhydride, CH₂Cl₂; v. C₁₁H₂₃OH, HBTU, DMAP, TEA, DMF.

Scheme 6

Reagents: i. BnOH, p-TSA, reflux; ii. (a) BnOH, HBTU, DMAP, DMF (b) 30% Piperidine, CH₂Cl₂; iii. (a) PhCH₂OCOCl, Na₂CO₃, Dioxane, H₂O (b) CF₃COOH; iv. (a) (Boc)₂O, TEA, MeOH (b) H₂, Pd(OH)₂/C, MeOH, 60 psi.

Scheme 7

Reagents: i. PS-Carbodiimide, PS-DMAP, TEA, CH₂Cl₂; ii. 30% Piperidine, CH₂Cl₂; iii. C₁₁H₂₃COCl, pyridine; iv. H₂, Pd(OH)₂/C, MeOH, 60 psi; v. CF₃COOH.