WO2024224089A1

WO2024224089A1 - Desmuramylpeptide monoesters as nod2 agonists and use thereof

Info

Publication number: WO2024224089A1
Application number: PCT/GB2024/051100
Authority: WO
Inventors: David John Fox; David John Grainger; Jonathan Richard Heal; Nigel Ramsden; Joseph Michael Sheridan; James Michael TOMLINSON
Original assignee: Imhotex Ltd
Current assignee: Imhotex Ltd
Priority date: 2023-04-27
Filing date: 2024-04-26
Publication date: 2024-10-31
Anticipated expiration: 2025-10-27

Abstract

The invention relates to novel analogues of muramyl dipeptide (MDP), desmuramylpeptides (DMPs), for example in a pharmaceutically acceptable salt, pharmaceutical compositions comprising these compounds, and their medical use, in particular for use in the treatment of Crohn's disease.

Description

DESMURAMYLPEPTIDE MONOESTERS AS NOD2 AGONISTS AND USE THEREOF

The present invention relates to novel analogues of muramyl dipeptide (MDP), desmuramylpeptides (DMPs), and their medical uses.

Nucleotide-binding Oligomerisation Domain (NOD) protein NOD2 is a member of an extended family of inflammatory and immune proteins in animals (NOD families). These proteins combine a central nucleotide -binding domain (NOD) with a C-terminal leucine- rich repeat (LRR) motif and an N-terminal caspase recruitment domain (CARD) or equivalent (Ohto, U. Front Immunol. 2022, 13, 953530 ). NOD2 is believed to be mainly expressed in a person’s peripheral blood monocytes (Ogura, Y et al. J Biol Chem. 2001, 276(7), 4812-4818; Segal, A.W. J Intern Med. 2019, 286, 373-388).

Genetic studies of patients with Crohn’s disease have identified NOD2 as the gene most strongly associated with the disease (Jostins, L et al. Nature. 2012, 491(7422), 119-124). The association of mutations on NOD2 with a predisposition to Crohn’s disease was identified by a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, of a known susceptibility region on chromosome 16 in 77 multiplex families. Mutations in this gene remain the most strongly associated genetic risk factor for Crohn’s disease (Hugot, J.P. et al. Nature. 2004, 411, 599-603).

NOD2 is hypothesised to play a role in Crohn’s disease via its involvement in a person’s innate immune response. Studies on patients with Crohn’s disease have identified the failure of a patient’s acute inflammatory response as being a common predisposition to Crohn’s disease (Marks, D.J.B. et al. Lancet. 2006, 367, 668-678).

It has been proposed that Crohn’s disease develops in three distinct phases (Sewell, G.W. et al. Opin Immunol. 2009, 21(5), 506-513). Firstly, a gastrointestinal infection allows faecal bowel contents access to vulnerable tissues within the bowel. Secondly, failure of the acute inflammatory response to tissue damage in Crohn’s disease patients results in failure to recruit immune cells to the inflammatory site, resulting, amongst other things, in the clearance of bacteria from the tissues being defective. Thirdly, the retained faecal products result in the characteristic chronic granulomatous inflammation and adaptive immune response, giving rise to the symptoms of Crohn’s disease.

NOD2 is understood to play a role in the secretion of pro-inflammatory molecules such as inflammatory cytokines (Boyle, J.P. et al. Open Biol. 2014, 4(12), 140178). In general, NOD proteins are understood to recognise a signal from an invading organism in their LRR domain that induces a polymerisation that triggers a signalling cascade which terminates in the production and release of pro -inflammatory molecules. NOD2 is understood to be activated by muramyl dipeptide (MDP), a component of the cell wall of both Gram negative and Gram positive bacteria. The current theoretical models hypothesise that in its resting state NOD2 is doubled back on itself in an auto -inhibited conformation in the cytoplasm until activated by the binding of MDP to its LRR domain. This is thought to overcome NOD2’s autoinhibition, allowing self-oligomerisation leading to production of pro -inflammatory molecules (e.g. pro-inflammatory cytokines) (Maekawa, S. et al. Nat Commun. 2016, 7, 711813). It has also been hypothesized that, whilst acute stimulation of the NOD2 receptor leads to secretion of pro-inflammatory mediators from a variety of cell types, chronic stimulation of the NOD2 receptor leads to tolerization of the cells to the effects of subsequent stimulation of both the NOD2 receptor and other pattern recognition receptors. (Hedl, M. et al. PNAS. 2007, 104(49), 119440 and Lessard, A-J. et al. Cell Reports. 2017, 20, 1830). This tolerization has been proposed to be a mechanism that could contribute to the restoration of homeostasis in inflamed tissues, and the failure of achieving tolerization may lead to the chronic inflammation observed in Crohn’s Disease.

Current therapies for the treatment of Crohn’s disease are immunosuppressive (Cushing, K. et al. JAMA. 2021, 325(1), 69-80). These drugs and biological treatments dampen down the secondary granulomatous and adaptive immune response in patients. Anti- TNF’s can be helpful but do not provide a comprehensive treatment with only one third of patients in remission after one year on these treatments (Ding, N.S. Pharmacol Ther. 2016, 43, 30 - 51). Immunosuppressant treatment further compromises the underlying innate immune deficit to mucosal damage, thereby increasing the likelihood of further infection and the influx of bowel contents into the tissues, and its impaired clearance. Thus, additional suppression of an already impaired inflammatory response could further impair the clearance of faecal material from the bowel wall, increasing the frequency of secondary inflammations and converting Crohn’s disease from a sporadic to a more chronic condition (Segal, A.W. J Intern Med. 2019, 286, 373-388).

There is therefore a need for a Crohn’s disease treatment that may modulate a patient’s innate immunity by acting as a modulator of pro -inflammatory mediator secretion in a subject. The applicants have identified novel desmuramylpep tides (DMP) compounds that are potent and selective agonists of NOD2.

In general, analogues of muramyl dipeptide (MDP) and desmuramylpeptides (DMPs) have at least two stereogenic centres on a dipeptide backbone. Herein, we refer to the stereogenic centre of the glutamic acid residue, analogue or derivative thereof as the “right” stereogenic centre and the stereogenic centre of the other amino acid residue as the “left” stereogenic centre (see the dipeptide of Formula (1) below for context).

Griffin, M. E. et al. (ACS Chem. Biol. 2023, 18, 1368-1377) describe N -arylpyrazole dipeptides as NOD2 agonists for use in immune checkpoint inhibitor therapy.

Gobec, M. et al. (J. Med. Chem. 2018, 61, 2707-2724) and Guzelj, S et al. (ACS Med. Chem. Lett. 2022, 13, 8, 1270 - 1277) describe desmuramylpeptide compounds as NOD2 agonists with nanomolar potency. Gobec, M. et al. (Eur. J. Med. Chem. 2016, 116, 1-12) also describe desmuramylpeptide compounds as NOD2 agonists.

Jakopin, Z. et. al. (Int. J. Mol. Sci. 2019, 20, 4265) describe an in vitro tool for functional characterization of NODI / NOD2 antagonists.

Blakskjicr, P. et. al. (J. Chem. Soc., Perkin Trans. 1, 2001, 1, 910-916) describe a method

of synthesising peptides, such as wherein the stereochemistry of the right stereogenic centre is undefined, by C-alkylation or C- allylation using glycyl radical intermediates. US4666890A describe an intermediate compound,

used in the synthesis of peptides for enhancing protective efficacy in infection.

Khan, F-A. et. al. (Eur. J. Org. Chem. 2021, 48, 6688-6699) describe desmuramylpeptide analogues, such

which are recognised by NOD2.

US4362716A describes dipeptides, wherein the right stereogenic centre is defined (L- alanine) and the left stereogenic centre is defined (D-glutamic acid or derivatives thereof), that are able to stimulate immune reactions. There is no indication of NOD2 activity of these compounds.

Summary of the invention

An objection of the present invention is to provide compounds useful in treating disorders and diseases (e.g. Crohn’s disease) wherein the treatment is affected or facilitated by the compounds acting as a potent and selective NOD2 agonists.

In one aspect the present invention, there is provided a compound (a dipeptide) of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from CO₂H, CONA¹ A², CN, CH₂OH, CH₂NHSO₂A³,CONHSO₂A⁵, 5- substituted 177-tetrazole or 3-substituted l,2,4-oxadiazol-5(47/)-one; wherein when R¹ is selected as CONA¹ A², A¹ and A² are each independently selected from H, alkyl groups or alkyl groups substituted with heteroatoms; wherein when R¹ is selected as CH2NHSO2A³ or CONHSO2A⁵, A³ and A⁵ are each alkyl groups; R² is an alkyl group which may optionally be further substituted with an aryl group; R³ is selected from an alkyl or aryl group, which may be optionally substituted with aryl, heteroaryl, alkyl, halogen or hydroxy groups; R³ may also be selected from a cyclic alkyl group or a heteroalkyl ring;

R⁴ is selected from Ar, CR⁵R⁶Ar, or NR⁷R⁸; wherein when R⁴ is selected as CR⁵R⁶Ar; Ar is selected from an aryl, a fused aryl or a heteroaryl ring system, which may be optionally substituted with alkyl, halogen, carboxylic acid or hydroxyl groups; R⁵ and R⁶ are each independently selected from H, alkyl, aryl, alkoxy, alcohol, amine, alkylamino or halogen groups; wherein R⁵ and R⁶ may be fused to form a carbocycle or heterocycle; wherein when R⁴ is selected as Ar, Ar is an aryl ring system which may optionally be substituted with alkyl or halogen groups; wherein when R⁴ is selected as NR⁷R⁸, R⁷ and R⁸ are each independently selected from H, alkyl, or aryl or benzyl groups which may be optionally substituted with halogen groups, wherein one or more of the following compounds are excluded from the invention:

Preferably, when R³ is methyl and R⁴ is CR⁵R⁶Ar, Ar is selected from a substituted aryl, a fused aryl, or a heteroaryl ring system, wherein the substituted aryl is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups.

Preferably, when R³ is methyl, R⁴ is not CR⁵R⁶Ar.

In another aspect of the invention there is provided a compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from CO₂H, CONA¹ A², CN, CH₂OH, CH₂NHSO₂A³, CONHSO₂A⁵, 5- substituted 177-tetrazole or 3-substituted l,2,4-oxadiazol-5(47/)-one; wherein when R¹ is selected as CONA¹ A², A¹ and A² are each independently selected from H, alkyl groups or alkyl groups substituted with heteroatoms; wherein when R¹ is selected as CH₂NHSO₂A³ or CONHSO₂A⁵, A³, and A⁵ are each alkyl groups;

R² is an alkyl group which may optionally be further substituted with an aryl group;

R³ is selected from an alkyl or aryl group, which may be optionally substituted with aryl, heteroaryl, alkyl, halogen or hydroxy groups; R³ may also be selected from a cyclic alkyl group or a heteroalkyl ring;

R⁴ is selected from Ar, CR⁵R⁶Ar, or NR⁷R⁸; wherein when R⁴ is selected as CR⁵R⁶Ar; Ar is selected from an aryl, a fused aryl or a heteroaryl ring system, which may be optionally substituted with alkyl, halogen, carboxylic acid or hydroxyl groups;

R⁵ and R⁶ are each independently selected from H, alkyl, aryl, alkoxy, alcohol, amine, alkylamino or halogen groups; wherein R⁵ and R⁶ may be fused to form a carbocycle or heterocycle; wherein when R⁴ is selected as Ar, Ar is an aryl ring system which may optionally be substituted with alkyl or halogen groups; wherein when R⁴ is selected as NR⁷R⁸, R⁷ and R⁸ are each independently selected from H, alkyl, or aryl or benzyl groups which may be optionally substituted with halogen groups, and wherein when R³ is methyl and R⁴ is CR⁵R⁶Ar, one of R⁵ and R⁶ is not hydrogen.

In this aspect of the invention, preferably when R³ is methyl and R⁴ is CR⁵R⁶Ar, Ar is selected from a substituted aryl, a fused aryl, or a heteroaryl ring system, wherein the substituted aryl is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups.

In this aspect of the invention, preferably when R³ is methyl, R⁴ is not CR⁵R⁶Ar.

In a further aspect of the invention there is provided a compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

R⁵ and R⁶ are each independently selected from H, alkyl, aryl, alkoxy, alcohol, amine, alkylamino or halogen groups; wherein R⁵ and R⁶ may be fused to form a carbocycle or heterocycle; wherein when R⁴ is selected as Ar, Ar is an aryl ring system which may optionally be substituted with alkyl or halogen groups; wherein when R⁴ is selected as NR⁷R⁸, R⁷ and R⁸ are each independently selected from H, alkyl, or aryl or benzyl groups which may be optionally substituted with halogen groups, wherein when R³ is methyl and R⁴ is CR⁵R⁶Ar, Ar is a selected from a substituted aryl, a fused aryl, or a heteroaryl ring system, wherein the substituted aryl is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups.

In this aspect of the invention, preferably R³ is methyl and R⁴ is CR⁵R⁶Ar, and one of

R⁵ and R⁶ is not hydrogen.

In all aspects of the invention, preferably R³ may be selected from cyclopropyl, cyclobutyl, cyclohexyl, Cf cyclopropyl, Ctbcyclobutyl, CH(CH3)C2Hs where the chiral centre can have either the (R) or (S) configuration, CH(CH3)0H where the chiral centre can have either the (R) or (S) configuration, ethyl, n-butyl, CH2CH(CH3)2, CH2PI1 (benzyl), 4-trifluoromethyl benzyl, 4-methyl benzyl or CH2-2-pyridyl. Most preferably, R³ is selected from CH(CH3)2 or C(CH3)3.

In all aspects of the invention, preferably, when R⁴ is selected as CR⁵R⁶Ar, Ar is selected from an aryl, a fused aryl or a heteroaryl ring system, which is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups, wherein the substitution is in the 4-position.

In all aspects of the invention, preferably, when R⁴ is selected as CR⁵R⁶Ar, at least one of R⁵ or R⁶ is an alkyl group.

In all aspects of the invention, preferably, when R⁴ is selected as CR⁵R⁶Ar and a stereocentre is present in CR⁵R⁶Ar, the stereocentre has an (S) configuration. In all aspects of the invention, most preferably, when R⁴ is selected as CR⁵R⁶Ar, both of R⁵ and R⁶ are alkyl groups which may be fused to form a carbocycle.

In all aspects of the invention, preferably, R¹ is CO2H. Preferably, R² is CH3, CH2CH3, CH2CH2CH3 or CH2PI1. Most preferably, R² is CH2CH3.

Unless otherwise defined, all the technical and scientific terms used have the same meaning as that usually understood by an ordinary specialist in the field to which the invention belongs.

The compounds according to the present invention (i.e. the compounds of Formula (1)) may be selected from the following list:

1. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)-5- ethoxy-5 -oxopentanoic acid

2. Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate

3. (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -oxopentanoic acid

5. (R)-5-ethoxy-4-((S)-2-(3-(4-fluorophenyl)ureido)-3,3-dimethylbutanamido)-5- oxopentanoic acid

6. Ethyl N2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanoyl)-N5-(methylsulfonyl)-D-glutaminate

7. Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-(methylsulfonamido)pentanoate

8. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -ethoxy-5 -oxopentanoic acid

9. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-methoxy-5 -oxopentanoic acid

10. (R)-5-(benzyloxy)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -oxopentanoic acid

11. (R)-5-ethoxy-4-((S)-2-(2-(4-fluorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -oxopentanoic acid 12. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)- 5 -oxo-5 -propoxypentanoic acid

13. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)- 5 -isopropoxy-5 -oxopentanoic acid

14. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)- 5 -methoxy-5 -oxopentanoic acid

15. Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-phenylalanyl-D-glutaminate

16. (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -isopropoxy-5 -oxopentanoic acid

17. (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -oxopentanoic acid

18. Ethyl ((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanoyl)- D-glutaminate

19. Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl)-N5-(2- hydroxyethyl)-D-glutaminate

20. (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2-methylpropanamido)-3- methylbutanamido)-5 -oxopentanoic acid

21. (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -oxopentanoic acid

22. Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate

23. Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-hydroxypentanoate

24. Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl)-N5,N5-dimethyl- D-glutaminate

25. Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-4-cyanobutanoate.

The above list of compounds of the present invention may also be represented by the following structural formulae:

According to a further aspect of the invention, the compounds according to the present invention (i.e. the compounds of Formula (1)) are most preferably selected from the following list:

The present invention also relates to a pharmaceutical composition comprising one or more of the compound(s) according to the present invention (e.g. compounds of Formula (1)). Preferably, the pharmaceutical composition comprises a pharmaceutically or therapeutically acceptable excipient or carrier.

It has been surprisingly found that compounds according to the present invention are potent and selective agonists of N0D2. By acting as agonist of N0D2, the compounds according to the present invention are capable of modulating proinflammatory mediator secretion in a subject, and are thus capable of modulating innate immunity in a subject.

Compounds with the stereochemical configuration shown in Formula (1), which is the same as in the natural agonist MDP, were found to be much more active in the HEK-blue hNOD2 assay than compounds with a stereochemical configuration different to that shown in Formula (1). We believe the absolute configuration of both stereogenic centres of the dipeptide backbone is important, because changing either of them leads to compounds with significantly reduced activity. A further aspect of the invention is the compound or pharmaceutical composition according to the present invention for use as a medicament for the treatment of a disease or disorder, preferably for the treatment of Crohn’s disease.

A further aspect of the invention is the compound according to the present invention for use as potent and selective N0D2 agonist. A further aspect of the invention is the compound according to the present invention for use as a medicament capable of modulating innate immunity in a subject. A further aspect of the present invention is the compound according to the present invention for use as a modulator of pro-inflammatory mediator secretion in a subject. The use may be in the treatment of a disease or disorder (e.g. in the treatment of Crohn’s disease).

The invention also encompasses a method of treating a disease or disorder, comprising the step of administering a compound according to the present invention or the pharmaceutical composition according to the present invention to a subject in need of the same. Preferably the disease or disorder being treated is Crohn’s disease. In the method, treatment is affected or facilitated by the compound of the invention acting as a potent and selective N0D2 agonist.

Comparative compounds

The following comparative compound is also disclosed:

4. IHX1199 Ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3 -methylbutanamidojbutanoate .

The above comparative compound may also be represented by the following structural formula:

Particular non-limiting examples of the present invention will now be described.

Experimental

General Method 1

Compounds of formula (2) depicted below were prepared using the following synthetic procedure.

Example 1 : (R)-4-((S)-2-(2-(4-chlorophenvl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid Step 1

To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5 -oxopentanoic acid (1 equiv.) in DMF was added K2CO3 (3 equiv.) and iodoethane (2 equiv.). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by reverse phase chromatography to afford 5 -(tert-butyl) 1 -ethyl ((benzyloxy)carbonyl)-D-glutamate as a yellow oil. Step 2

To a solution of 5 -(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-D-glutamate (1 equiv.) in EtOH was added 10% Pd/C (0.4 equiv.). The reaction mixture was stirred at room temperature under a hydrogen atmosphere overnight. The mixture was filtered and concentrated to afford 5 -(tert-butyl) 1-ethyl D-glutamate as a colourless oil, which was used directly in the next step.

Step 3

To a solution of ((benzyloxy)carbonyl)-L-valine (1 equiv.) in CH2CI2 was added HATU (1.3 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then 5 -(tert-butyl) 1-ethyl D-glutamate (1 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by preperative TLC to afford 5 -(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-L-valyl-D-glutamate as a white solid.

Step 4

To a solution of 5 -(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-L-valyl-D-glutamate (1 equiv.) in EtOH was added 10% Pd/C (0.4 equiv.). The reaction mixture was stirred at room temperature under a hydrogen atmosphere overnight. The mixture was filtered and concentrated to afford 5 -(tert-butyl) 1-ethyl L-valyl -D-glutamate as a colourless oil, which was used directly in the next step.

Step 5

To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in CH2CI2 was added HATU (1.3 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then 5 -(tert-butyl) 1-ethyl L-valyl -D-glutamate (1 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford 5 -(tert-butyl) 1-ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D- glutamate as a white solid.

Step 6 To a solution of 5 -(tert-butyl) 1 -ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl- D-glutamate (1 equiv.) in CH2CI2 was added TFA (2 mL). The reaction mixture was stirred at room temperature for three hours, then the solvent was removed under vacuum to afford (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5 -ethoxy-5 -oxopentanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 12.2 (1H, s), 8.27 (1H, d), 7.45 - 7.28 (4H, m), 6.86 (1H, d), 4.29 - 4.02 (4H, m), 2.27 (2H, t), 2.02 - 1.87 (2H, m), 1.84 - 1.71 (1H, m), 1.49 (3H, s), 1.46 (3H, s), 1.18 (3H, t), 0.81 (3H, d), 0.73 (3H, d).

LCMS m/z = 455.2 [M+H]⁺

General Method 2

Compounds of formula (3) depicted below were prepared using the following synthetic procedure.

Example 2: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate

To a solution of (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5 -ethoxy-5 -oxopentanoic acid (1 equiv., prepared as for Example 1) in CH2CI2 was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then NH4CI (2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl (2-(4- chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate as a white solid.

'H NMR (400 MHz, DMSO-t/d) 8 8.26 (1H, d), 7.42 - 7.29 (4H, m), 7.25 (1H, s), 6.82 (1H, d), 6.77 (1H, s), 4.15 (2H, t), 4.11 - 4.03 (2H, m), 2.09 (2H, t), 1.97 - 1.84 (2H, m), 1.80 - 1.68 (1H ,m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.70 (3H, d).

LCMS m/z = 454.3 [M+H]⁺

General Method 3

Compounds of formula (4) depicted below were prepared using the following synthetic procedure.

Example 3: (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid

Step 1

To a solution of 1 -(tert-butyl) 5-methyl ((benzyloxy)carbonyl)-D-glutamate (1 equiv.) in EtOH was added Pd/C (0.4 equiv.). The reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford crude 1 -(tert-butyl) 5-methyl D-glutamate which was used without further purification.

Step 2

To a solution of (S)-2-(((benzyloxy)carbonyl)amino)-3, 3 -dimethylbutanoic acid (1 g, 4.61 mmol) in CH2CI2 was added HATU (1 equiv.) and the reaction mixture was stirred at room temperature for 30 minutes. 1 -(tert-butyl) 5-methyl D-glutamate (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford 1 -(tert-butyl) 5-methyl ((S)-2-(((benzyloxy)carbonyl)amino)- 3 ,3 -dimethylbutanoyl)-D-glutamate .

Step 3

To a solution of 1 -(tert-butyl) 5-methyl ((S)-2-(((benzyloxy)carbonyl)amino)-3,3- dimethylbutanoyl)-D-glutamate.(l equiv.) in EtOH was added Pd/C (0.4 equiv.). The reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford crude 1 -(tert-butyl) 5- methyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate which was used without further purification.

Step 4

To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in DMF was added HATU (1 equiv.) and the reaction mixture stirred at room temperature for 30 minutes. 1 -(tert-butyl) 5 -methyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for two hours at room temperature. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine and concentrated. The crude residue was purified by prep-HPLC to afford 1 -(tert-butyl) 5-methyl ((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanoyl)-D-glutamate.

Step 5

To a solution of 1 -(tert-butyl) 5-methyl ((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanoyl)-D-glutamate (1 equiv.) in CH3CN and H2O was added LiOH (2 equiv.) at 0 °C. The reaction mixture was stirred at room temperature for three hours. The reaction solution was purified directly by reverse phase chromatography to afford (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 12.19 (1H, s), 8.39 (1H, d), 7.45 - 7.30 (4H, m), 6.38 (1H, d), 4.34 (1H, d), 4.12 - 3.95 (1H, m), 2.27 (2H, m), 1.99 - 1.86 (1H, m), 1.83 - 1.68 (1H, m), 1.52 (3H, s), 1.46 (3H, s), 1.40 (9H, s), 0.83 (9H, s).

LCMS m/z = 497.4 [M+H]⁺

General Procedure 4

Compounds of formula (5) depicted below were prepared using the following synthetic procedure.

Comparative Example 4: Ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3-methylbutanamido)butanoate

Step 1

To a solution of (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid (410 mg, 0.903 mmol) in tertbutanol was added NEt3 (3 equiv.), DPPA (2 equiv.) and (Boc)2O (5 equiv.). The reaction was stirred at 85 °C under a nitrogen atmosphere for 48 hours. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers was dried over Na2SO4, filtered and concentrated. The residue was purified by reverse phase chromatography to afford ethyl (R)-4-((tert-butoxycarbonyl)amino)-2-((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)butanoate.

Step 2

To a solution of ethyl (R)-4-((tert-butoxycarbonyl)amino)-2-((S)-2-(2-(4-chlorophenyl)- 2-methylpropanamido)-3-methylbutanamido)butanoate (1 equiv.) in CH2CI2 was added a solution of HC1 in dioxane (4M, 10 equiv.). The reaction mixture was stirred at room temperature for three hours. The solvent was removed under vacuum to afford ethyl (R)-4-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate hydrochloride salt which was used without purification.

Step 3

To a suspension of (R)-4-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate hydrochloride salt (1 equiv.) in CH2CI2 was added NEt? (3 equiv.) and acetyl chloride (2 equiv.) The reaction solution was stirred at room temperature for four hours. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by reverse phase chromatography to afford ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate as a white solid.

'H NMR (400 MHz, DMSO-</₆) 8 8.26 (1H, d), 7.84 (1H, s), 7.34 (4H q)„ 6.83 (1H, d), 4.18 (2H, dt), 4.11 - 4.03 (2H, m), 3.06 (1H, t), 3.01 - 2.94 (1H, m), 1.93 - 1.80 (2H, m), 1.78 (3H, s), 1.74 - 1.65 (1H, m), 1.46 (6H, d), 1.16 (3H, t), 0.79 (3H, d), 0.71 (3H, d).

LCMS m/z = 468.2 [M+H]⁺

General Procedure 5

Compounds of formula (6) depicted below were prepared using the following synthetic procedure.

Example 5: (R)-5-ethoxv-4-((S)-2-(3-(4-fluorophenyl)ureido)-3,3- dimethylbutanamido)-5-oxopentanoic acid

Step 1

To a solution of 5 -(tert-butyl) 1-ethyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate (1 equiv., prepared as for Example 1) in CH2CI2 at 0 °C was added DIPEA (4 equiv.) and (4-fluorophenyl)carbamic chloride (1.5 equiv.). The mixture was stirred at room temperature for two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford 5 -(tert-butyl) 1-ethyl ((S)-2-(3-(4- fLuorophenyl)ureido)-3,3-dimethylbutanoyl)-D-glutamate.

Step 2

To a solution of 5 -(tert-butyl) 1-ethyl ((S)-2-(3-(4-fhiorophenyl)ureido)-3,3- dimethylbutanoyl)-D-glutamate (1 equiv.) in CH2CI2 was added TFA (2 mL). The reaction mixture was stirred at room temperature for three hours, then the solvent was removed under vacuum to afford (R)-5-ethoxy-4-((S)-2-(3-(4-fluorophenyl)ureido)-3,3- dimethylbutanamido)-5-oxopentanoic acid. 'H NMR (400 MHz, DMSO-t/d) 8 8.75 (1H, s), 8.62 (1H, d), 7.44 - 7.36 (2H, m), 7.14 0 7.05 (2H, m), 6.40 (1H, d), 4.29 - 4.20 (2H, m), 4.14 - 4.03 (2H, m), 2.35 (2H, t), 2.03

- 1.93 (1H, m), 1.90 - 1.77 (1H, m), 1.18 (3H, t), 0.95 (9H, s).

LCMS m/z = 426.2 [M+H]⁺

General Procedure 6

Compounds of formula (7) depicted below were prepared using the following synthetic procedure.

Example 6: Ethyl N2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanoyl)-N5-(methylsulfonyl)-D-glutaminate

To a solution (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5 -ethoxy-5 -oxopentanoic acid (1 equiv., prepared as for Example 1) in CH2CI2 was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then methanesulfonamide (2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl N2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanoyl)-N5- (methylsulfonyl)-D-glutaminate.

'H NMR (400 MHz, DMSO-t/d) 8 8.47 (1H, d), 7.38 (2H, d), 7.33 (2H, d), 6.31 (1H, d), 4.31 (1H, d), 4.17 - 4.09 (1H, m), 4.07 (3H, q), 3.16 (3H, s), 2.35 - 2.26 (3H, m), 2.01 - 1.88 (1H, m), 1.84 - 1.71 (1H, m), 1.49 (3H, s), 1.45 (3H, s), 1.17 (3H, t), 0.80 (9H, s). LCMS m/z = 546.3 [M+H]⁺

General Procedure 7

Compounds of formula (8) depicted below were prepared using the following synthetic procedure.

Example 7: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-(methylsulfonamido)pentanoate

Step 1

To a solution (S)-2-((tert-butoxycarbonyl)amino)-3, 3 -dimethylbutanoic acid (1 equiv.) in CH2CI2 was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then ethyl (R)-2-amino-5- (((benzyloxy)carbonyl)amino)pentanoate 2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl (R)-5- (((benzyloxy)carbonyl)amino)-2-((S)-2-((tert-butoxycarbonyl)amino)-3,3- dimethylbutanamido)pentanoate.

Step 2

To a solution of ethyl (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-((tert- butoxycarbonyl)amino)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in CH2CI2 was added a solution of HC1 in dioxane (4 M, 16 equiv.). The reaction mixture was stirred at room temperature for three hours. The solvent was removed under vacuum to afford crude ethyl (R)-2-((S)-2-amino-3,3-dimethylbutanamido)-5- (((benzyloxy)carbonyl)amino)pentanoate hydrochloride salt which was used directly without purification.

Step 3 To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in DMF was added HATU (1 equiv.) and the reaction mixture stirred at room temperature for 30 minutes, ethyl (R)-2-((S)-2-amino-3,3-dimethylbutanamido)-5- (((benzyloxy)carbonyl)amino)pentanoate hydrochloride salt (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for two hours at room temperature. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine and concentrated. The crude residue was purified by prep-HPLC to afford ethyl (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanamido)pentanoate.

Step 4

To a solution of (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in EtOH was added Pd/C (0.4 equiv.). The reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford ethyl (R)-5-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)pentanoate which was used without further purification.

Step 5

To a solution of ethyl (R)-5-amino-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in CH2CI2 was added methanesulfonyl chloride (1.5 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature overnight. EtOAc and water were added and the organic phase was washed with water, dried over Na2SO4 and the solvent was removed under vacuum to afford ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-(methylsulfonamido)pentanoate. 'H NMR (400 MHz, DMSO-t/d) 8 8.45 (1H, d) 7.45 - 7.31 (4H, m), 6.99 (1H, t), 4.36 (1H, d), 4.18 - 4.04 (3H, m), 2.97 - 2.91 (2H, m), 2.89 (3H, s), 1.80 - 1.70 (1H, m), 1.68 - 1.57 (1H, m), 1.47 -1.40 (9H, m), 1.18 (3H, t), 0.82 (9H, s).

LCMS m/z = 532.4 [M+H]⁺

Example 8: (R)-4-((S)-2-(2-(4-chlorophenvl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-ethoxy-5-oxopentanoic acid The compound was prepared using the procedure as for Example 1 with (S)-2- ((benzyloxy)carbonyl)-3 ,3 -dimethylbutanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 8.46 (1H, d), 7.43 - 7.32 (4H, m), 6.36 (1H, d), 4.34 (1H, d), 4.22 - 4.14 (1H, m), 4.09 (2H, q), 2.29 (2H, t), 2.01 - 1.91 (1H, m), 1.83 - 1.72 (1H, m), 1.51 (3H, s), 1.46 (3H, s), 1.18 (3H, t), 0.83 (9H, s).

LCMS m/z = 469.2 [M+H]⁺

Example 9: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-methoxy-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with iodomethane and (S)-2-((benzyloxy)carbonyl)-3, 3 -dimethylbutanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 8.48 (1H, d), 7.44 - 7.31 (4H, m), 6.35 (1H, d), 4.34 (1H, d), 4.26 - 4.18 (1H, m), 2.29 (2H, t), 2.02 - 1.91 (1H, m), 1.84 - 1.71 (1H, m), 1.51 (3H, s), 1.46 (3H, s), 0.82 (9H, s).

LCMS m/z = 455.2 [M+H]⁺

Example 10: (R)-5-(benzyloxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with benzyl bromide and (S)-2-((benzyloxy)carbonyl)-3, 3 -dimethylbutanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 8.50 (1H, d), 7.42 - 7.30 (9H, m), 5.18 - 5.08 (2H, m), 4.38 - 4.25 (2H, m), 2.29 (2H, t), 2.06 - 1.95 (1H, m), 1.87 - 1.74 (1H, m), 1.50 (3H, s), 1.44 (3H, s), 0.80 (9H, s).

LCMS m/z = 531.2 [M+H]⁺

Example 11 : (R)-5-ethoxy-4-((S)-2-(2-(4-fluorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with (S)-2- ((benzyloxy)carbonyl)-3, 3 -dimethylbutanoic acid and 2-(4-fluorophenyl)-2- methylpropanoic acid.

'H NMR (400 MHz, DMSO-t/e) 8 12.11 (1H, s), 8.44 (1H, d), 7.42 - 7.29 (2H, m), 7.21 - 7.07 (2H, m), 6.27 (1H, d), 4.32 (1H, d), 4.22 - 4.02 (3H, m), 2.27 (2H, t), 2.02 - 1.89 (1H, m), 1.84 - 1.69 (1H, m), 1.50 (3H, s), 1.46 (3H, s), 1.17 (3H, t), 0.80 (9H, s). LCMS m/z = 453.2 [M+H]

Example 12: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-oxo-5-propoxypentanoic acid

The compound was prepared using the procedure as for Example 1 with 1 -iodopropane. 'H NMR (400 MHz, DMSO-tfe) 8 12.20 (1H, s), 8.30 (1H, d), 7.43 - 7.29 (4H, m), 6.86 (1H, d), 4.29 - 4.13 (2H, m), 4.09 - 3.93 (2H, m), 2.28 (2H, t), 2.04 - 1.86 (2H, m), 1.84 - 1.72 (1H, m), 1.65 - 1.55 (2H, m), 1.49 (3H, s), 1.46 (3H, s), 0.89 (3H, t), 0.81 (3H, d), 0.73 (3H, d).

LCMS m/z = 469.3 [M+H]⁺

Example 13: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-isopropoxy-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with 2-iodopropane. 'H NMR (400 MHz, DMSO-t/d) 8 12.17 (1H, s), 8.26 (1H, d), 7.44 - 7.32 (4H, m), 6.89 (1H, d), 4.97 (1H, m), 4.23 - 4.12 (2H, m), 2.29 (2H, t), 2.02 - 1.87 (2H, m), 1.83 - 1.71 (1H, m), 1.50 (3H, s), 1.48 (3H, s), 1.20 (6H, t), 0.83 (3H, d), 0.74 (3H, d).

LCMS m/z = 491.2 [M+Na]⁺

Example 14: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-methoxy-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with iodomethane. 'H NMR (400 MHz, DMSO-t/e) 8 12.19 (1H, s), 8.30 (1H, d), 7.44 - 7.29 (4H, m), 6.85 (1H, d), 4.30 - 4.12 (2H, m), 3.64 (3H, s), 2.27 (2H, t), 2.04 - 1.87 (2H, m), 1.83 - 1.71 (1H, m), 1.49 (3H, s), 1.46 (3H, s), 0.80 (3H, d), 0.73 (3H, d).

LCMS m/z = 441.4 [M+H]⁺

Example 15: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-phenylalanyl-D- glutaminate

The compound was prepared using the procedure as for Example 2 with (R)-4-((S)-2-(2- (4-chlorophenyl)-2-methylpropanamido)-3-phenylpropanamido)-5 -ethoxy-5 - oxopentanoic acid. 'H NMR (400 MHz, DMSO-t/d) 8 8.29 (1H, d), 7.33 - 7.08 (11H, m), 6.81 (1H, s), 4.67 - 4.59 (1H, m), 4.27 (1H, m), 4.17 - 4.07 (2H, m), 2.99 (1H, dd), 2.86 (1H, dd), 2.12 (2H, t), 2.02 - 1.91 (1H, m), 1.85 - 1.73 (1H, m), 1.35 (3H, s), 1.33 (3H, s), 1.21 (3H, t).

LCMS m/z = 502.2 [M+H]⁺

Example 16: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-isopropoxy-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with (S)-2- ((benzyloxy)carbonyl)-3, 3 -dimethylbutanoic acid and 2-iodopropane.

'H NMR (400 MHz, DMSO-t/d) 8 12.20 (1H, s), 8.39 (1H, d), 7.46 - 7.34 (4H, m), 6.26 (1H, d), 4.93 - 4.83 (1H, m), 4.31 (1H, d), 4.23 - 4.12 (1H, m), 2.36 - 2.20 (2H, m), 202 - 1.90 (1H, m), 1.86 - 1.73 (1H, m), 1.52 (3H, s), 1.47 (3H, s), 1.18 (6H, d), 0.84 (9H, s).

LCMS m/z = 483.4 [M+H]⁺

Example 17: (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 3 with ((benzyloxy)carbonyl)-L-valine.

'H NMR (400 MHz, DMSO-t/d) 8 12.19 (1H, s), 8.19 (1H, d), 7.44 - 7.31 (4H, m), 6.89 (1H, d), 4.21 - 4.07 (2H, m), 2.27 (2H, t), 2.01 - 1.86 (2H, m), 1.82 - 1.69 (1H, m), 1.51 (3H, s), 1.48 (3H, s), 1.42 (9H, s), 0.83 (3H, d), 0.74 (3H, d).

LCMS m/z = 483.2 [M+H]⁺

Example 18: Ethyl ((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanoyl)-D-glutaminate

The compound was prepared using the procedure as for Example 2 with (R)-4-((S)-2-(2- (4-chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanamido)-5-ethoxy-5- oxopentanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 8.46 (1H, d), 7.43 - 7.32 (4H, m), 7.27 (1H, s), 6.79 (1H, s), 6.34 (1H, d), 4.33 (1H, d), 4.18 - 4.03 (3H, m), 2.13 (2H, t), 1.99 - 1.87 (1H, m), 1.83 - 1.72 (1H, m), 1.50 (3H, s), 1.47 (3H, s), 1.18 (3H, t), 0.82 (9H, s). LCMS m/z = 468.2 [M+H]

Example 19: Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl)-N5-(2- hydroxyethyl)-D-glutaminate

The compound was prepared using the procedure as for Example 2 with_2-aminoethan- l-ol

'H NMR (400 MHz, DMSO-t/d) 8 8.28 (1H, d), 7.81 (1H, t), 7.43 - 7.31 (4H, m), 6.84 (1H, d), 4.66 (1H, t), 4.22 - 4.04 (4H, m), 3.16 - 3.06 (2H, m), 2.13 (2H, t), 2.00 - 1.87 (2H, m), 1.84 - 1.71 (1H, m), 1.49 (2H, s), 1.47 (3H, s), 1.19 (3H, t), 0.81 (3H, d), 0.72 (3H, d).

LCMS m/z = 498.2 [M+H]⁺

Example 20 : (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2-methylpropanamido)-3- methylbutanamido)-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with 2-(4- hydroxyphenyl)-2-methylpropanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 12.16 (1H, s), 9.30 (1H, s), 8.29 (1H, d), 7.13 (2H, d), 6.72 (2H, d), 6.48 (1H, d), 4.26 - 4.04 (4H, m), 2.27 (2H, t), 2.21 - 1.84 (2H, m), 1.81 - 1.71 (1H, m), 1.43 (6H, s), 1.18 (3H, t), 0.79 (3H, d), 0.69 (3H, d).

LCMS m/z = 437.4 [M+H]⁺

Example 21 : (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2-methylpropanamido)- 3,3-dimethylbutanamido)-5-oxopentanoic acid

The compound was prepared using the procedure as for Example 1 with (S)-2-((tert- butoxycarbonyl)amino)-3,3-dimethylbutanoic acid and 2-(4-hydroxyphenyl)-2- methylpropanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 9.34 (1H, s), 8.48 (1H, d), 7.15 (2H, d), 6.74 (2H, d), 6.04 (1H, d), 4.29 (1H, d), 4.23 - 4.03 (3H, m), 2.29 (2H, t), 2.01 - 1.90 (1H, m), 1.84 - 1.71 (1H, m), 1.45 (3H, s), 1.42 (3H, s), 1.19 (3H, t), 0.79 (9H, s).

LCMS m/z = 451.4 [M+H]⁺

Example 22: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate The compound was prepared using the procedure as for Example 2 with (R)-4-((S)-2-(2- (4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)-5 -ethoxy-5 - oxopentanoic acid.

'H NMR (400 MHz, DMSO-t/d) 8 8.26 (1H, d), 7.42 - 7.29 (4H, m), 7.25 (1H, s), 6.82 (1H, d), 6.77 (1H, s), 4.15 (2H, t), 4.11 - 4.03 (2H, m), 2.09 (2H, t), 1.97 - 1.84 (2H, m), 1.80 - 1.68 (1H, m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.70 (3H, d).

LCMS m/z = 454.3 [M+H]⁺

Example 23: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-hydroxypentanoate

The compound was prepared using the procedure as for Example 1 with ethyl (R)-2- ((S)-2-amino-3-methylbutanamido)-5-hydroxypentanoate.

'H NMR (400 MHz, DMSO-t/d) 8 8.19 (1H, d), 7.34 (4H, q), 6.83 (1H, d), 4.46 (1H, t), 4.20 - 4.03 (4H, m), 3.37 (2H, d), 1.95 - 1.85 (1H ,m), 1.79 - 1.68 (1H ,m), 1.61 - 1.51 (1H, m), 1.45 (6H, d), 1.43 - 1.30 (2H, m), 1.16 (3H, t), 0.78 (3H, d), 0.70 (3H, d).

LCMS m/z = 441.3 [M+H]⁺

Example 24: Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl)-N5,N5- dimethyl-D-glutaminate

The compound was prepared using the procedure as for Example 2 with (R)-4-((S)-2-(2- (4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)-5 -ethoxy-5 - oxopentanoic acid and dimethylamine.

'H NMR (400 MHz, DMSO-t/d) 8 8.24 (1H, d), 7.34 (4H, q), 6.86 (1H, d), 4.26 - 4.17 (1H, m), 4.17 - 4.03 (3H, m), 2.88 (3H, s), 2.80 (3H, s), 2.32 - 2.23 (2H, m), 2.00 - 1.89 (2H ,m), 1.81 - 1.70 (1H, m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.71 (3H, d) LCMS m/z = 482.2 [M+H]⁺

Example 25: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-4-cyanobutanoate

The compound was prepared using the procedure as for Example 1 with ethyl (R)-2- ((S)-2-amino-3-methylbutanamido)-4-cyanobutanoate. 'H NMR (400 MHz, DMSO-t/d) 8 8.53 (1H, d), 7.41 - 7.30 (4H, m), 6.36 (1H, d), 4.29 (1H, d), 4.22 - 4.14 (1H, m), 4.09 (2H, q), 2.49 - 2.45 (2H, m), 2.08 - 1.98 (1H, m), 1.93 - 1.81 (1H, m), 1.50 (3H, s), 1.44 (3H, s), 1.17 (3H, t), 0.82 (9H, s).

LCMS m/z = 450.3 [M+H]⁺

Example 26: Compound activity in HEK-Blue™ hNOD2 cells

All HEK-Blue™ cell lines were purchased from Invitrogen.

A dose-reponse curve for each test compound is generated. A dose-response curve is generated on each plate for MDP (control activating ligand) and may be generated for MDP control (a non-activating ligand). Cells treated with the appropriate vehicle (0.05% DMSO) are also included in each plate.

Initial cell culture procedure:

Cell vials are thawed in a 37°C water bath and cells are transfered to a sterile 15 ml tube containing 15 ml pre-warmed DMEM 10% FBS medium. The tube is centrifuged at 200 x g for 5 minutes. The supernatant is removed and the cells resuspended with 15 ml DMEM 10% FBS medium without selective antibiotics. The vial contents are transfered to a 75 cm³ tissue culture flask and the flask placed in and incubator and cultured in a humidified atmosphere at 37°C and 5% CO2.

Cell maintenance:

Cells are maintained and subcultured in growth medium supplemented with 30 pg/ml of blasticidin and 100 pg/ml of Zeocin™. Cells are passaged twice when an 80% confhiency is reached. Medium is aspirated and the cells washed with 10 ml PBS. The cells are detached with 5 ml of pre-warmed PBS by pipetting up and down. The PBS cell suspension is diluted 5 -fold and cells counted in a haemocytometer. 1.5 x 10⁶ cells are seeded per 75 cm³ tissue culture flask in 15 ml pre-warmed DMEM 4.5 g/L glucose, and 10% FBS. The required selection antibiotics are added. After 2-3 days cells are ready to perform screening experiments. Preparation of compound pre-dilution and master plates:

HEK-Blue™ detection medium solutions are prepared following the manufacture’s instructions and warmed in a 37°C water bath. 30 mM DMSO stocks of test compounds are diluted 1.5-fold in DMSO and 5 pl added to compound the pre-dilution plate. 95 pl of HEK-Blue detection medium solution is added to all the wells with compound. Test compound solutions from the pre-dilution plate are diluted 50-fold in the master plate.

Preparation of test plates:

25 pl of each test compound and sequential dilutions of the control ligands (MDP and MDP control) are trans fered from the master plate to the 384-well test plate. The test plate is incubated at 37°C for at least 1 hour, whilst preparing the cells for loading.

Detection of SEAP activity:

The 150 cm³ tissue culture flasks with HEK-Blue™ cells are removed from the incubator growth medium is aspirated. Cells in each flask are gently rinsed with prewarmed 10 ml PBS. 5 ml pre- warmed PBS is added to the 150 cm³ tissue culture flasks and cells are detached by pipetting up and down. The cell suspension is trans fered to 50 cm³ tube and incubated at 37°C while determining the total number of cells harvested. A cell suspension of 5 x 10⁵ cells per ml is prepared by diluting the PBS cell suspension in HEK-Blue™ Detection medium. The cell suspension is transfered into a reservoir and 25 pl of the cell suspension is added to the relevant wells of the 384-microwell test plate. The plate is incubated the plates at 37 °C in 5% CO2 for 16-18 h. SEAP activity is quantified using a spectrophotometer at 655 nm.

Results:

Compounds of the invention gave the following ECso’s:

The compound of the comparative example gave the following ECso’s:

Example 27: Compound activity in HEK-Blue™ Null2 cells

A similar general procedure as Example 26 is used to screen compounds in HEK- Blue™ Null2 cells with the exception that Null2 cells are maintained and subcultured in growth medium supplemented with 100 pg/ ml of Zeocin™.

Compounds of the invention were typically inactive in the assay.

Example 28: Compound activity in HEK-Blue™ hNODl cells

A similar general procedure as Example 26 is used to screen compounds in HEK- Blue™ hNODl cells with the exceptions that C12 iE-DAP is used as the control activing ligand, no non-activating ligand is used and test plates were incubated at room temperature while preparing the cells for loading.

Compounds of the invention were typically inactive in the assay.

Example 29: Compound activity in HEK-Blue™ Nulll cells A similar general procedure as Example 27 is used to screen compounds in HEK- Blue™ Nulll cells with the exception that Nulll cells are maintained and subcultured in growth medium supplemented with 100 pg/ ml of Zeocin™.

Compounds of the invention were typically inactive in the assay.

Example 30: Compound activity in HEK-Blue™ mNOD2 cells

A similar general procedure as Example 27 is used to screen compounds in HEK- Blue™ mN0D2 cells.

Results:

Compounds of the invention gave the following ECso’s.

Example 31: Compound activity in THP-1 cells

THP-1 cells are plated with a cell density of, for eample, 8 x 10⁵ cells/ml and incubated with test compounds at a DMSO concentration of < 1% at 37°C for 20 hours. Plates are then centrifuged at 100 x g for 10 minutes and 150 pl of supernatant harvested for assessement of compound activity. Compound activity is assessed using, for example, a DuoSet IL-8/CXCL8 ELISA (R&D systems, DY208) for the determination of secreted IL-8 or a Luminex 65plex detection kit (ThermoFisher Scientific, ProcartaPlex, EPX650-10065-901) for the detection of a range of secreted cytokines and chemokines. Cell viability can addtionally be determined using, for example, a CellTiterGlo™ viability kit (Promega, G7571) with 50 pl CellTiterGlo™ being added to each well and incubated at room temperature for 10 minutes. Luminscence can be read on a FluoStar plate reader. The N0D2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce one (heterogenous) or two (homogenous) copies of a single N0D2 mutations such as L1007fs, G908R or R702W that are known to cause Crohn’s Disease.

The N0D2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce one copy of two different N0D2 mutations (compound hetereogenous) such as L1007fs, G908R and R702W that are known to cause Crohn’s Disease.

The N0D2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce 1 (hetero) or 2 (homo) copies of N0D2 mutations such as R334Wor N670K that are known to cause Blau Syndrome.

Compounds of the present invention can be tested for activity in these modified THP-1 cells using the general methods described above.

Example 32: Compound activity in human monocytes.

Healthy volunteers who had no history or family history of Inflammatory Bowel Disease, were not taking anti-inflammatory medication, were not pregnant and were aged between 18-70 years old were recruited. Patient’s aged between 18-70 years old with clinically and histologically proven Crohn’s disease who had previously been genotyped for their NOD2 status (Inflamm Bowel Dis. 2012 Nov;18(l l):2120-7. doi: 10.1002/ibd.22952.) were recruited. Crohn’s disease patients who were homozygous or compound heterozygous for R702W, G908R and p.L1007fs were excluded. Approximately 40 mL of blood was collected from participants in EDTA coated vacutainers. Vacutainers were inverted immediately after collection, with samples left on a roller prior to processing no more than 2 hours after collection. Vacutainers were centrifuged at 500 g for 10 minutes to separate plasma. Peripheral blood mononuclear cells (PBMC) were then isolated from buffy coat using density gradient separation. Monocytes were purified from isolated PBMC using Magnetic-activated cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) at 4 °C as per manufacturers protocol. An automated cell counter (Bio-Rad, California, USA) was used to estimate monocyte purity and recovery, with an additional round of MACS performed in instances where monocyte purity appeared <90%. Purified monocytes were then resuspended in RPMI (+ 10% FBS, 50 pg/mL Pen/strep and 1 mM pyruvate) at 2.78xl0⁵ cells/mL prior to being used in downstream assays.

Test compounds were initially diluted in sterile DMSO, prior to dilution in RPMI to achieve a final DMSO concentration of 0.05% at working concentration of each compound. The assay was plated in a 96-well plate, with working concentration of each compound reached in 200 pL of RPMI containing 5.0xl0⁴ primary monocytes. Compounds and controls were tested in triplicate. In a sub-group of participants, compounds were also tested in the presence of 100 pg/mL sLPS. Furthermore, 100 ng/mL MDP was used as a positive control. The assay was incubated for 24 hours at 37 °C in 5% CO2. After incubation, the assay was briefly centrifuged at 200 g for 2 minutes to pellet cells and 100 pL of the supernatant was collected and immediately frozen at -80 °C.

Cell metabolism after 24-hour treatment was assessed using WST-1 reagent (Abeam. Cambridge, UK) as per manufacturer’s instructions. Briefly, 10 pL of WST-1 reagent was added to treated cells contained in 100 pL of RPMI (+ 10% FBS, 50 pg/mL Pen/strep and 1 mM pyruvate) media with care taken to avoid direct light exposure. The plate was gently shaken to mix the reagent prior to placing in a 37 °C incubator in 5% CO2 for 2 hours. WST-1 reagent in media was used as a blank control. The assay was read at 480 nm on a Clariostar plate reader (BMG Labtech, Ortenberg, Germany) after incubation. Metabolic activity was quantified as a percentage of activity compared to untreated control cells.

ELISA was used to quantify IL-8 (R&D Systems, Minneapolis, USA, Cat Number DY208) and IL-ip (Biolegend, California, USA, Cat Number DY208) in cell supernatants collected from the drug treatment assay. Cell supernatants were thawed on ice prior to being used in ELISA conducted using manufacturer’s protocol. Optical density was determined using a Clariostar plate-reader.

Compounds of the invention can be tested for activity in these human monocytes using the general methods described above. Example 33: Compound exposure after oral dosing in mice.

C57B1/6J female mice were dosed with 100 mg/kg of the compounds of the invention by oral gavage in PBS or aqueous sodium hydrogencarbonate. 9 mice were dosed per compound. Blood:water, liver and caecum samples were collected 4 hours post-dosing and were stored for LC-MS/MS analysis. Tissues were separated from their contents and frozen separately.

Results:

Concentrations of compounds of the invention in various tissues are shown below:

BLQ = Below Limit of Quantification

Example 34: Mouse and human hepatocyte stability of the compounds of the invention

The intrinsic clearances (CLint) and half-lives of the compounds of the invention were measured in either a hepatocyte suspension of cryopreserved male C57BL6 mouse hepatocytes or a mixed hepatocyte suspension of cryopreserved human hepatocytes. Briefly, the compound was incubated with hepatocyte suspensions at 37°C over a time course and the remaining compound at each time point was assessed by mass spectrometry (UPLC-MS/MS).

Example 35: Plasma Protein Binding of compounds of the invention. The extent to which compounds of the invention bound to plasma proteins such as albumin and alpha- 1 acid glycoprotein within human, rat or mouse plasma was determined by rapid equilibrium dialysis. Compounds were incubated at 5pM for 4 hours at 37°C.

Example 36: Activity of compounds of the invention against the HERG channel

Compounds of the invention were tested for inhibition of cardiac potassium (hERG) channels using the QPatch automated patch clamp system (Sophion, Denmark). The compounds were screened at eight concentrations (using 0.5-log unit dilutions) from a top concentration of 30 pM, against a minimum of three separate cells. Each eight-point concentration-response curve was constructed using cumulative single sample additions of each concentration to the same cell.

Example 37: Activity of compounds of the invention against a panel of enzymes, ion channels and receptors

Compounds of the invention were tested against the DiscoverX SAFETY scan E/IC50 ELECT - 78 assay panel.

Example 38: Permeability of compounds of the invention in Caco 2 cells

Caco 2 cells are used as an in vitro model of the human intestinal epithelium and permit assessment of the intestinal permeability of potential drugs. Compounds of the invention were added to either the apical or basolateral side of a confluent monolayer of Caco 2 cells and permeability was measured by monitoring the appearance of the test compound on the opposite side of the monolayer using LC MS/MS. The efflux ratio (ER) was calculated from the ratio of B A and A B permeabilities.

Example 39: Reversible Inhibition of CYPs by compounds of the invention.

The inhibition of individual CYPs by compounds of was assessed using human liver microsomes in combination with specific probe substrates.

Claims

1. A compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

R⁵ and R⁶ are each independently selected from H, alkyl, aryl, alkoxy, alcohol, amine, alkylamino or halogen groups; wherein R⁵ and R⁶ may be fused to form a carbocycle or heterocycle; wherein when R⁴ is selected as Ar, Ar is an aryl ring system which may optionally be substituted with alkyl or halogen groups; wherein when R⁴ is selected as NR⁷R⁸, R⁷ and R⁸ are each independently selected from H, alkyl, or aryl or benzyl groups which may be optionally substituted with halogen groups, wherein one or more of the following compounds are excluded from the invention:

2. The compound according to claim 1, wherein when R³ is methyl and R⁴ is CR⁵R⁶Ar, one of R⁵ and R⁶ is not hydrogen.

3. The compound according to claim 1 or claim 2, wherein when R³ is methyl and R⁴ is CR⁵R⁶Ar, Ar is selected from a substituted aryl, a fused aryl, or a heteroaryl ring system, wherein the substituted aryl is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups.

4. The compound according to claim 1 or claim 2, wherein when R³ is methyl, R⁴ is not CR⁵R⁶Ar.

5. A compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from CO₂H, CONA¹ A², CN, CH₂OH, CH₂NHSO₂A³, CONHSO₂A⁵, 5- substituted 177-tetrazole or 3-substituted l,2,4-oxadiazol-5(47/)-one; wherein when R¹ is selected as CONA¹ A², A¹ and A² are each independently selected from H, alkyl groups or alkyl groups substituted with heteroatoms; wherein when R¹ is selected as CH2NHSO2A³ or CONHSO2A⁵, A³, and A⁵ are each alkyl groups;

R² is an alkyl group which may optionally be further substituted with an aryl group; R³ is selected from an alkyl or aryl group, which may be optionally substituted with aryl, heteroaryl, alkyl, halogen or hydroxy groups; R³ may also be selected from a cyclic alkyl group or a heteroalkyl ring;

6. The compound according to claim 5, wherein when R³ is methyl and R⁴ is CR⁵R⁶Ar, Ar is selected from a substituted aryl, a fused aryl, or a heteroaryl ring system, wherein the substituted aryl is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups.

7. The compound according to claim 5, wherein when R³ is methyl, R⁴ is not CR⁵R⁶Ar.

8. A compound of Formula (1):

or a pharmaceutically acceptable salt thereof, wherein:

9. The compound according to claim 8, wherein when R³ is methyl, R⁴ is not CR⁵R⁶Ar.

10. The compound according to claim 8, wherein R³ is methyl and R⁴ is CR⁵R⁶Ar, and one of R⁵ and R⁶ is not hydrogen.

11. The compound according to any of claims 1, 5 or 8, wherein R³ is selected from cyclopropyl, cyclobutyl, cyclohexyl, Cfhcyclopropyl, Cfhcyclobutyl, CH(CH3)C₂Hs where the chiral centre can have either the (R) or (S) configuration, CH(CH3)0H where the chiral centre can have either the (R) or (S) configuration, ethyl, n-butyl, CH₂CH(CH₃)₂, CH₂Ph (benzyl), 4-trifluoromethyl benzyl, 4-methyl benzyl or CH₂-2- pyridyl.

12. The compound according to any of claims 1, 5 or 8, wherein R³ is selected from CH(CH₃)₂ or C(CH₃)₃.

13. The compound according to any preceding claim, wherein when R⁴ is selected as CR⁵R⁶Ar, Ar is selected from an aryl, a fused aryl or a heteroaryl ring system, which is substituted with alkyl, halogen, carboxylic acid or hydroxyl groups, wherein the substitution is in the 4-position.

14. The compound according to any preceding claim, wherein when R⁴ is selected as CR⁵R⁶Ar, at least one of R⁵ or R⁶ is an alkyl group.

15. The compound according to any preceding claim, wherein when R⁴ is selected as CR⁵R⁶Ar and a stereocentre is present in CR⁵R⁶Ar, the stereocentre has an (S) configuration.

16. The compound according to any preceding claim, wherein when R⁴ is selected as CR⁵R⁶Ar, both of R⁵ and R⁶ are alkyl groups which may be fused to form a carbocycle.

17. The compound according to any proceeding claim, wherein R¹ is CO₂H.

18. The compound according to any proceeding claim, wherein R² is selected from CH3, CH₂CH₃, CH₂CH₂CH₃ or CH₂Ph.

19. The compound according to any preceding claim, wherein R² is CH₂CH3.

20. The compound according to any of claims 1, 5 or 8, selected from compounds of the following structural formulae:

21. The compound according to any of claims 1, 5 and 8, selected from compounds of the following structural formulae:

22. A compound according to any of claims 1 to 21 for use as a medicament for the treatment of a disease or disorder.

23. A compound according to any of claims 1 to 21 for use as a potent and selective N0D2 agonist.

24. A compound according to any of claims 1 to 21 for use as a medicament capable of modulating innate immunity in a subject.

25. A compound according to any of claims 1 to 21 for use as a modulator of pro- inflammatory mediator secretion in a subject.

26. A compound according to any of claims 1 to 21 for use in the treatment of Crohn’s disease.

27. A pharmaceutical composition comprising a compound according to any of claims 1 to 21 and a pharmaceutically or therapeutically acceptable excipient or carrier.

28. A method of treating a disease or disorder, comprising the step of administering a compound according to any of claims 1 to 21 or pharmaceutical composition according to claim 27 to a subject in need of the same.

29. A method of modulating innate immunity in a subject, comprising the step of administering a compound according to any of claims 1 to 21 or pharmaceutical composition according to claim 27 to a subject in need of the same.

30. A method of treating Crohn’s disease, comprising the step of administering a compound according to any of claims 1 to 21 or pharmaceutical composition according to claim 27 to a subject in need of the same.

31. The method according to any of claims 28 to 30 wherein the treatment is affected or facilitated by the compound acting as a potent and selective NOD2 agonist.

32. Use of a compound according to any of claims 1 to 21 in the treatment of a disease or disorder.

33. Use of a compound according to any of claims 1 to 21 in modulating innate immunity in a subject.

34. Use of a compound according to any of claims 1 to 21 in the treatment of Crohn’s disease.

35. The use according to any of claims 32 to 34 as a potent and selective NOD2 agonist.

36. Use of a compound according to any of claims 1 to 21 in the manufacture of a medicament for the treatment of a disease or disorder.

37. Use of a compound according to any of claims 1 to 21 in the manufacture of a medicament capable of modulating innate immunity in a subject.

38. Use of a compound according to any of claims 1 to 21 in the manufacture of a medicament for the treatment of Crohn’s disease.

39. The use according to any of claims 36 to 38 as a potent and selective NOD2 agonist.