BACKGROUND

Efforts on the discovery of vaccine adjuvants have led to the examination of structure-activity relationships in a variety of innate immune stimuli, including small molecule agonists of Toll-like receptors (TLRs) such as TLR2 (Wu, W. et al.J. Med. Chem.2010, 53, 3198-3213; Agnihotri, G. et al.J. Med. Chem.2011, 54, 8148-8160; Salunke, D. B. et al.J. Med. Chem.2013; Salunke, D. B. et al.J. Med. Chem.2012, 55, 3353-3363 and Salyer, A. C. et al.PloS one2016, 11, e0149848), TLR7 (Nuhn, L. et al.Proceedings of the National Academy of Sciences of the United States of America2016, 113, 8098-8103; Ganapathi, L. et al.PloS one2015, 10, e0134640; Hood, J. D. et al.Human Vaccines2010, 6, 1-14; Shukla, N. M. et al.Bioorg. Med. Chem. Lett.2009, 19, 2211-2214; Shukla, N. M. et al.Bioorg. Med. Chem. Lett.2011, 21, 3232-3236; Shukla, N. M. et al.J. Med. Chem.2010, 53, 4450-4465; Shukla, N. M. et al.J. Med. Chem.2012, 55, 1106-1116; Shukla, N. M. et al.Bioorg. Med. Chem. Lett.2010, 20, 6384-6386; Shukla, N. M. et al.PloS one2012, 7, e43612; Yoo, E. et al.Organic&biomolecular chemistry2013, 11, 6526-6545 and Yoo, E. et al.J. Med. Chem.2014, 57, 7955-7970), TLR8 (Yoo, E. et al.J. Med. Chem.2014, 57, 7955-7970; Kokatla, H. P. et al.Organic&biomolecular chemistry2013, 11, 1179-1198; Salunke, D. B.; Yoo, E. et al.J. Med. Chem.2012, 55, 8137-8151; Kokatla, H. P. et al.J. Med. Chem.2013, 56, 6871-6885; Kokatla, H. P. et al.Chem Med Chem2014, 9, 719-723; Beesu, M. et al.J. Med. Chem.2016, 59, 8082-8093; Beesu, M. et al.J. Med. Chem.2014, 57, 7325-7341; Beesu, M. et al.J. Med. Chem.2015, 58, 7833-7849 and Beesu, M. et al.J. Med. Chem.2016, 59, 3311-3330), NOD1 (Agnihotri, G. et al.J. Med. Chem.2011, 54, 1490-1510), as well as C—C chemokine receptor type 1 (CCR1) (Ukani, R. et al.Bioorg. Med. Chem. Lett.2012, 22, 293-295). Crystal structures of several TLRs (Zhang, Z. et al.FEBS letters2017, 591, 3167-3181; Shimizu, T.Current opinion in structural biology2017, 47, 52-59 and Botos, I. et al.Structure(London, England: 1993) 2011, 19, 447-459) confirm a common structural motif in the TLRs: these are type I integral membrane receptors containing an N-terminal extracellular leucine-rich repeat (LRR) domain, a transmembrane domain, and a C-terminal cytoplasmic domain. Ligand recognition occurs via the LRR domain (Narayanan, K. B. et al.Apoptosis: an international journal on programmed cell death2015, 20, 196-209 and Leonard, J. N. et al.Methods in molecular biology(Clifton, N.J.) 2009, 517, 55-67), while the cytoplasmic domain known as the Toll/IL-1 receptor (TIR) region activates downstream signaling cascades by interacting with adaptor proteins such as MyD88, Mal, TRIF, and TRAM (Narayanan, K. B. et al.Apoptosis: an international journal on programmed cell death2015, 20, 196-209; Watters, T. M. et al.Immunology and cell biology2007, 85, 411-419; Miyake, K. et al.International immunology2018, 30, 43-51 and Brikos, C. et al.Handbook of experimental pharmacology2008, 21-50). The crystal structures also emphasize that ligand-induced dimerization, and the consequent apposition of the C-terminal regions of the two TLR protomers is the common mechanism of activation of TLRs (Zhang, Z. et al.FEBS letters2017, 591, 3167-3181 and Song, D. H. et al.Immunological reviews2012, 250, 216-229).

The ubiquitous presence of the 24-residue LRR motif in all of the TLRs (Botos, I et al.Structure(London, England: 1993) 2011, 19, 447-459 and Leonard, J. N. et al.Methods in molecular biology(Clifton, N.J.) 2009, 517, 55-67) suggests the possibility that small-molecule agonists may engage innate immune receptors such as TLR3 and TLR9 for which only canonical nucleic acid agonists are currently known. A high-throughput screen (HTS) utilized a multiplexed Poly-TLR/NLR readout (Salyer, A. C. et al.PloS one2016, 11, e0149848), to examine cytokine (TNF-α/IL-1β) and interferon (IFN-α/β and IFN-γ) induction in porcine whole blood.

Currently there is a need for exploring the biological effects of NOD2-active compounds as agonists of TLRs and NLRs, including identifying novel, non-canonical immunostimulatory small molecules for innate immune receptors.

SUMMARY

In one aspect the present invention provides compounds having NOD2-agonistic activity that are useful as vaccine adjuvant potentiators.

Accordingly the invention provides a compound of formula I:



or a salt thereof, wherein:

R1is selected from the group consisting of H, C1-C20alkyl, and C2-C20alkenyl, where;

R2is phenyl or a 5-6 membered ring heteroaryl, where phenyl or 5-6 membered ring heteroaryl is substituted with one or more groups independently selected from the group consisting of F, Cl, —CN, C1-C12alkyl, C2-C12alkenyl, —(C1-C6alkyl)-NRaRb, —C(═NH)NH(OH), —C(═NH)NH2, —C(═O)NRaRb, —C(═O)NRa—NRaRb, —C(═O)NH(C1-C6alkyl)-NRaRb, —C(═O)ORa, —NRaRb, —NO2, —ORa, —OC(═O)Ra, —SRa, —S(O)Ra, —S(O)2Ra, —S(O)2NRa, and —S(O)3H;

each R3is independently selected from the group consisting of F, Cl, —CN, —NRaRb, —NO2, —ORa, and —(C1-C6alkyl)-NRaRb;

A is CR4or N, where R4is selected from H, F, Cl, —CN, —NRaRb, —NO2, —ORa, and —(C1-C6alkyl)-NRaRb;

Raand Rbare independently selected from H, OH, phenyl, benzyl, 5-6 membered ring, and C1-C6alkyl, where phenyl, benzyl or 5-6 membered ring is optionally substituted with one or more groups independently selected from the group consisting of F, Cl, —CN, C1-C12alkyl, C2-C12alkenyl, —(C1-C6alkyl)-NRaRb, —C(═NH)NH(OH), —C(═NH)NH2, —C(═O)NRaRb, —C(═O)NRa—NRaRb, —C(═O)NH(C1-C6alkyl)-NRaRb, —C(═O)ORa, —NRaRb, —NO2, —ORa, —OC(═O)Ra, —SRa, —S(O)Ra, —S(O)2Ra, —S(O)2NRa, and —S(O)3H;

each alkyl and alkenyl are optionally and independently substituted with one or more groups independently selected from the group consisting of F, Cl, —CN, —NRaRb, —NO2, and —ORa, —OC(═O)Ra, —SRa, —S(O)Ra, —S(O)2Ra, —S(O)2NRa, and —S(O)3H; or

Raand Rbare taken together to form a 4-6 membered ring heterocycle with N;

n is 0, 1, 2, 3.

The invention also provides a pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

The invention also provides a compound of formula I, wherein stimulating the immune system in the animal comprises regulating the secretion of cytokines or chemokines in human blood, activating the nucleotide-binding oligomerization domain 2 (NOD2) receptor, or activating the Toll-like receptor (TLR7/8) receptor.

The invention also provides a method for stimulating immune activity in an animal comprising administering a compound of formula I, or a pharmaceutically acceptable salt thereof, to the animal.

The invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in medical therapy.

The invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for stimulating immune activity.

The invention also provides a compound of formula I, or a pharmaceutically acceptable salt thereof, used as vaccine adjuvant potentiators.

The invention also provides a use of a compound of formula I, or a pharmaceutically acceptable salt thereof, to prepare a medicament for stimulating immune activity in an animal.

The invention also provides processes and intermediates disclosed herein that are useful for preparing a compound of formula I or a salt thereof.

