Structure-activity relationships in human toll-like receptor 7-active imidazoquinoline analogues

Abstract

Engagement of toll-like receptors serve to link innate immune responses with adaptive immunity and can be exploited as powerful vaccine adjuvants for eliciting both primary and anamnestic immune responses. TLR7 agonists are highly immunostimulatory without inducing dominant proinflammatory cytokine responses. A structure-activity study was conducted on the TLR7-agonistic imidazoquinolines, starting with 1-(4-amino-2-((ethylamino)methyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol as a lead. Modifications of the secondary amine of the C2 ethylaminomethylene side chain are poorly tolerated. The 4-amino group must be retained for activity. Replacement of the imidazole ring of the scaffold with triazole or cyclic urea led to complete loss of activity. A systematic exploration of N(1)-benzyl-C2-alkyl substituents showed a very distinct relationship between alkyl length and TLR7-agonistic potency with the optimal compound bearing a C2-n-butyl group. Transposition of the N(1) and C2 substituents led to the identification of an extremely active TLR7-agonistic compound with an EC(50) value of 8.6 nM. The relative potencies in human TLR7-based primary reporter gene assays were paralleled by interferon-alpha induction activities in whole human blood models.

Figure 1

Structures of Imiquimod and 2.

Figure 2

Alkyl chain length dependence of TLR7-agonistic activity profiles of the N¹-benzyl-C2-alkyl compounds (right panel). The activity of 2 (reference compound) is shown on the left. Error bars represent standard deviations obtained on quadruplicate samples.

Figure 3

Dose-dependent induction of IFN-α in whole human blood by 2, 31, and 34. Plasma IFN-α was assayed in triplicate by ELISA.

Figure 4

TLR7-agonistic activities of active compounds in a human TLR7-specific reporter gene assay. Error bars represent standard deviations obtained on quadruplicate samples.

Figure 5

Correlation of TLR7-agonistic potency and hydrophobicity (measured as retention time on C₁₈ reverse-phase chromatography).

Scheme 1

Syntheses of 2-imidazolyl sidechain-modified analogues of 2.

Scheme 2

Syntheses of 2-imidazolyl sidechain-modified analogues of 2.

Scheme 3

Syntheses of 1-benzyl-2-(alkyl)-1H-imidazo[4,5-c]quinolin-4-amines.

Scheme 4

Syntheses of 4-substituted-1-benzyl-2-(alkyl)-1H-imidazo[4,5-c]quinolines.

Scheme 5

Syntheses of C-2/N-1 substituent-swapped 31/2 hybrids.

Scheme 6

Syntheses of imidazole-modified analogues.