Toll-like receptor-8 agonistic activities in C2, C4, and C8 modified thiazolo[4,5-c]quinolines

Abstract

Toll-like receptor (TLR)-8 agonists typified by the 2-alkylthiazolo[4,5-c]quinolin-4-amine (CL075) chemotype are uniquely potent in activating adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds could be promising candidate vaccine adjuvants, especially for neonatal vaccines. Alkylthiazoloquinolines with methyl, ethyl, propyl and butyl groups at C2 displayed comparable TLR8-agonistic potencies; activity diminished precipitously in the C2-pentyl compound, and higher homologues were inactive. The C2-butyl compound was unique in possessing substantial TLR7-agonistic activity. Analogues with branched alkyl groups at C2 displayed poor tolerance of terminal steric bulk. Virtually all modifications at C8 led to abrogation of agonistic activity. Alkylation on the C4-amine was not tolerated, whereas N-acyl analogues with short acyl groups (other than acetyl) retained TLR8 agonistic activity, but were substantially less water-soluble. Immunization in rabbits with a model subunit antigen adjuvanted with the lead C2-butyl thiazoloquinoline showed enhancements of antigen-specific antibody titers.

Figure 1

TLR8 and TLR7 agonistic potencies of the C2-alkyl thiazoloquinoline homologues. Data points represent means and standard deviations of EC₅₀ values derived from dose-response profiles and are computed on quadruplicates.

Figure 2

TLR8- and TLR7-specific NF-κB induction profiles of branched-chain and trifluoromethyl analogues of 8c and 8d. Means and SD obtained from quadruplicate samples are shown.

Figure 3

EC₅₀ values of proinflammatory cytokine induction in human PBMCs by analogues of 8c and 8d. Representative data from three independent experiments are shown.

Figure 4

TLR8 induction by C4-amides of 8c (Panel A) and 8d (Panel B). Data points represent means and standard deviations on quadruplicates.

Figure 5

Dose-response profiles of proinflammatory cytokine induction in hPBMCs by C4-amides of 8c (Panel A) and 8d (Panel B). Representative data from three independent experiments are presented. Vehicle controls (not shown) elicited undetectable levels of cytokines.

Figure 6

Box-plots of anti-bovine α-lactalbumin IgG titers in cohorts of three rabbits immunized with α-lactalbumin adjuvanted with either 8c or 8d. Means and medians of titers are represented by □ and — symbols within the box, respectively, and the X symbols indicate the 1% and 99% percentile values.

Scheme 1

Syntheses of C2-alkylthiazoloquinoline analogues. Reagents: (i) HCl, HON=CHCH₂NO₂, H₂O; (ii) (CH₃CO)₂O, CH₃COOK; (iii) Pt/C, H₂, DMF; (iv) RCOCl, Et₃N, CH₂Cl₂: DMF(10:1); (v) P₂S₅, pyridine; (vi) m-CPBA, CHCl₃; (vii) (a) benzoyl isocyanate, CH₂Cl₂, (b) NaOCH₃, MeOH.

Scheme 2

Modification at the C8 position. Reagents: (i) HNO₃, H₂SO₄; (ii) Zn, NH₄COOH, MeOH; (iii) NaNO₂, CH₃COOH, NaN₃; (iv) alkyne, CuSO₄, sodium ascorbate, THF, H₂O (v) TBAF, THF.

Scheme 3

Synthesis of C8 N-alkyl and N-acyl analogues. Reagents: (i) For 13a, C₆H₁₃I, K₂CO₃, DMF; For 13b, C₃H₇COCl, Et₃N, CH₂Cl₂.

Scheme 4

Synthesis of 8-bromothiazoloquinoline. Reagents: (i) NBS, NH₄OAc, CH₃CN.

Scheme 5

Syntheses of C4 N-alkyl and N-acyl analogues. Reagents: (i) For 15a 15b and 15c, RX, NaH, THF; For 15d, 2,2,2-trifluoroethylformate, Et₃N; For 15e-i, RCOCl, Py; For 15j-l, RCO₂H, HBTU, Et₃N; For 15m-p, ROCOCl, Et₃N, CH₂Cl₂; For 15q, ClSO₂NCO, NaHCO₃; For 15r-v, RSO₂Cl, CH₂Cl₂; For 15w, ClP(O)OEt₂, CH₂Cl₂.

Scheme 6

Syntheses of C4 guanidino analog. Reagents: (i) POCl₃, 100 °C; (ii) guanidine hydrochloride, NaH, 1,4-dioxane.

Scheme 7

Syntheses of C4 N-alkyl and N-acyl analogues of 8d. Reagents: (i) 2,2,2-trifluoroethylformate, Et₃N; For 18a; RCOCl, pyridine for 18b and 18c.