GB2574862A

GB2574862A - Azaheterocyclic small molecule modulators of human STING

Info

Publication number: GB2574862A
Application number: GB201810172A
Authority: GB
Inventors: Banerjee Monali; Middya Sandip; Basu Sourav; Ghosh Rajib; Cameron Pryde David; Yadav Dharmendra; Shrivastava Ritesh; Surya Arjun
Original assignee: Curadev Pharma Ltd
Current assignee: Curadev Pharma Ltd
Priority date: 2018-06-21
Filing date: 2018-06-21
Publication date: 2019-12-25
Also published as: GB201810172D0

Abstract

New compounds as modulators of STING protein, and the use of such compounds in therapy Compounds of formula (I) are as disclosed herein. Pharmaceutically acceptable salts and prodrugs of (I) are encompassed. Most preferably, Q is C=O. Most preferably, X1-X3 are each carbon atoms. Y is, preferably, C1-C6 alkyl. Both L and Y may be methylene. Most preferably, Z is NR10, e.g. (I) is a benzene ring fused to a partially-saturated triazine; e.g. a tetrahydrobenzo[e][1,2,4]triazine-6-carbonamide moiety. Claimed are pharmaceutical compositions comprising (I), and a process for producing such compositions. Compounds of formula (I) may be used in therapy, e.g. for modulating the stimulator of interferon genes (STING) protein. (I) may be used to treat diseases such as cancer (sarcoma, carcinoma etc.), infection, neurodegenerative disease, cardiovascular disease etc. A conjugate may be formed through linking (I) and a targeting moiety. Also disclosed are compounds not within the scope of (I) where, e.g. the carboxamide unit is replaced with an ester group.

Description

Azaheterocyclic Small Molecule Modulators of Human STING

The present invention relates to small molecules for use in modulating the Stimulator of Interferon Genes (STING) protein. Accordingly, the small molecules maybe for use 5 in the treatment of diseases, such as cancer and microbial infections, and so on. The invention extends to the compounds per se pharmaceutical compositions, methods of making the compounds and methods of modulating the STING protein.

The human immune system may generally be divided into two arms, referred to as the 10 ‘innate immune system’ and the ‘adaptive immune system’. The innate arm is mainly responsible for an initial inflammatory response via a number of factors such as cytokines, chemokines and complement factors. These factors act upon a number of different cell types including mast cells, macrophages, dendritic cells and natural killer cells. The adaptive arm involves a delayed and longer lasting response to challenge via 15 antibody production together with CD8+ and CD4+ T-cell responses that are critical for immunological memory.

Research has been conducted for many years on how the immune system can recognise and eliminate malignant tumors (Parish et. al., Immunol and Cell Biol, 2003, 81,10620 113). One of the pioneers in this area is William Coley, who in the late 1800’s noted that a cancer patient had a complete remission of their cancer after acute infection with the bacteria Streptococcus pyogenes. Subsequent studies with Coley’s toxin and with bacille Calmette-Guerin (BCG) for cancer immunotherapy provided some clinical success but by no means offered a panacea for tumor treatment (Coley, Am J Med Sci., 25 1893,105, 487-511). Through the 1900’s, opinions fluctuated on the benefits of immunotherapy, with theories of acquired immunological tolerance (Burnet,, Lancet, 1967,1,1171-1174 and Matzinger, Ann. Rev. Immunol., 1994,12, 991-1045 and Smyth et. al., Nat Immunol., 2.001, 2, 293-299) and tumor-associated antigens (Rosenberg et. al., Immunity, 1999,10, 281-287) gaining support with the emergence of the innate 30 immune system as an important mediator of immunity (Lanier, Nat Med. 2001, z,

1178-1180 and Mayardomo et al., Nat Med. 1995,1,1297-1302 and Medzhitov et al., Trends Microbiol., 2000, 8, 452-456 and Akira et. al., Nat. Immunol., 2001, 2, 675680). The detection of pathogen-associated molecular patterns (PAMPs) such as nucleic acids is now recognized as a central strategy by which the innate immune 35 system senses microbes and tumor-associated antigens to then initiate protective responses (Barbalat et. al., Annu. Rev. Imunol., 2011, 29.185-214).

- 2 As described above, innate immunity is initiated when PAMPs or damage-associated molecular patterns (DAMPs) are detected by pattern recognition receptors which include TLRs, NOD-like receptors and RIG-I-like receptors. These pattern recognition 5 receptors respond to DAMPs and PAMPs by up-regulating Type-1 interferons and cytokines. Cytosolic nucleic acids are known PAMPs/DAMPs and engage the STING protein to stimulate the innate immune system and promote an antitumor response. Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of cyclic dinucleotides (CDNs). CDNs are second messenger signalling molecules produced by diverse bacteria and consist of two ribonucleotides that are connected via phosphodiester bonds to make a cyclic structure. CDNs Cyclo-di(GMP), cyclo-di(AMP) and hybrid cyclo-(AMP/GMP) derivatives all bind to STING with subsequent activation of the interferon pathway (Gao et. al., Cell, 2013,153,1094-1107; Zhang et. al., Mol. Cell, 2013,51,226-235). The canonical s’-3’ phosphodiester linkage is recognised along with various other linkage isomers (notably the 5’-2’ linkage, e.g. c[G(2’,5’)pA(3’,5’)p]) which all bind to STING with various affinities (Shi et. al., PNAS, 2015,112,19478952). These observations have been corroborated by structural studies (Gao et. al., Cell, 2013,154, 748-762) of various linkage isomers of CDNs bound to the human and mouse STING proteins.

One possible mechanism by which traditional vaccine adjuvants, such as alum, potentiate an immune response is through the release of DAMPs. Adjuvants, such as alum, trigger the release of host cell DNA, which can promote a Th2 response, induce T cell responses and the production of IgGi and IgE. Ideally, adjuvants should be molecularly defined and able to enhance the magnitude and timeframe of a specific immune response to an antigen that offers protection against intracellular pathogens and/or reduce tumor burden.

Activation of the STING protein can create an activated or primed immune system, similarly to that generated by an adjuvant. This may produce a protective or prophylactic state upon challenge or re-challenge by intracellular pathogens or by tumors which inhibits the growth or propagation of intracellular pathogens or tumors.

It can also be appreciated that when a STING activator is administered therapeutically 35 to a system in which tumors/pathogens are present it can act beneficially in two different, but related, ways. Firstly, by direct shrinkage of tumors/pathogen eradication

-3through up-regulation of Type-I interferons and cytokines to act directly upon the tumor/pathogens, as described above. Secondly, a STING activator will also induce a lasting immune response, such that re-challenge or re-inoculation with a pathogen or tumors will be resisted both through a general activation of the immune system and through a latent antigen-specific response to said pathogen or tumor.

Tumor immunosurveillance does occur with, for example, thriving tumors having been immunoselected to evade immune elimination and indeed, the crucial role that the innate immune system plays in tumor clearance puts Coley’s original findings in a new 10 light. It is now clear that fragments of cyclic nucleotides, oligonucleotides and double stranded motifs can all activate the innate immune system through toll-like receptors (Horscroft, J.Antimicrob. Ther., 2.012., 67(4), 789-801 and Diebold et al., Science, 2004, bob, 1529-1531), RIG-I like receptors (Pichlmair et. al., Science, 2006, B14, 9971001) and stimulator of IFN genes (STING) adaptor proteins (Burdette et. al., Nat.

Immunol., 2013,14(1), 19-26).

This developing knowledge has stimulated considerable research into possible therapeutic applications of immunomodulation via some of these target classes.

STING has emerged more recently as a critical signalling molecule in the innate response to cytosolic nucleic acid molecules (Burdette and Vance, Nat. Immunol, 2013, 14,19-26). STING plays a role in the transcriptional induction of Type I interferons and coregulated genes in response to nucleic acids in the cytosol. Studies in STINGdeficient mice have confirmed the role of STING in innate responses to cytosolic nucleic-acid ligands, particularly double stranded DNA and bacterial nucleic acids based on a cyclic dinucleotide structure (Ishikawa et. al., Nature, 2009, 461, 788-792). STING has a critical role in the innate response to many bacterial, viral and eukaryotic pathogens (Watson et. al., Cell, 2012,150, 803-815; de Almeida et. al., PLoS One, 2011, 6, 623135; Holm et. al, Nat. Immunol, 2012,13, 737-743; Stein et. al., J. Virol.,

2012, 86.4527-4537; Sharma et. al., Immunity, 2011, 35,194-207).

STING is broadly expressed throughout the body in both immune cells and nonimmune cells, for example in the spleen, heart, thymus, placenta, lung and peripheral leukocytes, indicating a role in triggering the innate immune system in response to

PAMPs/DAMPs (Sun et. al., PNAS, 2009,106, 8653-8658). Its expression in immune cells leads to rapid amplification of the initial immune signal and maturation of APCs.

-4It is expressed in several transformed cell lines including HEK293 human embryonic kidney cells, A549 adenocarcinomic human alveolar basal epithelial cells, THP-i monocytic cells and U937 leukemic monocytic lymphoma cells.

STING also has a central role in certain autoimmune disorders initiated by inappropriate recognition of self DNA (Gall et. al., Immunity, 2.012., 36,120-131) and has been proposed to sense membrane-fusing events associated with viral entry, in a manner independent of the sensing of nucleic acids (Holm et. al., Nat. Immunol., 2012,13, 737-743)·

STING is comprised of an N-terminal transmembrane domain, a central globular domain and a C-terminal tail. The protein forms a symmetrical dimer in the ligand bound state, with the cyclic dinucleotides binding at a dimer interface binding pocket. Binding of CDNs to STING activates a cascade of events whereby the protein recruits and activates IkB kinase (IKK) and TANK-binding kinase (TBK1), which following their phosphorylation activate nuclear transcription factors (NFkB) and interferon regulatory factor 3 (IRF3), respectively. These activated proteins translocate to the nucleus to induce transcription of the genes that encode Type I interferon and cytokines for promoting intercellular immune system defense. Sequence variations are known between human and mouse STING proteins, and betweenSTING proteins within the human population. Several naturally occurring variant alleles have been identified.

Derivatives of the CDN class are currently being developed as antitumor agents upon intratumoral injection (Corrales et.al., Cell Rep., 2015,19,1018-1030). The xanthenebased small molecule 5,6-dimethyl-xanthenone acetic acid (DMXAA) was initially identified as a small molecule exhibiting immune modulatory activities through induction of cytokines and disrupting tumor vascularization in mouse xenograft models (Baguley and Ching, Int. J. Radiat. Oncol. Biol. Phys., 2002, S4,1503-1511). This promising efficacy led to its investigation in a Phase II clinical trial against non-small cell lung carcinoma but subsequently failed its endpoints. The mechanism of DMXAA’s activity against murine tumors was eventually ascribed to its activity as a murine

STING activator. Its failure in human clinical trials was due to the fact that DMXAA was only capable of activating mouse STING and not human STING (Lara et. al., J.

-5Clin. Oncol., 2011, 29, 2965-2971; Conlon et. al., J. Immunol., 2013,190, 5216-5225). This lack of human activity has hampered all further attempts to develop this agent as a tumor therapy. Recently, a related small molecule io-carboxymethyl-9-acridanone (CMA) (Caviar et. al., EMBOJ., 2013,32,1440-1450) has been found to bind to mouse

STING, but also not to human STING. Both DMXAA and CMA have been shown to bind two molecules of each ligand to the STING dimer at a region close to the dimer interface.

Accordingly, there remains a need in the art for improved therapies for treating diseases, such as cancer, which can be refractory to traditional therapeutic approaches. Immunologic strategies show promise for the treatment of cancer, and there is a need to develop improved compositions and methods in this field. In particular, there is a need for compounds that modulate the human STING protein, as well as methods for treating diseases that can benefit from such modulation.

The present invention has arisen from the inventors work in attempting to identify STING protein modulators.

Hence, in a first aspect of the invention, there is provided a compound of formula (I):

CD or a pharmaceutically acceptable salt or prodrug thereof, wherein:

XGs CR¹ orN;

X²is CR²orN;

X³isCR³orN;

Z is NR¹⁰ or CR^1OR^U;

Q is C=O, S=O, S0₂, C=S or CFUR⁵;

L is optionally substituted G-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C2-C6 alkynyl, C=O, S=O, S0₂,

-CH₂C(0)-, -CH2CONH-, or -CONH-;

-6Y is an optionally substituted Ci-Ce alkyl, an optionally substituted C₂-C6 alkenyl, an optionally substituted C₂-C6 alkynyl, an optionally substituted C₃-C6 cycloalkyl, or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle;

R¹, R² and R³ are each independently selected from the group consisting of H, halogen,

CN, hydroxyl, COOH, CONR¹²R¹³, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted mono or bicyclic C3-C6 cycloalkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl group, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic 3 to 8 membered heterocycle, optionally substituted aryloxy, optionally substituted heteroaryloxy, and optionally substituted heterocyclyloxy;

R4 and R⁵ are each independently selected from the group consisting of H, halogen, optionally substituted Ci-Ce alkyl and optionally substituted C3-C6 cycloalkyl; or R⁴ and

R⁵ together with the atom to which they are attached form a spirocyclic ring;

R⁶ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted C3-C6 cycloalkyl or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle;

R⁷ is H, optionally substituted Ci-Ce alkyl, optionally substituted sulfonyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C₂-C& alkenyl or optionally substituted C₂-C& alkynyl;

R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic C₃-Ce cycloalkyl or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle;

R9 is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C₂-C& alkenyl, H, hydroxyl, C0NR¹²R¹³, sulfonyl, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce thioalkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted heterocycle, optionally substituted aryloxy, and an optionally substituted heteroaryloxy;

when Z is NR¹⁰ then R¹⁰ is selected from the group consisting of optionally substituted Ci-Ce alkyl, H, hydroxyl, optionally substituted Ci-Ce thioalkyl, optionally substituted

Ci-Ce alkylsulfonyl, optionally substituted C3-C6 cycloalkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy,

-7optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted C₅Cio aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted heterocycle, optionally substituted aryloxy, and an optionally substituted heteroaryloxy;

when Z is CR¹⁰Rⁿ then R¹⁰ and R¹¹ are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, H, hydroxyl, CONR¹²R¹³, sulfonyl, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce thioalkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C₃-Ce cycloalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted C₅C10 aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted heterocycle, optionally substituted aryloxy, an optionally substituted heteroaryl oxy, CN, halogen, azido and C0₂H; or R¹⁰ and R¹¹ together with the atoms to which they are attached can combine to form an optionally substituted spirocyclic ring;

or, when Z is NR¹⁰ or CR¹⁰Rⁿ, then R⁹ andR¹⁰ together with the atoms to which they are attached can combine to form an optionally substituted fused ring; and

R¹² and R¹³ are each independently selected from the group consisting of H, halogen,

CN, hydroxyl, COOH, C0NH₂, NH₂, NHCOH, optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted mono or bicyclic C3-C6 cycloalkyl, optionally substituted C₂-C6 alkenyl, optionally substituted C₂-C6 alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl group, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic 3 to 8 membered heterocycle, optionally substituted aryloxy, optionally substituted heteroaryloxy, and optionally substituted heterocyclyloxy.

The inventors have found that the compounds of formula (I) are useful in therapy or as a medicament.

Hence, in a second aspect, there is provided a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, for use in therapy.

The inventors have also found that compounds of formula (I) are useful in modulating the Stimulator of Interferon Genes (STING) protein.

-8Hence, in a third aspect, there is provided a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, for use in modulating the Stimulator of Interferon Genes (STING) protein.

Preferably, the compound of formula (I) is for use in activating, or agonising, the STING protein.

Advantageously, the compounds of the invention modulate the major human polymorphs of the human STING protein. There are several STING polymorphs reported, but the 5 polymorphs listed below are the major ones which comprise almost 99% of the total human population. Accordingly, the STING protein may be a wild type polymorph (WT/R232), a HAQ polymorph, a REF polymorph (H232), an AQ polymorph or a Q polymorph. As shown in Figure 1, the wild type polymorph has arginines at the 71, 232 and 293 positions and a glycine at the 230 position, the HAQ polymorph has a histidine at the 71 position, an alanine at the 230 position, an arginine at the 232 position and a glutamine at the 293 position, the REF polymorph has arginines at the 71 and 293 positions, a glycine at the 230 position and a histidine at the 20 232 position, the AQ polymorph has arginines at the 71 and 232 positions, an alanine at the 230 position and a glutamine at the 293 position, and the Q polymorph has arginines at the 71 and 232 positions, a glycine at the 230 position and a glutamine at the 293 position.

By modulating the STING protein, it is possible to treat, ameliorate or prevent cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immunemediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, mood disorder, sleep disorder, cerebrovascular disease, peripheral artery disease or cardiovascular disease.

Accordingly, in a fourth aspect there is provided a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, for use in treating, ameliorating or preventing a disease selected from cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immune35 mediated disorder, central nervous system disease, peripheral nervous system disease,

-9neurodegenerative disease, mood disorder, sleep disorder, cerebrovascular disease, peripheral artery disease or cardiovascular disease.

Preferably, the disease is cancer.

In a fifth aspect, there is provided a method of modulating the Stimulator of Interferon Genes (STING) protein in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.

Preferably, the method comprises activating the STING protein.

The STING protein maybe a wild type polymorph, a HAQ polymorph, a REF polymorph, an AQ polymorph or a Q polymorph.

In a sixth aspect, there is provided a method of treating, ameliorating or preventing a disease selected from cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immune-mediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, mood disorder, sleep disorder, cerebrovascular disease, peripheral artery disease or cardiovascular disease, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.

Preferably, the disease is cancer.

It maybe appreciated that the term “preventing” can mean “reducing the likelihood of’.

The neurodegenerative disease may be Alzheimer’s disease or dementia. The viral disease maybe Hepatitis. The parasitic infection maybe malaria. The mood disorder maybe depression. The sleep disorder maybe insomnia.

In one preferred embodiment, the disease is cancer. The cancer maybe selected from the group consisting of colorectal cancer, aero-digestive squamous cancer, lung cancer, brain cancer, liver cancer, stomach cancer, sarcoma, leukaemia, lymphoma, multiple

- 10 myeloma, ovarian cancer, uterine cancer, breast cancer, melanoma, prostate cancer, bladder cancer, pancreatic carcinoma or renal carcinoma.

In an alternative preferred embodiment, the disease is a viral infection. The viral infection may be a hepatitis C virus (HCV) infection.

The following definitions are used in connection with the compounds of the present invention unless the context indicates otherwise.

Throughout the description and the claims of this specification the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions.

“Optional” or “optionally” means that the subsequently described event, operation or circumstances can or cannot occur, and that the description includes instances where the event, operation or circumstance occurs and instances where it does not.

The term “alkyl” as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, the alkyl group includes one to six carbon atoms, i.e. Ci-Ce alkyl. Ci-Ce alkyl includes for example methyl, ethyl, n-propyl (1-propyl) and isopropyl (2-propyl, i-methylethyl), butyl, pentyl, hexyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl. An alkyl group can be unsubstituted or substituted with one or more of halogen, OH, Ci-Ce alkoxy, NR^R^, CONR^R^, CN, COOH, C₅-Ci₀ aryl, 5 to 10 membered heteroaryl, C₃-Ce cycloalkyl and 3 to 8 membered heterocycle. Accordingly, it will be appreciated that an optionally substituted Ci-Ce alkyl may be an optionally substituted Ci-Ce haloalkyl, i.e. a Ci-Ce alkyl substituted with at least one halogen, and optionally further substituted with one or more of OH, Ci-Ce alkoxy, NR^R^, CONR^R¹?-, CN, COOH, C₅-Ci₀ aryl, 5 to 10 membered heteroaryl, C3-C6 cycloalkyl and 3 to 8 membered heterocycle. The

- 11 optionally substituted Ci-Ce alkyl may be a polyfluoroalkyl. R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

The term “halo” includes fluoro (-F), chloro (-C1), bromo (-Br) and iodo (-1).

The term “polyfluoroalkyl” may denote a Ci-C₃ alkyl group in which two or more hydrogen atoms are replaced by fluorine atoms. The term may include perfluoroalkyl groups, i.e. a Ci-C₃ alkyl group in which all the hydrogen atoms are replaced by fluorine io atoms. Accordingly, the term Ci-C₃ polyfluoroalkyl includes, but is not limited to, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 3,3,3trifluoropropyl, 2,2,3,3,3-pentafluoropropyl, and 2,2,2-trifluoro-i(trifluoromethyl)ethyl.

“Alkoxy” refers to the group R¹⁴-O- where R¹⁴ is an optionally substituted Ci-Ce alkyl group or an optionally substituted C₃-Ce cycloalkyl group. Exemplary Ci-Ce alkoxy groups include but are not limited to methoxy, ethoxy, n-propoxy (1-propoxy), nbutoxy and tert-butoxy. An alkoxy group can be unsubstituted or substituted with one or more of halogen, OH, 0P(0)(0H)₂, alkoxy, NR¹²R¹³, C0NR¹²R^1!, CN, COOH, aryl, heteroaryl, cycloalkyl and heterocycle. R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Thioalkyl” refers to the group R¹⁴-S- where R¹⁴ is an optionally substituted Ci-Ce alkyl group or an optionally substituted C₃-Ce cycloalkyl group. A thioalkyl group can be unsubstituted or substituted with one or more of halogen, OH, alkoxy, NR¹²R¹³, CONR¹²R¹³, CN, COOH, aryl, heteroaryl, cycloalkyl and heterocycle. R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Aryl” refers to an aromatic 5 to 10 membered hydrocarbon group. Examples of a C₅C10 aryl group include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, tetrahydronaphthyl and indanyl. An aryl group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, OH, 0P(0)(0H)₂, optionally substituted Ci-Ce alkoxy, NR¹²R¹³, CONR¹²R¹³, CN, COOH,

C00R¹², 0C(0)R¹², 0C(0)0R¹², OC(O)NR¹²R¹³, N0₂, azido, aryloxy, heteroaryloxy, 5 to membered heteroaryl, 3 to 8 membered heterocycle, S0₂R¹² and NHCOR¹². R¹² and

- 12 may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

The term “bicycle” or “bicyclic” as used herein refers to a molecule that features two fused rings, which rings are a cycloalkyl, heterocyclyl, or heteroaryl. In one embodiment, the rings are fused across a bond between two atoms. The bicyclic moiety formed therefrom shares a bond between the rings. In another embodiment, the bicyclic moiety is formed by the fusion of two rings across a sequence of atoms of the rings to form a bridgehead. Similarly, a “bridge” is an unbranched chain of one or more atoms connecting two bridgeheads in a polycyclic compound. In another embodiment, the bicyclic molecule is a “spiro” or “spirocyclic” moiety. The spirocyclic group may be a C₃-C6 cycloalkyl or a mono or bicyclic 3 to 8 membered heterocycle which is bound through a single carbon atom of the spirocyclic moiety to a single carbon atom of a carbocyclic or heterocyclic moiety. In one embodiment, the spirocyclic group is a cycloalkyl and is bound to another cycloalkyl. In another embodiment, the spirocyclic group is a cycloalkyl and is bound to a heterocyclyl. In a further embodiment, the spirocyclic group is a heterocyclyl and is bound to another heterocyclyl. In still another embodiment, the spirocyclic group is a heterocyclyl and is bound to a cycloalkyl. A spirocyclic group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, OH, optionally substituted Ci-C₆ alkoxy, NR¹²R¹³, CONFUR¹?-, CN, COOH, N0₂, azido, and NHCOR¹². R¹² and R¹³may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Alkoxycarbonyl” refers to the group alkyl-O-C(O)-, where alkyl is a Ci-Ce alkyl. An alkoxycarbonyl group can be unsubstituted or substituted with one or more of halogen, OH, NR¹²R¹³, CN, Ci-Ce alkoxy, COOH, C₅-Ci₀ aryl, 5 to 10 membered heteroaryl or C3-C6 cycloalkyl. R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Aryloxy” refers to the group Ar-O- where Ar is a mono or bicyclic optionally substituted C₅-Ci₀ aryl group, as defined above.

“Cycloalkyl” refers to a non-aromatic, saturated, partially saturated, monocyclic, bicyclic or polycyclic hydrocarbon 3 to 6 membered ring system. Representative examples of a C3-C6 cycloalkyl include, but are not limited to, cyclopropyl,

-13cyclobutyl, cyclopentyl, cyclohexyl. A cycloalkyl group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, CN, hydroxyl, COOH, CONR¹²R¹³, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce alkoxy, azido, aryloxy, heteroaryloxy,, mono or bicyclic optionally substituted C₅5 Cio aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic 3 to 8 membered heterocycle, optionally substituted C3-C6 cycloalkyl or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle. R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Heteroaryl” refers to a monocyclic or bicyclic aromatic 5 to 10 membered ring system in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen. Examples of 5 to 10 membered heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole,

1,3,4-oxadiazole, 1,2,4-triazole, 1- methyl-i,2,4-triazole, iH-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, Nmethylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic 5 to 10 membered heteroaryl groups include those where a phenyl, pyridine, pyrimidine, pyrazine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring. A heteroaryl group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, OH, CN, NR¹²R¹³, azido, COOH, Ci-C₆ alkoxycarbonyl, CONR¹²R¹³, C00R¹², 0C(0)R¹², 0C(0)0R¹²,

OC(O)NR¹²R¹³, N02, NHCOR¹² and S02R¹². R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Heterocycle” or “heterocyclyl” refers to 3 to 8 membered monocyclic, bicyclic or bridged molecules in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen. A heterocycle may be saturated or partially saturated. Exemplary 3 to 8 membered heterocyclyl groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,635 tetrahydropyridine-i-yl, tetrahydropyran, pyran, morpholine, piperazine, thiane, thiine, piperazine, azepane, diazepane, oxazine. A heterocyclyl group can be

-14unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, optionally substituted Ci-Ce alkoxy, OH, NR¹²R¹³, COOH, Ci-Ce alkoxycarbonyl, CONR¹²R¹³, N02, NHCOR¹², mono or bicyclic optionally substituted C5-C10 aryl and S02R¹². R¹² and R¹³ may each independently be selected from the group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Alkenyl” refers to olefinically unsaturated hydrocarbon groups which can be unbranched or branched. In certain embodiments, the alkenyl group has 2 to 6 carbons, i.e. it is a C₂-Ce alkenyl. C₂-Ce alkenyl includes for example vinyl, allyl, 10 propenyl, butenyl, pentenyl and hexenyl. An alkenyl group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, OH, optionally substituted Ci-C₆ alkoxy, NR¹²R¹³, CONR¹²R¹³, S0₂R¹², NHCOR¹², CN, COOH, C5-C10 aryl, 5 to 10 membered heteroaryl, C3-C6 cycloalkyl, aryloxy, heteroaryloxy, and 3 to 8 membered heterocycle. R¹² and R¹³ may each independently be selected from the 15 group consisting of H, halogen and optionally substituted Ci-Ce alkyl.

“Alkynyl” refers to acetylenically unsaturated hydrocarbon groups which can be unbranched or branched. In certain embodiments, the alkynyl group has 2 to 6 carbons, i.e. it is a C₂-Ce alkynyl. C₂-Ce alkynyl includes for example propargyl, 20 propynyl, butynyl, pentynyl and hexynyl. An alkynyl group can be unsubstituted or substituted with one or more of optionally substituted Ci-Ce alkyl, halogen, OH, Ci-Ce alkoxy, NR¹²R¹³, CONR¹²R¹³, S02R¹², NHCOR¹³, CN, COOH, C5-C₁₀ aryl, 5 to 10 membered heteroaryl, C3-C6 cycloalkyl, aryloxy, heteroaryloxy, and 3 to 8 membered heterocycle. R¹² and R¹³ may each independently be selected from the group consisting 25 of H, halogen and optionally substituted Ci-Ce alkyl.

“Alkylsulfonyl” refers to the group alkyl-S0₂- where alkyl is an optionally substituted Ci-Ce alkyl, and is as defined as above.

“Heteroaryloxy” refers to the group heteroaryl-O- where the heteroaryl is a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, and is as defined above.

“Heterocyclyloxy” refers to the group heterocycle-O- where heterocycle is an optionally substituted mono or bicyclic 3 to 8 membered heterocycle, and is as defined 35 as above.

-ι₅A complex of the compound of formula (I) may be understood to be a multi-component complex, wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. The complex may be other than a salt or solvate. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together - see Chem Commun, 17,1889-1896, by O.

Almarsson and M. J. Zaworotko (2004), incorporated herein by reference. For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975), incorporated herein by reference.

The term “pharmaceutically acceptable salt” may be understood to refer to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, adepic, aspartic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 225 naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4methylbicyclo[2.2.2]-oct-2-ene-i-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminium ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine,

-16chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, Nmethylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.

Pharmaceutically acceptable salts may include, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride, hydrobromide and hydroiodide, carbonate or bicarbonate, sulfate or bisulfate, borate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, palmitate, pamoate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, tosylate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, camsylate, citrate, cyclamate, benzoate, isethionate, esylate, formate, 3-(415 hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methylsulphate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-i-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluceptate, gluconate, glucoronate, hexafluorophosphate, hibenzate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate, xinofoate and the like.

Hemisalts of acids and bases may also be formed, for example, hemisulphate salts.

The skilled person will appreciate that the aforementioned salts include ones wherein the counterion is optically active, for example D-lactate, or racemic, for example DLtartrate.

For a review on suitable salts, see “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Pharmaceutically acceptable salts of compounds of formula (I) maybe prepared by one or more of three methods:

(i) by reacting the compound of formula (I) with the desired acid or base;

(ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula (I) using the desired acid or base; or (iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.

The term “solvate” maybe understood to refer to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D₂0, de-acetone and de-DMSO.

A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates - see Polymorphism in

Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995), incorporated herein by reference. Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline, including polymorphs of said crystalline material.

The term ‘amorphous’ refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical

-18properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order (‘glass transition’).

The term ‘crystalline’ refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order (‘melting point’).

The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described 15 as ‘thermotropic’ and that resulting from the addition of a second component, such as water or another solvent, is described as ‘lyotropic’. Compounds that have the potential to form lyotropic mesophases are described as ‘amphiphilic’ and consist of molecules which possess an ionic (such as -COONa⁺, -COOK+, or -SO3 Na⁺) or non-ionic (such as -N-N⁺(CH3)₃) polar head group. For more information, see Crystals and the Polarizing 20 Microscope by N. H. Hartshorne and A. Stuart, 4^th Edition (Edward Arnold, 1970), incorporated herein by reference.

In one embodiment, Z may be CR^1OR^U. Accordingly, the compound may be represented by Formula (Ia):

(Ia)

R¹⁰ and R¹¹ may independently be an optionally substituted Ci-Ce alkyl, H, optionally substituted C2-C6 alkenyl or a C₃-Ce cycloalkyl. More preferably, R¹⁰ and R¹¹ are each 30 independently an optionally substituted Ci-Ce alkyl, H, optionally substituted C2-C6 alkenyl or a C₃-Ce cycloalkyl, even more preferably an optionally substituted Ci-C₃ alkyl or a C₃-Ce cycloalkyl, and most preferably R¹⁰ is methyl, ethyl, propyl, allyl or

-19cyclopropyl and R¹¹ is H. The Ci-Ce or C1-C3 alkyl may be optionally substituted with one or more substituents selected from a halogen, OH and/or CN. In one embodiment, R¹⁰ is methyl and R¹¹ is H.

Alternatively, R⁹ and R¹⁰ together with the atoms to which they are attached can combine to form an optionally substituted fused ring. Preferably, R⁹ and R¹⁰ together with the atoms to which they are attached form a five or six-membered ring, most preferably a five-membered ring. Preferably R¹¹ is H.

Alternatively, R¹⁰ and R¹¹ together with the atoms to which they are attached can combine to form an optionally substituted spirocyclic ring. Preferably, R¹⁰ and R¹¹together with the atoms to which they are attached form a five or six-membered ring.

In a preferred embodiment, Z is NR¹⁰. Accordingly, the compound maybe represented 15 by Formula (Ib):

R⁹ (Ib)

Preferably, R¹⁰ is an optionally substituted Ci-Ce alkyl, H, an optionally substituted C220 Ce alkenyl or a C3-Ce cycloalkyl. More preferably, R¹⁰ is a Ci-Ce alkyl, a C2-C6 alkenyl, or a C3-Ce cycloalkyl, even more preferably a Ci-C₃ alkyl, a C₂-C₃ alkenyl or a C₃-Ce cycloalkyl, and most preferably R¹⁰ is methyl, ethyl, propyl, allyl or cyclopropyl.

Alternatively, R⁹ and R¹⁰ together with the N atoms to which they are attached can combine to form an optionally substituted fused ring. Preferably, R⁹ and R¹⁰ together with the N atoms to which they are attached form a five or six-membered ring, most preferably a five-membered ring.

Q maybe C=O, S0₂, S=O, CR⁴R⁹ or C=S. Accordingly, the compound maybe represented by any one of Formula (I-I-I) to (I-I-V);

R⁹ (l-l-IV)

(l-l-V)

In a preferred embodiment, Q is C=0, S0₂ or CR⁴R⁵. Preferably, Q is C=0 or CR⁴R⁵. Preferably, R⁴ and R⁵ are each independently selected from the group consisting of H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C₃-C6 cycloalkyl or R⁴and R⁵ together with the atom to which they are attached form a spirocyclic ring. Accordingly, R⁴ and R⁵ may both be H. Alternatively, R⁴ and R⁵ may both be methyl or R⁴ may be methyl and R⁵ may be H.

Most preferably, Q is C=0.

Compounds of formula (I) may include one or more stereogenic centers and so may exist as optical isomers, such as enantiomers and diastereomers. All such isomers and mixtures thereof are included within the scope of the present invention.

In one embodiment, Z is CR^1OR^U. Accordingly, the compound maybe a compound of formula (I)-ent 1 or (I)-ent 2:

(I)-ent 1

(I)-ent 2

Alternatively or additionally, Q is CR⁴R⁵. Accordingly, the compound may be a compound of formula (I)-ent 3 or (I)-ent 4:

R⁹ (I) ent.3

R⁹ (I) ent.4

In one embodiment, when Q is CR⁴R⁴ and Z is CR¹⁰Rⁿ, the compound could possess two chiral centres, and could be a compound of formula (lA-I)-ent 1, formula (lA-I)-ent 5 2, formula (lA-I)-ent 3 or formula (lA-I)-ent 4:

(I_A-I)-ent 1

(I_A-I)-ent 2

(I_A-I)-ent 3

(I_A-I)-ent 4

It will be understood that the above compounds may exist as enantiomers and as diastereoisomeric pairs. These isomers also represent further embodiments of the invention.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).

Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be

- 22 separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.

Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from o to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine.

Concentration of the eluate affords the enriched mixture.

Mixtures of stereoisomers may be separated by conventional techniques known to those skilled in the art; see, for example, “Stereochemistry of Organic Compounds” by E. L. Eliel and S. H. Wilen (Wiley, New York, 1994).

In one embodiment, one or two of X¹, X² and X³ is N. Accordingly, X¹ may be N, X² may be CR² and X³ may be CR³, X¹ may be CR¹, X² may be N and X³ may be CR³ or X¹ may be CR¹, X² may be CR² and X³ may be N. Accordingly, the compound may be a compound of any one of Formula (I-I-I-I) to (Ι-Ι-Ι-ΠΙ):

Preferably X² is CR². Accordingly, X¹ may be CR¹ or N and X³ may be CR³ or N. X¹ may be N, X² may be CR² and X³ may be CR³, or X¹ may be CR¹, X² may be CR² and X³ may be N, or X¹ may be N, X² may be CR² and X³ may be N. Preferably, R² is H, halogen, OH, CN, optionally substituted C1-C3 alkyl, optionally substituted C₂-C₃ alkenyl, optionally substituted C₂-C₃ alkynyl, or optionally substituted C1-C3 alkoxy. The C1-C3 alkyl, C₂-C₃ alkenyl or C₂-C₃ alkynyl may be optionally substituted with one or more substituents selected from a halogen, OH, CN and/or NR¹²R¹³. Preferably, R¹² and R¹³are both H. The optionally substituted C1-C3 alkoxy may be substituted with one or more substituents selected from OH and COOH. More preferably, R² is H, OH, halogen, methyl or OMe. Most preferably, R² is H.

-23Preferably, R¹ and/or R3, in embodiments where they are present, are independently H, halogen or Ci-C₃ alkyl. More preferably, R¹ and/or R3, in embodiments where they are present, are independently H, halogen or methyl. Most preferably, R¹ and/or R³, in embodiments where they are present, are H.

In a preferred embodiment, X¹ is CR¹, X² is CR² and X³ is CR³. R¹, R² and R³ may each independently be selected from the group consisting of H, halogen, and optionally substituted Ci-Ce alkyl. Preferably, R² is H, halogen, OH, CN, optionally substituted CiC₃ alkyl, optionally substituted C₂-C₃ alkenyl, optionally substituted C₂-C₃ alkynyl, or optionally substituted Ci-C₃ alkoxy. The Ci-C₃ alkyl, C₂-C₃ alkenyl or C₂-C₃ alkynyl may be optionally substituted with one or more substituents selected from a halogen, OH, CN and/or NR¹²R¹³. Preferably, R¹² and R¹³ are both H. The optionally substituted CiC₃ alkoxy may be substituted with one or more substituents selected from OH and COOH. More preferably, R² is H, OH, halogen, methyl or OMe. Most preferably, R² is

H. Preferably, R¹ and R³ are each independently selected from the group consisting of

H, halogen, and Ci-C₃ alkyl. More preferably, R¹ and R³ are each independently selected from the group consisting of H, halogen, and methyl. Most preferably, R¹ and R³ are each H. In a most preferred embodiment, R¹, R² and R³ are H.

L may be C=O or S0₂. However, in a preferred embodiment, L is optionally substituted

Ci-Ce alkyl, -CH₂C(0)- or -CH₂C0NH-. Preferably, L is optionally substituted Ci-C₃alkyl, more preferably -CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, C(Me)H, CF₂ or C(H)F and most preferably -CH₂-.

Preferably, R⁶ is a mono or bicyclic optionally substituted C5-Ci0 aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl. Most preferably, R⁶ is a mono or bicyclic optionally substituted C5-Ci₀ aryl.

R⁶ may comprise between 1 and 5 substituents. The or each substituent maybe independently selected from the list consisting of halogen, optionally substituted Ci-Ce alkyl, CN, optionally substituted Ci-Ce alkoxy, azido, CONR¹²R¹³, OH, C00R¹², 0C(0)R¹², NR¹²R¹³, 0C(0)0R¹², OC(O)NR¹²R¹³ and 0P(0)(0H)₂. Preferably, the or each substituent is selected from the list consisting of halogen, Ci-Ce alkyl, CN, OMe, OEt, OCF₃, 0CH₂0H, 0CH₂CH₂0H, 0CH₂CH₂CH₂0H, CF₃, azido, C0NH₂, OH and

0P(0)(0H)₂, NH₂ and NHCH₂CH₂0H.

-24Preferably, R⁶ is an optionally substituted C₅-Ci₀ aryl, wherein the C₅-Ci₀ aryl is a phenyl or a naphthyl. Most preferably, the C₅-Ci₀ aryl is phenyl. Preferably, the C₅-Ci₀aryl is substituted with between i and 5 substituents. The between 1 and 5 substituents may be as defined above. Preferably, the C₅-Ci₀ aryl is substituted with methyl, ethyl, propyl, azido, OMe, OEt, OH, 0CH₂0H, 0CH₂CH₂0H, 0CH₂CH₂ CH₂0H, 0P(0)(0H)₂, NH₂, NHCH₂CH₂0H or halogen. Preferably, the C₅-Ci₀ aryl is substituted with at least one halogen and/or OH. Accordingly, the C₅-Ci₀ aryl maybe substituted by 1 or 2 halogens. Preferably, the or each halogen is fluorine or chlorine.

Alternatively, R⁶ may be a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl selected from the group consisting of an optionally substituted pyridine, an optionally substituted pyrazole, an optionally substituted thiazole and an optionally substituted isoxazole. Preferably, the R⁶ is an optionally substituted pyridine. Preferably, the mono or bicyclic optionally substituted 5 to 10 membered heteroaryl is substituted with between 1 and 5 substituents. The between 1 and 5 substituents may be as defined above. Preferably, the mono or bicyclic optionally substituted 5 to 10 membered heteroaryl is substituted with at least one halogen and/or OH. Accordingly, the mono or bicyclic optionally substituted 5 to 10 membered heteroaryl may be substituted by 1 or 2 halogens. Preferably, the or each halogen is fluorine or chlorine, 20 preferably flourine.

R⁷ is preferably H or an optionally substituted Ci-Ce alkyl, more preferably H or a C1-C3 alkyl, and most preferably R⁷ is H.

Preferably, Y is an optionally substituted Ci-Ce alkyl, more preferably a C1-C3 alkyl, even more preferably-CH₂-, -CH₂CH₂-, -CH₂CH₂CH₂-, -CH(CH₃)-, -CH(F)- or -CF₂- and most preferably -CH₂-.

In some embodiments, R⁸ may be an optionally substituted C₃-Ce cycloalkyl or C₃-Ce heterocyclyl. R⁸ may be an optionally substituted C& cycloalkyl or an optionally substituted 6 membered heterocycle. Accordingly, R⁸ may be optionally substituted cyclohexane, an optionally substituted piperidine or an optionally substituted piperazine. The C& cycloalkyl or 6 membered heterocycle may be substituted with an optionally substituted Ci-Ce alkyl or a mono or bicyclic optionally substituted C₅-Ci₀ aryl. Preferably, the C& cycloalkyl or 6 membered heterocycle is substituted with a

-25phenyl or a C1-C3 alkyl substituted with a phenyl, more preferably the C& cycloalkyl or 6 membered heterocycle is substituted with a phenyl or -CH₂-phenyl.

Alternatively, in a preferred embodiment, R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl. Accordingly, R⁸ may be an optionally substituted phenyl, an optionally substituted pyridine, an optionally substituted naphthyl, an optionally substituted furanyl, an optionally substituted benzofuranyl, an optionally substituted thiophene, an optionally substituted pyridofuran, an optionally substituted benzoxazole or an optionally substituted benzothiazole. The mono or bicyclic C₅-Ci₀ aryl or the mono or bicyclic 5 to 10 membered heteroaryl may be substituted with between 1 and 5 substituents. The or each substituent may independently be selected from the list consisting of optionally substituted Ci-Ce alkyl, halogen, OH, Ci-Ce alkoxy, CONR^R^, CN, NH₂, 0P(0)(0H)₂ and azido.Preferably, the or each substituent is independently selected from the list consisting of Ci-Ce alkyl, halogen, OH, OMe, OEt, OCF₃, CF₃, C0NH₂, CN and azido. More preferably, the mono or bicyclic C₅-Ci₀ aryl or the mono or bicyclic 5 to 10 membered heteroaryl may be substituted with at least one Ci-Ce alkyl or halogen, even more preferably by at least one Ci-C₃ alkyl or halogen, and most preferably by at least one methyl or fluorine.

In a preferred embodiment, R⁸ is an optionally substituted benzofuranyl. Preferably, R⁸ is an unsubstituted benzofuranyl.

In an alternative preferred embodiment, R⁸ is an optionally substituted furanyl. The furanyl may be an unsubstituted furanyl. Alternatively, the furanyl may be substituted. Preferably, the furanyl is substituted with at least one of Ci-C₃ alkyl or halogen, more preferably at least one of methyl or fluorine and most preferably with one methyl group.

In an alternative preferred embodiment, R⁸ is an optionally substituted phenyl. The phenyl may be unsubstituted. Alternatively, the phenyl may be substituted. Preferably, the phenyl is substituted with at least one of Ci-C₃ alkyl or halogen, more preferably at least one of methyl or fluorine and most preferably with 1, 2 or 3 fluorines.

R⁹ may be an optionally substituted Ci-Ce alkyl, H, optionally substituted C₂-Ce alkenyl or a C₃-Ce cycloalkyl. More preferably, R⁹ is an optionally substituted Ci-Ce alkyl, H,

- 26 optionally substituted C₂-C6 alkenyl or a C₃-C6 cycloalkyl, even more preferably an optionally substituted C1-C3 alkyl or a C₃-Ce cycloalkyl, and most preferably R⁹ is methyl, ethyl, propyl, allyl or cyclopropyl. The Ci-Ce or Ci-C₃ alkyl maybe optionally substituted with one or more substituents selected from a halogen, OH and/or CN.

In a preferred embodiment, X¹ may be CR¹. X² may be CR². X³ may be CR³. Z may be CR¹⁰Rⁿ or NR¹⁰. Q maybe C=O or CR⁴R⁵. L maybe an optionally substituted Ci-C₃alkyl. Y maybe an optionally substituted Ci-Ce alkyl. R¹, R² and R³ may each be independently selected from the group consisting of H, halogen, CN, optionally substituted Ci-Ce alkyl, and optionally substituted mono and bicyclic C₃-Ce cycloalkyl. R4 and Rs may each be independently selected from the group consisting of H and Ci-Ce alkyl. R⁶ may be a mono or bicyclic optionally substituted C5-Ci0 aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl. R⁷ maybe H. R⁸ maybe a mono or bicyclic optionally substituted C5-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl or an optionally substituted mono or bicyclic C₃-Ce cycloalkyl. R⁹, R¹⁰ and R¹¹ may each be independently selected from the group consisting of optionally substituted Ci-Ce alkyl, H, hydroxyl, CONR¹²R¹³, sulfonyl, NR¹²R¹³, NHCOR¹², optionally substituted C₃-Ce cycloalkyl, optionally substituted C2-C6 alkenyl, and optionally substituted Ci-Ce alkoxy, or R⁹ and R¹⁰ together with the N atoms to which they are attached can combine to form an optionally substituted fused ring. Preferably, Z is NR¹⁰. Preferably, Q is C=O. Preferably L is a Ci-C3 alkyl, more preferably a C1-C2 alkyl, and most preferably -CH2-. Preferably Y is a Ci-C3 alkyl, more preferably a C1-C2 alkyl, and most preferably -CH2-. Preferably, R¹, R² and R³ are each H. Preferably, R⁶ is a mono or bicyclic substituted C5-Ci₀ aryl or a mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl are substituted with one or more substituents selected from a halogen, Ci-C₃alkoxy, OH, NH₂ and/or Ci-C₃ alkyl. Preferably, R⁶ is an optionally substituted phenyl or pyridinyl. Preferably, R⁸ is a mono or bicyclic optionally substituted C5-Ci0 aryl or mono or bicyclic optionally substituted 5 to 10 membered heteroaryl. More preferably, 30 R⁸ is a mono or bicyclic substituted C5-Ci₀ aryl or mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl is substituted with one or more substituents selected from Ci-Ce alkyl, halogen, OH, Ci-Ce alkoxy, CONR¹²R¹³, CN and/or azido. Preferably, R⁸ is optionally substituted phenyl, optionally substituted furanyl or optionally substituted benzofuranyl. Preferably, R⁹ and R¹⁰ are each independently a Ci-Ce alkyl, H, optionally substituted C₂-C6 alkenyl or a C₃-Ce

-27cycloalkyl, even more preferably a Ci-C₃ alkyl or a C₃-Ce cycloalkyl, and most preferably R9 and R¹⁰ are each independently methyl, ethyl, propyl, allyl or cyclopropyl.

In a more preferred embodiment, X¹ may be CR¹. X² may be CR². X³ may be CR³. Z may 5 be NR¹⁰. Q may be CO. L may be C1-C2 alkyl. Y may be Ci-C₂ alkyl. R¹, R² and R³ may each be independently selected from the group consisting of H, halogen, CN, optionally substituted Ci-Ce alkyl, optionally substituted mono and bicyclic C₃-Ce cycloalkyl. R⁶may be a mono or bicyclic optionally substituted C5-Ci0 aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl. R⁷ maybe H. R⁸ maybe a mono or 10 bicyclic optionally substituted C5-Ci₀ aryl or mono or bicyclic optionally substituted 5 to membered heteroaryl. R⁹ and R¹⁰ may each be independently selected from the group consisting of Ci-Ce alkyl, C2-Ce alkenyl and H. Preferably L is -CH2-. Preferably Y is -CH2-. Preferably, R¹, R² and R³ are each H. Preferably, R⁶ is a mono or bicyclic substituted C5-Ci₀ aryl or a mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl are substituted with one or more substituents selected from a halogen, a Ci-C₃ alkoxy, OH, NH₂ and/or a Ci-C₃ alkyl.

Preferably R⁶ is an optionally substituted phenyl or pyridinyl. Preferably, R⁸ is a mono or bicyclic substituted C₅-Ci₀ aryl or mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl is substituted with one or 20 more substituents selected from Ci-Ce alkyl, halogen, OH, Ci-Ce alkoxy, CONR¹²R¹³, CN and/or azido. Preferably, R⁸ is optionally substituted phenyl, optionally substituted furanyl or optionally substituted benzofuranyl. Most preferably, R⁸ is a phenyl substituted with at least one halogens. The or each halogen is preferably fluorine.

Preferably, R⁹ and R¹⁰ are each independently a Ci-C₃ alkyl or a C₂-C₄ alkenyl, and most 25 preferably are methyl or allyl.

In a preferred embodiment, X¹ is CR¹; X² is CR²; X³ is CR³; Z is CR^1OR^U; Q is CO; L is CH₂-; Y is -CH₂-; and R⁷ is H.

In a further preferred embodiment, X¹ is CR¹; X² is CR²; X³ is CR³; Z is CR^1OR^U; Q is

CR⁴R⁵; L is C=O; Y is -CH₂-; and R⁷ is H.

In a further preferred embodiment, X¹ is CR¹; X² is CR²; X³ is CR³; Z is CR^1OR^U; Q is CR4R5; L is S0₂; Y is -CH₂-; and R⁷ is H.

-28In a further preferred embodiment, X¹ is CR¹; X² is CR²; X³ is CR³; Z is NR¹⁰; Q is CO; L is -CH₂-; Y is -CH₂-; and R⁷ is H.

In a further preferred embodiment, X¹ is CR¹; X² is CR²; X³ is N; Z is NR¹⁰; Q is CO; L is 5 -CH₂-; Y is -CH₂-; and R⁷ is H.

It will be appreciated that an ‘agonist’, an ‘effector’ or an activator, as it relates to a ligand and STING, comprises a molecule, combination of molecules, or a complex, that stimulates STING. Conversely, an ‘antagonist’, as it relates to a ligand and STING, comprises a molecule, combination of molecules, or a complex, that inhibits, counteracts, downregulates, and/or desensitizes STING. ‘Antagonist’ encompasses any reagent that inhibits a constitutive activity of STING. A constitutive activity is one that is manifest in the absence of a ligand/STING interaction. ‘Antagonist’ also encompasses any reagent that inhibits or prevents a stimulated (or regulated) activity of STING.

Preferably, the compound of formula (I) is an activator of the STING protein.

It will be appreciated that the compounds described herein or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof maybe used in a 20 medicament which may be used in a monotherapy (i.e. use of the compound alone), for modulating the STING protein and/or treating, ameliorating or preventing a disease.

Alternatively, the compounds or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof may be used as an adjunct to, or in combination with, known therapies for modulating the STING protein and/or treating, ameliorating or preventing a disease.

Accordingly, in one aspect, a second therapeutic agent maybe administered with a compound of Formula (I). The compound of Formula (I) maybe administered before, 30 after, and/or together with the second therapeutic agent. The second therapeutic agent may comprise an antiviral agent, an anti-inflammation agent, conventional chemotherapy, an anti-cancer vaccine and/or hormonal therapy. Alternatively, or additionally, the second therapeutic agent may comprise a B7 costimulatory molecule, interleukin-2, interferon-g, GM-CSF, a CTLA-4 antagonist (such as Ipilimumab and 35 tremilimumab), an IDO inhibitor or IDO/TDO inhibitor (such as Epacadostat and

GDC-0919), a PD-i inhibitor (such as Nivolumab, Pembrolizumab, Pidilizumab, AMP-29224, and MDX-1106), a PD-L1 inhibitor (such as Durvalumab, Avelumab and Atezolizumab), an OX-40 ligand, a LAG3 inhibitor, a CD40 ligand, a 41BB/CD137 ligand, a CD27 ligand, Bacille Calmette-Guerin (BCG), liposomes, alum, Freund’s complete or incomplete adjuvant, a TLR agonist (such as Poly I:C, MPL, LPS, bacterial 5 flagellin, imiquimod, resiquimod, loxoribine and a CpG dinucleotide) and/or detoxified endotoxins.

Methods for co-administration with an additional therapeutic agent are well known in the art (Hardman et. al. (eds.), Goodman and Gilman’s The Pharmacological Basis of 10 Therapeutics, 10^th ed., 2001, McGraw-Hill New York, NY; Poole and Peterson (eds.),

Pharmacotherapeutics for Advanced Practice: A Practical Approach, 2001, Lippincott, Williams and Wilkins, Philadelphia, PA; Chabner and Longo (eds.), Cancer Chemotherapy and Biotherapy, 2001, Lippincott, Williams and Wilkins, Philadelphia, PA).

In one aspect, the disease is cancer and a chemotherapeutic agent may be administered with a compound of Formula (I). The chemotherapeutic agent maybe selected from a group further consisting of a cancer vaccine, a targeted drug, a targeted antibody, an antibody fragment, an antimetabolite, an antineoplastic, an antifolate, a toxin, an 20 alkylating agent, a DNA strand breaking agent, a DNA minor groove binding agent, a pyrimidine analogue, a ribonucleotide reductase inhibitor, a tubulin interactive agent, an anti-hormonal agent, an immunomodulator, an anti-adrenal agent, a cytokine, radiation therapy, a cell therapy, cell depletion therapy such as B-cell depletion therapy and a hormone therapy. Alternatively or additionally, the chemotherapeutic agent may 25 comprise abiraterone, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, bleomycin, cachectin, cemadotin, chlorambucil, cyclophosphamide, docetaxol, doxetaxel, carboplatin, cysplatin, cytarabine, dactinomycin, daunorubicin, decitabine, doxorubicin, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea, streptozocin, mitomycin, methotrexate, taxanes, tamoxifen, vinblastine, vincristine 30 and/or vindesine.

The compound of Formula (I) maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, 35 capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be

-30administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is welltolerated by the subject to whom it is given.

Medicaments comprising the compounds described herein may be used in a number of ways. Suitable modes of administration include oral, intra-tumoral, parenteral, topical, inhaled/intranasal, rectal/intravaginal, and ocular/aural administration.

Formulations suitable for the aforementioned modes of administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays, liquid formulations and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methyl cellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986, by Liang and Chen (2001).

For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 35 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples

-31of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkylsubstituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate.

Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tablet formulation.

Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene 10 glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.

Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.

Exemplary tablets contain up to about 80% drug, from about 10 weight % to about weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant. Tablet blends maybe compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in

-32“Pharmaceutical Dosage Forms: Tablets”, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

Suitable modified release formulations for the purposes of the invention are described in US Patent No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in “Pharmaceutical Technology On-line”, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298.

The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a 20 sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical 25 techniques well known to those skilled in the art.

The solubility of compounds of formula (I) used in the preparation of parenteral solutions maybe increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents. Formulations for parenteral 30 administration may be formulated to be immediate and/or modified release.

Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of 35 such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.

-33The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, 5 dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers maybe incorporated - see, for example, J Pharm Sci, 88 (io), 955-958, by Finnin and Morgan (October 1999).

Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, 20 atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1,1,1,2tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.

The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This maybe achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.

-34Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as L-leucine, mannitol, 5 or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution formulation for use in an atomiser using electrohydrodynamics 10 to produce a fine mist may contain from ipg to 2omg of the compound of the invention per actuation and the actuation volume may vaiy from ΐμΐ to ιοομί. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.

Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or “puff’ containing from ipg to toomg of the compound of formula (I). The overall daily dose will typically be in the range ipg to 2oomg which may be administered in a single dose 25 or, more usually, as divided doses throughout the day.

The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, microbicide, vaginal ring or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used 30 as appropriate.

The compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses

-35and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

The compounds of the invention may also be administered directly to a site of interest by injection of a solution or suspension containing the active drug 10 substance. The site of interest may be a tumour and the compound may by administer via intratumoral injection. Typical injection solutions are comprised of propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which maybe used instead of propylene glycol include glycerol and polyethylene glycol.

The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned 20 modes of administration.

Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the 25 cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gammacyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.

It will be appreciated that the amount of the compound that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the compound, and whether it is being used as a monotherapy, or in a combined therapy. The frequency of administration will also be influenced by the half-life of the compound within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound

-36in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, for administration to a human, the total daily dose of the compounds of the invention is typically in the range toopg to tog, such as img to ig, for example tomg to 500mg. For example, oral administration may require a total daily dose of from 25mg to 25omg. The total daily dose may be administered in single or divided doses and may, 10 at the physician’s discretion, fall outside of the typical range given herein. These dosages are based on an average human subject having a weight of about 6okg to 70kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.

However, it is appreciated by those skilled in the art that for agents that modulate the immune system, both the dose and the frequency of administration may be different to those of more traditional therapies. In particular, for agents that stimulate the immune system, for example through modulation of STING, they maybe administered in small doses, and quite infrequently, for example twice weekly, weekly or monthly. Smaller doses may also be effective when administered topically to a small area of skin.

The compound may be administered before, during or after onset of the disease to be treated.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations comprising the compounds according to the invention and precise therapeutic regimes (such as daily doses of the compounds and the frequency of administration). The inventors believe that they are the first to describe a pharmaceutical composition for treating a disease, based on the use of the compounds of the invention.

Hence, in a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a compound according to the first aspect, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

-37The invention also provides, in an eighth aspect, a process for making the composition according to the seventh aspect, the process comprising contacting a therapeutically effective amount of a compound of the first aspect, or a pharmaceutically acceptable 5 salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

A “subject” maybe a vertebrate, mammal, or domestic animal. Hence, compounds, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.

A “therapeutically effective amount” of compound is any amount which, when administered to a subject, is the amount of drug that is needed to treat the target disease, or produce the desired effect, i.e. modulate the STING protein.

For example, the therapeutically effective amount of compound used may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is preferred that the amount of compound is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle maybe a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents (i.e. the compound according to the first, second and third aspects) according to the invention. In tablets, the active compound maybe mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably

-38contain up to 99% of the active compound. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle maybe a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.

The compound according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.

Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.

fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The compound maybe prepared as a sterile solid composition that maybe dissolved or 30 suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The compound and compositions of the invention may be administered in the form of a sterile solution or suspension containing other solutes or suspending agents 35 (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its

-39anhydrides copolymerized with ethylene oxide) and the like. The compounds used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

It will be known to those skilled in the art that active drug ingredients may be converted into a prodrug, which is a metabolically labile derivative that is converted within the body into the active drug substance. Also included within the scope of the invention are prodrugs which are compounds of formula (I) which contain metabolically or hydrolytically labile moieties which in vivo are converted into the active drug of formula (I). The processes by which the prodrug is converted into the active drug substance include, but are not limited to, ester hydrolysis, phosphate ester hydrolysis, S-oxidation, N-oxidation, dealkylation and metabolic oxidation as described in Beaumont et. al., Curr. Drug Metab., 2003,4, 461-485 and Huttenen et. al., Pharmacol. Revs., 2011, 63, 750-771. Such prodrug derivatives may offer improved solubility, stability or permeability compared to the parent drug substance, or may better allow the drug substance to be administered by an alternative route of administration, for example as an intravenous solution.

Also included within the scope of the invention are soft drugs or antedrugs which are compounds of formula (I) which contain metabolically or hydrolytically labile moieties which in vivo are converted into inactive derivatives. The processes by which the active drug substance is converted into an inactive derivative include, but are not limited to, 25 ester hydrolysis, S-oxidation, A-oxidation, dealkylation and metabolic oxidation as described for example in Pearce et al., Drug Metab. Dispos., 2.006, 34.1035-1040 and B. Testa, Prodrug and Soft Drug Design, in Comprehensive Medicinal Chemistry II, Volume 5, Elsevier, Oxford, 2007, pp. 1009-1041 and Bodor, N. Chem. Tech. 1984,14, 28-38.

The invention also extends to a conjugate of a compound of formula (I).

Accordingly, in a further aspect of the invention, there is provided a conjugate of formula (VI):

wherein, C is a compound of formula (I);

L¹ is a linker;

T is a targeting moiety; and a is an integer between i and to.

Such conjugates may be designed to specifically target certain cell types or tumor types via the targeting moiety, which directs the compound of formula (I) to just those cells or tumors and deliver the STING activator in a cell-specific manner. The principle of this targeted delivery will be known to those skilled in the art as being closely related to ADC (antibody-drug conjugate) technology, for example as described in Polakis, P., Pharmacol. Revs., 2016, 68, 3-19. The linker will then be designed to cleave and the active compound would then diffuse into the cell and contact the STING protein.

T may comprise an antibody, an antibody fragment, a nucleic acid based molecule, a carbohydrate, a peptide or a modified peptide.

In one embodiment, T comprises an antibody or antibody fragment. The antibody or antibody fragment may be designed to target the Human Epidermal Growth Factor Receptor (EGFR), a plasminogen activator, a cytotoxic T-lymphocyte associated antigen (CTLA) such as CTLA-4, vascular endothelial growth factor (VEGF), neurotrophic factors such as BDNF, a nerve growth factor, platelet-derived growth factor (PDGF), transforming growth factor (TGF), EpCAM, FLT3, PSMA, PSCA, STEAP, CEA, folate receptor, the CD33/CD30/CD79/CD22 receptors, the SLC34A2 gene product, the mesothelin protein, the EphA2 tyrosine kinase, the Muci/Muci6 cell-surface antigens, ALK, AFP, brc-abl, caspase-8, CD20, CD40, CD123, CDK4, c-kit, cMET, ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, Her2, OX4O, p53, PAP, PAX3, PAX5, Ras, Rho or any other tumor antigen known to those skilled in the art.

The invention extends to both whole antibodies, as well as to antigen-binding fragments or regions of the corresponding full-length antibody.

-41The antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L). Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulphide bridge.

Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain.

The variable region includes several hypervariable regions, or Complementarity Determining Regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity for the antigen or variant or fragment thereof (e.g. an epitope). On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs. Antibody fragments may include a bi-specific antibody (BsAb) or a chimeric antigen receptor (CAR).

The constant region consists of one of five heavy chain sequences (μ, γ, ζ, a, or ε) and one of two light chain sequences (k or λ). The heavy chain constant region sequences 20 determine the isotype of the antibody and the effector functions of the molecule.

Preferably, the antibody or antigen-binding fragment thereof is isolated or purified.

In one preferred embodiment, the antibody or antigen-binding fragment thereof 25 comprises a polyclonal antibody, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be generated in a rabbit, mouse or rat.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. Preferably, 30 the antibody is a human antibody. As used herein, the term “human antibody” can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence 35 identity when compared to a reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human

-42antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids.

The term “human monoclonal antibody” can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells.

The term “humanised antibody” can mean an antibody from a non-human species (e.g. 10 mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.

The antibody may be a recombinant antibody. The term “recombinant human antibody” can include a human antibody produced using recombinant DNA technology.

The term “antigen-binding region” can mean a region of the antibody having specific binding affinity for its target antigen or a variant or fragment thereof. Preferably, the fragment is an epitope. The binding region may be a hypervariable CDR or a functional portion thereof. The term “functional portion” of a CDR can mean a sequence within 20 the CDR which shows specific affinity for the target antigen. The functional portion of a

CDR may comprise a ligand which specifically binds to the target antigen or a fragment thereof.

The term “CDR” can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CD Rs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.

The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

-43The term “functional fragment” of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. A functional activity can also be, for example, an effector function provided by an antibody constant region. The term “functional fragment” is 5 also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments such as

F(ab')₂; single chain Fv (scFv); and Fc fragments.

The term “VL fragment” can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CD Rs. A VL fragment can further include light chain constant region sequences.

The term “VH fragment” can means a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

The term “Fd fragment” can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-1. The “Fd fragment” does not include the light chain, or the second and third constant regions of the heavy chain.

The term “Fv fragment” can mean a monovalent antigen-binding fragment of a human 25 monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.

The term “Fab fragment” can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid 35 residues from about 110 to about 220 of the heavy and light chains.

-44The term “Fab' fragment” can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a

Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

The term “F(ab')₂ fragment” can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')₂ fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

The term “single chain Fv (scFv)” can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

The term “bispecific antibody (BsAb)” can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

One skilled in the art knows that the exact boundaries of a fragment of an antibody are 20 not important, so long as the fragment maintains a functional activity. Using wellknown recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.

The antigen-binding fragment thereof may comprise or consist of any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab')₂ and Fc fragment.

The antigen-binding fragment thereof may comprise or consist of any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody. The appropriate number and combination of VH and VL antigen 35 binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the antigen-binding

-45fragment. Functional fragments or antigen-binding fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc 10 fragment. Pepsin cleavage, for example, results in a bivalent F (ab') fragment. An F (ab')₂ fragment of the invention may be further reduced using, for example, DTT or 2mercaptoethanol to produce two monovalent Fab' fragments.

Functional or antigen-binding fragments of antibodies produced by proteolysis maybe 15 purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments maybe removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

The antibody or antigen-binding fragment thereof may be produced by recombinant methodology. Preferably, one initially isolates a polynucleotide encoding desired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. Preferably, such regions 25 can particularly include the antigen binding regions of the heavy and light chains, preferably the antigen binding sites, most preferably the CD Rs.

The polynucleotide encoding the antibody or antigen-binding fragment thereof according to the invention maybe produced using methods known to those skilled in 30 the art. The polynucleotide encoding the antibody or antigen-binding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.

As used herein, the term “immunospecificity” can mean the binding region is capable of immunoreacting with the target antigen, or a variant or fragment thereof, by

-46specifically binding therewith. The antibody or antigen-binding fragment thereof can selectively interact with an antigen with an affinity constant of approximately io³ to 10’ ¹³ M'¹, preferably io^-6 to io^-9 M^_1, even more preferably, io¹⁰ to io^-12 M^_1.

The term “immunoreact” can mean the binding region is capable of eliciting an immune response upon binding with SEQ ID No:3, or an epitope thereof.

The term “epitope” can mean any region of an antigen with the ability to elicit, and combine with, a binding region of the antibody or antigen-binding fragment thereof.

io

In one embodiment, T comprises a nucleic acid based molecule. The nucleic acid base molecule may be an aptamer. The nucleic acid based molecule may target the CD33/CD34 or PSMA tumor antigens, or any other tumor antigen known to those skilled in the art, for example as described in Orava, E., Biochem. Biophys. Acta, 2.010, 15 17q8, 2190-2200.

Aptamers are nucleic acid or peptide molecules that assume a specific, sequencedependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand. Typically, aptamers may comprise either single20 or double-stranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA). Peptide aptamers consist of a short variable peptide domain, attached at both ends to a protein scaffold. Aptamers may be used to bind both nucleic acid and non-nucleic acid targets.

Suitable aptamers may be selected from random sequence pools, from which specific aptamers maybe identified which bind to the selected antigen with high affinity. Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Preferred methodologies for producing aptamers include those disclosed in WO 2004/042083.

-47In an alternative embodiment, T comprises a peptide or a modified peptide. The peptide or modified peptide may comprise the RGD sequence motif, as described in Mousavizadeh, A., Colloids Surfaces B., 2017,158, 507-517.

L¹ may comprise a carbonate, a carbamate, an ester, an amide, a urea and/or a lactam functional group (Beck, A. et. al., Nat. Revs. Drug Disc., 2017,16,315-337). Said linkers will be known to those skilled in the art as either ‘stable’ linkers which are resistant to degradation in cells and in the systemic circulation or ‘conditionally labile’ linkers which are designed to degrade in cells and/or in the systemic circulation following a defined trigger event, which may be a change in pH or a metabolic process such as ester or amide hydrolysis. Specific hydrolysis processes have been described, such as the peptidase cleavage of a dipeptide e.g. the valine-citrulline dipeptide moiety contained in the clinically precedented ADC brentuximab vedotin or the hydrolysis of a labile hydrazone moiety in gemtuzumab ozogamicin. Non-cleavable linkers include that contained in the clinically precedented ADC trastuzumab emtansine.

a maybe 1, 2, 3,4,5, 6, 7, 8, 9 or 10.

L¹ may comprise an extended chain of carbon atoms or heteroatoms, for example a linear or branched polyethylene glycol (PEG) chain, an optionally substituted natural or unnatural sequence of amino acids or a linear or branchedoptionally substituted alkyl chain. The linked may be viewed as comprising an optionally substituted backbone, and the backbone of carbon atoms and/or heteroatoms. The backbone may consist of between 2 and too atoms, more preferably between 10 and 80 atoms or between 20 and 60 atoms. The backbone atoms may define one or more optionally substituted C₅C10 aryl, optionally substituted 5 to 10 membered heteroaryl, optionally substituted C₃C& cycloalkyl and/or optionally substituted 3 to 8 membered heterocycle rings within the backbone. The backbone atoms may consist of carbon, nitrogen and/or oxygen atoms. The backbone atoms maybe substituted with H, OH, =0, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C₃-Ce cycloalkyl and/or optionally substituted Ci-Ce alkoxy. L¹ may also contain a functional group handle that allows the STING modulator to be chemically combined with the targeting moiety via a covalent bond. For example thiol groups, or cysteine residues may be bonded to the linker or spacer group via a maleimide group. Alternative conjugation chemistries include lysine reactive groups, such as succinyl esters, pentafluorophenyl esters, β-lactam amides, isocyanates, and isothiocyanates; azide reactive groups, such as alkynes and strained

-48alkynes; cysteine reactive groups, such as maleimides, o-haloacetamides, pyridyl disulfides and vinyl sulfoxides; and ketone reactive groups, such as hydroxylamines, hydrazines and acyl hydrazides.

Linkers may be joined to a compound of formula (I) through a C atom, an O atom, a N atom or a S atom and may be functionalised with groups that include, but are not limited to, the following;

io

Linkers maybe cleavable, non-cleavable, hydrophilic or hydrophobic. A cleavable linker 5 can be sensitive to enzymes and may be cleaved by enzymes such as proteases. For example, a cleavable linker can be a valine-citrulline linker or a valine-alanine linker. For example;

-50A non-cleavable linker maybe protease insensitive.

L¹ may include alkyl chains (for example n-hexyl, n-pentyl, n-butyl, n-propyl), heteroatom containing chains (for example ethyloxy, propyloxy, butyloxy, pentyloxy, hexyoxy, ethylene dioxy, polyethylene glycol (PEG)), amino acids (gycinyl, alaninyl, aminopropanoic acid, aminobutanoic acid, aminopentanoic acid, aminohexanoic acid) and peptide units.

The inventors have found that compounds of the current invention may be functionalised in various locations with a variety of linkers and spacers to provide conjugate molecules. Said linkers may include self-immolating groups (for example a paminobenzyl ether or amine and/or a valine-citrulline unit) that are designed to release the parent STING modulator upon a hydrolytic event, for example following amide, peptide or carbamate hydrolysis.

The scope of the invention includes all pharmaceutically acceptable isotopicallylabelled compounds of the invention wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as ²H and ³H, carbon, such as ^UC, ¹³C and ¹⁴C, chlorine, such as ³⁶C1, fluorine, such as ¹⁸F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵0,¹⁷0 and ¹⁸0, phosphorus, such as ³²P, and sulphur, such as ³⁵S.

Certain isotopically-labelled compounds of the invention, for example those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready 30 means of detection. Substitution with isotopes such as deuterium, i.e. ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence maybe preferred in some circumstances. Substitution with positron emitting isotopes, such as ⁿC, ¹⁸F, ¹⁵0 and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

-₅ιIsotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

In accordance with a further aspect of the invention, there is provided a compound of the formula (II), (III), (IV) or (V):

R⁹

Formula (II)

Formula (III)

Formula (IV)

Formula (V)

IO wherein, X¹, X², X3, Q, L, R⁶, R⁷, R⁸, R⁹ and Z are as defined in the first aspect; and

R is H or a Ci-Ce alkyl, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.

It will be appreciated that compounds of formula (II) and (III) may be used to synthesise compounds of formula (I) and that compounds of formula (LV) and (V) may be used to synthesise compounds of formula (II) and (III).

Preferably, R is H or methyl, ethyl, benzyl or tert-butyl. More preferably, R is H or methyl.

The compound of formula (II) maybe selected from:

1O

The compound of formula (IV) maybe selected from:

The compound of formula (V) may be selected from:

All features described herein (including any accompanying claims, drawings and abstract), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show embodiments of the same may 10 be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:Figure 1 shows some of the major polymorphisms of human STING.

General Schemes

General Scheme i

Compounds of formula (I) may be prepared from compounds of formula (II) and (III) using an amide bond forming reaction, as shown below.

(Il)

Amidation o ^H (III) (i)

(l)

Typical conditions employ activation of the carboxylic acid of the compound of formula (II) using a suitable organic base and a suitable coupling agent. Preferred coupling agents are either i-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) with hydroxybenzotriazole (HOBt), propanephosphonic acid anhydride (T₃P), 1-54[bis(dimethylamino)methylene]-iH-i,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 2-(iH-benzotriazol-i-yl)-i,i,3,3-tetramethyluronium hexafluorophosphate (HBTU) or benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP). Preferred organic 5 bases comprise either Ν,Ν-diisopropyl ethylamine (DIPEA) or triethylamine (TEA) in a suitable organic solvent such as dichloromethane (DCM), dimethylformamide (DMF), dimethylacetamide (DMA) or acetonitrile (MeCN). The reaction may be shaken or stirred at room temperature.

Compounds of formula (II) may be synthesised according to the below methods.

Compounds of formula (III) are commercially available or may be synthesized by those skilled in the art. In particular, methods of synthesising compounds of formula (II) are described in General Schemes 2 to 4 below.

General Scheme 2

Compounds of formula (II) may be synthesized from esters of formula (IV), where R is methyl, ethyl, benzyl or tert-butyl, by a hydrolysis reaction.

The compound of Formula (IV) may be reacted with a suitable alkali or base to cause it to undergo hydrolysis and provide a compound of formula (II). The suitable alkali or base maybe LiOH, KOH, NaOH or K₂CO₃, and the reaction maybe conducted in an aqueous solution.

General Scheme 3

Alternatively, compounds of formula (II) can be obtained from a halide of formula (V) as shown in the general scheme below.

-55R⁶

R⁶

⁰ halo. r. .. mc *3 J,X

YG Q Cyanation Hydrolysis HO ' Ό

R⁹ R9R (V) (VI)(II)

First the compound of formula (V) undergoes a cyanation reaction to give a compound of formula (VI). This could be conducted using CuCN or ZnCN₂ in a polar solvent at elevated temperatures with a suitable catalyst. The polar solvent could be N-methyl-2pyrrolidone (NMP), dimethylformamide (DMF), dimethylacetamide (DMA) or acetonitrile (MeCN) and the catalyst could be, for example, tetrakzstriphenylphosphine palladium(o).

The compound of formula (VI) may then undergo hydrolysis to give the compound of formula (II). In particular, the hydrolysis reaction could use an aqueous solution of an alkali such as NaOH, LiOH and KOH, or an acid, such as HC1, at an elevated temperature.

General Scheme 4

In a further alternative process, the compound of formula (V) may undergo a direct carbonylation reaction to produce a compound of formula (II), as shown below.

_R⁶ „ . ^R⁶

Ο ν' halo. ,X? N

V⁵ Q Carbonylation HO rr Q

X2 1 7 ------------* _X2. 17 ^X<X¹^N'^{Z Xx}X¹^N'^Z

R⁹R (V)(II)

The reaction could be conducted using CO gas in the presence of a suitable catalyst in 20 an appropriate polar solvent. The catalyst may be a Pd, Rh, Ir or Fe catalyst, and the solvent may be NMP, DMF, DMA or MeCN with the reaction carried out in the presence of a suitable nucleophile such as water to produce the acids (II). It will be appreciated by those skilled in the art that carrying out the same reaction in the presence of alcohols instead of water provides an alternative means to prepare esters of 25 formula (IV).

-56General Scheme 5

Compounds of formulae (IV), (V) and (VI) may be synthesized by those skilled in the art via an alkylation/acylation/sulfonylation reaction of (VIII) with a compound of formula (VII), where G is a leaving group such as an optionally substituted alkylaryl(het), alkyl, aryl(het), cycloalkyl, alkylcycloalkyl halide, triflate or tosylate.

R⁹ (VIII) g^xL'r⁶ (VII) (iii)

General Scheme 6

A compound of formula (IX) may be prepared in a six or seven-step process, as shown below, from a compound of formula (XVIII), where R is methyl, ethyl, benzyl or tert-

Firstly, the compound of formula (XVIII) may undergo a nucleophilic substitution reaction with a hydrazine derivative (XVII) in a suitable solvent such as dimethyl 15 sulfoxide (DMSO) or dimethylformamide (DMF) to provide a compound of formula (XVI).

Secondly the electrophiles R⁹ and/or R¹⁰ maybe introduced sequentially, firstly on the nitrogen atom adjacent to the carbonyl in (XVI) and then the anilinic nitrogen atom.

By using excess molar equivalents in the first alkylation step, both nitrogen atoms may

-57be alkylated in the same step to give a compound of formula (XIV). The alkylation can be achieved using a suitable base such as i,8-diazabicyclo[5.4.o]undec-7-ene (DBU), NaH, TEA, DIPEA, K₂CO₃, Cs₂CO₃or KHCO₃. The solvent used maybe DMF, acetone, MeCN or tetrahydrofuran (THF).

Thirdly, the nitro group on the compound of formula (XIV) may then be reduced to an amino group by using a reducing agent, such as Fe/AcOH, Zn/HCl, Zn/NH₄C1, Zn/HCOONH₄, SnCl₂/HCl, Pd/C/H₂ or Na₂S₂O₄/K₂CO₃ in a suitable solvent, such as EtOAc, EtOH, MeOH, THF or MeCN. The ensuing amino compound undergoes in-situ 10 cyclization resulting in the formation of a compound of formula (XII), however when R is tert-butyl then this cyclization is impeded, and the corresponding amino ester of formula (XIII) may be isolated. (XIII) can then be cyclized to give a compound of formula (XII) using suitable solvents like THF or MeCN in the presence of a base such as LiHMDS, NaHMDS or LDA.

Fourthly, the compound of formula (XII) may then undergo an alkylation/acylation/ sulfonylation reaction, as described in General Scheme 5, to give a compound of formula (XI). This compound may undergo a hydrolysis reaction, as described in General Scheme 2, to give a compound of formula (X). Finally, this compound may be 20 reacted with a compound of formula (III), as described in General Scheme 1, to give a compound of formula (IX).

It will be appreciated that the compound of formula (IX) is a compound of formula (I) where Q is C=O and Z is NR¹⁰.

General Scheme 7

Alternatively, a compound of formula (XIX) maybe prepared in a six-step process, as shown below, from a compound of formula (XVHI), where R is methyl, ethyl, benzyl or tert-butyl.

Firstly, the compound of formula (XVffi) undergoes a nucleophilic substitution reaction with a compound of formula (XXV), where R is methyl or ethyl to produce a compound of formula (XXIV). The nucleophilic substitution reaction may be conducted in the presence of a mild base such as DBU, NaH, TEA, DIPEA, K₂CO₃,

Cs₂CO₃ or KHCO₃. The solvent used may be 1,4-dioxane, acetone, MeCN, THF or DMF.

The nitro group on the compound of formula (XXIV) may then be reduced to an amino group by using a reducing agent, such as Fe/AcOH, Zn/HCl, Zn/NH₄C1, Zn/HCOONH₄, 10 SnCl₂/HCl or Pd/C/H₂, in a suitable solvent, such as EtOH, MeOH or THF. The ensuing amino compound undergoes in-situ cyclization resulting in the formation of a compound of formula (XXIII).

The compound of formula (XXIII) may then undergo an alkylation/acylation/ sulfonylation reaction, as described in General Scheme 5, to give a compound of formula (XXII).

The electrophile R⁹ may be introduced on the anilinic nitrogen atom of the compound (XXII) to give a compound of formula (XXI). This can be achieved using an alkylating 20 agent R⁹-halo (XV) and a suitable base or by using a suitable aldehyde under reductive formylation condition. The suitable base can be DBU, NaH, TEA, DIPEA, K₂CO₃, Cs₂CO₃ or KHCO₃. The solvent used may be DMF, acetone, MeCN or THF. For the formylation reaction, a suitable reducing agent can be sodium triacetoxyborohydride or sodium cyanoborohydride.

-59(XXI) may then undergo a hydrolysis reaction, as described in General Scheme 2, to give a compound of formula (XX). Finally, this compound may be reacted with a compound of formula (III), as described in General Scheme i, to give a compound of 5 formula (XIX).

It will be appreciated that the compound of formula (XIX) is a compound of formula (I) where Q is C=O and Z is CR^1OR^U.

io General Scheme 8

Further derivatization of compounds of formula (XXX) can be achieved as shown below.

Compounds of formula (XXIX) may be obtained by de-methylation of a compound of formula (XXX) using a suitable de-methylating agent such as BBr₃, BC1₃, A1C1₃ or HBr in a suitable solvent such as DCM or ethylene dichloride (EDC). (XXIX) maybe further alkylated with a suitable alkylating agent in the presence of mild base such as DBU, NaH, TEA, DIPEA, K₂CO₃, Cs₂CO₃or KHCO₃ in a solvent such as 1,4-dioxane, acetone, 20 MeCN, THF or DMF to generate a compound of formula (XXVIII).

Finally, the terminal ester group contained in compounds of formula (XXVffi) either can be reduced to a primary alcohol of formula (XXVI) with a suitable reducing agent such as L1AIH4 or NaBH4; or can be hydrolyzed as described in General Scheme 2, to 25 give a compound of formula (XXVII).

- 6ο General Scheme Q

A compound of formula (XXXI) may be prepared in a nine-step process, as shown below, from a compound of formula (XXXIX), where R is methyl, ethyl, benzyl or tertbutyl.

(XVII) o

R⁹-halo and/or R¹⁰-halo (XV) (v)

Firstly, the compound of formula (XXXIX) undergoes a nitration reaction with nitrating mixtures of H₂SO₄/HNO₃ or H₂SO₄/KNO₃to give a compound of formula (XXXVIII) which can be converted into a compound of formula (XXXVII) according 10 to the method described in General Scheme 6.

Next, the compound of formula (XXXVH) may undergo an alkylation reaction with reagents of formula R⁹-halo and/or R^lo-halo as described in General Scheme 6, to provide a compound of formula (XXXVI).

The bromo group of (XXXVI) can be substituted using nucleophilic reagents such as NaCN, Zn(CN)₂ or KCN, NaOR² or R²NH₂ with an appropriate transition metal catalyst and a suitable base in a suitable solvent. The transition metal catalyst may be a palladium catalyst, such as tetrakzstriphenylphosphine palladium(o) or Ataphose, the 20 base may be TEA or DIPEA and the solvent may be 1,4-dioxane, NMP, DMF, DMA or

MeCN. A compound of formula (XXXV) maybe formed in the case where the nucleophilic reagent is Zn(CN)₂.

-61This compound of formula (XXXV) may then undergo a nitro reduction followed by an in-situ cyclization reaction, as described in General Scheme 6, to give a compound of formula (XXXIII).

(ΧΧΧΙΠ) may then undergo an alkylation/acylation/sulfonylation reaction, as described in General Scheme 5, followed by a hydrolysis reaction, as described in General Scheme 2, to give a compound of formula (XXXII). Finally, this compound maybe reacted with a compound of formula (III), as described in General Scheme 1, to give a compound of formula (XXXI).

It will be appreciated that the compound of formula (XXXI) is a compound of formula (I) where Q is C=O and Z is NR¹⁰.

General Scheme 10

A compound of formula (XL) may be prepared in a six-step process, as shown below, from a compound of formula (XXXVI), where R is methyl, ethyl, benzyl or tert-butyl.

The compound of formula (XXXVI) can be synthesized as described in General Scheme 9 in which a bromo group can be substituted with a terminal alkyne (XLVI;

where X is NH, O and P is any protecting group) under Sonogashira reaction conditions to give a compound of formula (XLV) where P is a suitable protecting group. This reaction can most successfully be conducted in a sealed tube at an elevated temperature using a suitable transition metal catalyst, co-catalyst (typically Cui) and a suitable base in a suitable solvent. The transition metal catalyst maybe a palladium catalyst, such as tetrakzstriphenylphosphine palladium(o) or Pd(dba)₂, or Pd₂(PPh₃)₂Cl₂ the base may

- 62 be TEA or DIPEA and the solvent may be 1,4-dioxane, THF, NMP, DMF, DMA or MeCN.

The compound of formula (XLV) may then be reduced using a hydrogenation reaction with hydrogen gas and a suitable catalyst in a suitable solvent to provide a compound of formula (XLIV). The catalyst may be palladised charcoal and the solvent EtOH or MeOH. Alternatively, a compound of formula (XLV) can undergo a nitro group reduction followed by cyclization reaction, as described in General Scheme 6, to give a compound of formula (XLHI).

The compound of formula (XLIII) may then undergo an alkylation/acylation/sulfonylation reaction, as described in General Scheme 5, followed by a hydrolysis reaction, as described in General Scheme 2, to give a compound of formula (XLII). This compound may then be reacted with a compound of formula (III), as described in General Scheme 1, to give a compound of formula (XLI). Finally, the compound of formula (XLI) can undergo a de-protection reaction to give a compound of formula (XL). Those skilled in the art will recognize that TFA/DCM or HC1 maybe used for the de-protection of a tert-butyl or a tert-BOC group and that either bases or fluoride-based reagents such as TBAF may be used for de-protection of 20 silyl groups.

It will be appreciated that the compound of formula (XL) is a compound of formula (I) where Q is C=O and Z is NR¹⁰.

General Synthetic Procedures

General Procedure 1

Amidation

R⁹ R⁹ (II) ©

To a stirred solution of a carboxylic acid (II) (1.277 mmol) in a suitable solvent, such as DCM, DMF, DMA or MeCN (10 mL) was added amine (III) (1.2 eq.) and a coupling reagent, such as T₃P, HATU, EDCI, HOBT, BOP or HBTU (1.5 eq.), followed by addition of an organic base, such as DIPEA or TEA (2.0 eq.) drop wise to the solution and the

-63mixture allowed to stir at RT for 2-3 h. When UPLC or TLC showed completion of the reaction, the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with aqueous NaHCO₃ solution followed by dilute aqueous HC1 and finally with brine, and then dried over anhydrous Na₂SO₄. The 5 solvent was evaporated under reduced pressure to obtain the crude material which was purified by Combi-flash using mixtures of EtOAc in hexanes as eluent to afford a compound of formula (I) (70-80% yield) as a pale yellow solid. A similar procedure can be followed to synthesize all amides of formula (I).

General Purification and Analytical Methods

All final compounds were purified by either Combi-flash or prep-HPLC purification, and analysed for purity and product identity by UPLC or LCMS according to one of the below conditions.

Prep-HPLC

Preparative HPLC was carried out on a Waters auto purification instrument using either a YMC Triart C18 column (250 x 20 mm, 5 pm) or a Phenyl Hexyl column (250 x 21.2 mm, 5 pm) operating at between ambient temperature and 50 °C with a flow rate of 16.0 - 50.0 mL/min.

Mobile phase 1: A = 20mM Ammonium Bicarbonate in water, B = Acetonitrile;

Gradient Profile: Mobile phase initial composition of 80% A and 20% B, then to 60% A and 40% B after 3 min., then to 30% A and 70% B after 20 min., then to 5% A and 95% B after 21 min., held at this composition for 1 min. for column washing, then returned 25 to initial composition for 3 min.

Mobile phase 2: A = lomM Ammonium Acetate in water, B = Acetonitrile; Gradient Profile: Mobile phase initial composition of 90% A and 10% B, then to 70% A and 30% B after 2 min., then to 20% A and 80% B after 20 min., then to 5% A and 95% B after 21 30 min., held at this composition for 1 min. for column washing, then returned to initial composition for 3 min.

LCMS method

General 5 min method: Zorbax Extend C18 column (50 x 4.6 mm, 5pm) operating at 35 ambient temperature and a flow rate of 1.2 mL/min. Mobile phase: A = 10 mM

Ammonium Acetate in water, B = Acetonitrile; Gradient profile: from 90 % A and 10 %

-64B to 70 % A and 30 B in 1.5 min, and then to 10 % A and 90 % B in 3.0 min, held at this composition for 1.0 min, and finally back to initial composition for 2.0 min.

UPLC method

UPLC was carried out on a Waters auto purification instrument using a Zorbax Extend C18 column (50 x 4.6 mm, 5pm) at ambient temperature and a flow rate of i.5ml/min.

Mobile phase 1: A = 5 mM Ammonium Acetate in water, B = 5 mM Ammonium Acetate in 90:10 Acetonitrile/water; Gradient profile from 95% A and 5% B to 65% A and 35% B 10 in 2 min., then to 10% A and 90% B in 3.0 min., held at this composition for 4.0 min.

and finally back to the initial composition for 5.0 min.

Mobile phase 2: A = 0.05 % formic acid in water, B = Acetonitrile; Gradient profile from 98 % A and 2 % B over 1 min., then 90 % A and 10 % B for 1 min., then 2 % A and 15 98 % B for 2 min. and then back to the initial composition for 3 min.

General Procedure 2

Hydrolysis (ii)

To a stirred solution of ester (IV) (1.49 mmol) in a mixture of MeOH or THF (10 mL) and water (5 mL) was added LiOH, NaOH or KOH (2.0 eq.) at RT and the resulting reaction mixture was stirred at RT for 2-16 h. TLC showed complete consumption of the ester (IV), upon which the solvent was evaporated under reduced pressure and the resulting residue was washed with ether. The residue was then acidified with 1N HC1 to pH 2-4, which resulted in the formation of a precipitate, which was filtered and washed with water and then dried under reduced pressure at 5O-6o°C to afford the desired carboxylic acid of formula (II) (70-85% yield) as an off white solid.

-65General Procedure 3

g'^l'r⁶ (Vii) (iii)

Method i

To a stirred solution of a compound of formula (VIII) (2.77 mmol, 1.0 eq.) in DMF or

THF (4 mL/mmol) was added K₂CO₃, Cs₂CO₃, Na₂CO₃, NaOH or NaH (2.0 eq.) - in the case where NaOH was used, TBAB (0.1 eq.) was also added as a phase transfer catalyst - followed by addition of a compound of formula (VII) (1.5 eq.) and the mixture allowed to stir at RT for 0.5-1 h. The reaction was monitored by TLC. After completion of the reaction the reaction mixture was diluted with water, extracted with EtOAc, and the organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organics were evaporated under reduced pressure to obtain the crude product which was purified by Combi-flash using mixtures of EtOAc in hexanes as eluent to afford compounds of formula (IV) (80-90% yield) as colourless oils.

Method 2

Alternatively, to a stirred solution of a compound of formula (VHI) (2.77 mmol) in DCM or MeCN or THF (4 mL/mmol) was added TEA or DIPEA (2.0 eq.) followed by addition of a compound of formula (VII) (1.5 eq.) and the whole allowed to stir at RT for 0.5 to 1 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with water, extracted with EtOAc, and the combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organic layers were evaporated under reduced pressure to obtain the crude product which was purified by Combi-flash using mixtures of EtOAc in hexanes as eluent to afford compounds of formula (IV) (80-90% yield) as colourless oils.

General Procedure 4-a

(XVIII)

H

Η₂Ν^ζΝγ°Τ (XVII) ⁰ (iv-a)

-66To a stirred solution of a compound of formula (XVIII) (15.07 mmol, 1.0 eq.) in DMSO, DMF, MeCN or THF (1.67 mL/mmol, 25 mF) was added a compound of formula (XVII) (16.58 mmol, 1.1 eq.) and the whole reaction mass allowed to stir at room temperature for 3-5 h. The course of the reaction was monitored by TLC and or LCMS.

After completion of the reaction, the mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organic layers were evaporated under reduced pressure to give a compound of formula (XVI) (90-100% yield) as a yellow wax which solidified on cooling. The product was pure enough to use in the next step without any further purification.

General Procedure 4-b

X' naio (XVIII)

(XXV) ⁰ (iv-b)

To a stirred solution of a compound of formula (XVHI) (3.77 mmol, 1.0 eq.) in THF, DMF, DMSO or MeCN (2.7 mL/mmol, 10 mL) was added NaHCOs (2.0 eq.) followed by a compound of formula (XXV) (1.5 eq.) and the whole reaction mass allowed to stir at room temperature for 15-20 h. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction, the mixture was diluted with water and 20 extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organic layers were evaporated under reduced pressure to give a compound of formula (XXIV) (85-90% yield) as a yellow solid. The product was pure enough to use in the next step without any further purification.

General Procedure 5

R⁹-halo and/or R¹⁰-halo (XV) (v)

To a stirred solution of a compound of formula (XVI) (15.072 mmol, 1 eq.) in DMF,

THF, MeCN, acetone (1.5 mL/mmol, 23 mL) was added K₂CO₃, Cs₂CO₃, Na₂CO₃, or

-67NaH (2.1 eq.) at 0 to 5 °C under an inert atmosphere and the whole allowed to stir for 20-30 min. Then, a compound of formula (XV) (1.2 to 2.2 eq.) was added dropwise and the mixture allowed warming slowly to room temperature. The reaction mixture was further stirred at room temperature for 2-3 h. The course of the reaction was monitored 5 with TLC and/or LCMS. After completion of the reaction the reaction mixture was quenched with a saturated solution of NH₄C1, diluted with water, extracted with MTBE, and the combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organics were evaporated under reduced pressure to obtain a compound of formula (XIV) (75-80% yield) as a yellow semi solid. The product was pure enough 10 to use in the next step without any further purification.

General Procedure 6

O O ⁰ || ,R¹⁰ Reduction O ^10 ^XX¹ Ν'^Νγ°Υ (vi-a/b) ^X*X¹ γ^Νγ% (XIV) R⁹ O (XIH) r⁹ O

Option a (Reduction by Sodium dithionate)

To a stirred solution of a compound of formula (XIV) (1.0 mmol, 1.0 eq.) in a mixture of either MeCN:H₂0 or THF:H₂0 (12 mL/mmol, 2:1) was added sodium hydrosulphite (8.0 eq.), tetra butyl ammonium hydrosulphate (0.5 eq.) and potassium carbonate (6.0 eq.) at RT and then the mixture was stirred for 1 h. Progress of the reaction was monitored by TLC and or LCMS. After completion of the reaction, solvents were evaporated under reduced pressure to give an oily liquid which was dissolved in 1N HC1 and extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organics were filtered and evaporated under reduced pressure to give a compound of formula (XIII) (90-95% yield) as a yellowish solid.

Option b (Reduction by Pd/C/H?)

To a stirred solution of a compound of formula (XIV) (12.85 mmol, 1.0 eq.) in EtOAc, MeOH or EtOH (9.4 mL/mmol, 120 mL) was added Pd-C (50% w/w in water) (77.8 mg/mmol) under an inert atmosphere at room temperature. The reaction mixture was 30 purged with H₂ gas using balloon pressure and then allowed to further stir for 3-5 h at room temperature. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction the reaction mass was diluted with EtOAc, filtered

-68carefully through a bed of celite and washed with EtOAc 4-5 times until the mother liquor showed no compound remaining by TLC. Then the collected organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give a compound of formula (ΧΠΙ) (80-85 % yield) as a yellow semi solid. The product was 5 pure enough to use in the next step without any further purification.

General Procedure 7

O

(aiii; r\

Cyclization

LiHMDS (vii)

(XII) R⁹

To a stirred solution of a compound of formula (ΧΠΙ) (10.35 mmol, 1.0 eq.) in dry THF or diethyl ether (9.7 ml/mmol, 100 mL) was added LiHMDS (2.0 eq., 1M sol in THF) dropwise at 0-5 °C. The reaction mixture was slowly warmed to 40-45 °C and maintained at this temperature for 1 hr. The course of the reaction was monitored with TLC and/or LCMS. After completion of the reaction, the solvent was evaporated under reduced pressure from the reaction mixture and the ensuing residue diluted with EtOAc and a saturated solution of NH₄C1 and the resulting mixture was allowed to stir for 30 min. The mixture was further diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organics were evaporated under reduced pressure to give the crude product which was purified by Combi-flash chromatography using mixtures of EtOAc (30-40%) in hexanes as eluent to afford a compound of formula (XII) (80-85% yield) as a white solid.

General Procedure 8

(XXIII)

To a stirred solution of a compound of formula (XXIV) (1.06 mmol, 1.0 eq.) in glacial acetic acid (4 mL/mmol, 4.25 mL) was added Fe powder (5.0 eq.) at room temperature and the resulting reaction mixture was heated at 75-80 °C for 10-15 h. The progress of the reaction was monitored by TLC and or LCMS. After completion of the reaction the

-69mixture was diluted with ice cold water and extracted with DCM. The combined organics were dried over anhydrous Na₂SO₄and evaporated under reduced pressure to give a cyclized compound of formula (XXIII) (80-90 % yield) as a yellow solid. The product was pure enough to use in the next step without any further purification.

General Procedure Q

(XXII)

Reductive formylation (ix)

To a stirred solution of a compound of formula (XXII) (0.28 mmol, 1.0 eq.) in a polar 10 protic solvent such as EtOH or MeOH (3.6 mL/mmol) was added formalin solution (0.36 mL/mmol) (when R⁹ is methyl) and the resulting mixture was stirred at RT for 12 h. Sodium cyanoborohydride or sodium borohydride (2.0 eq.) and 1-2 drops of acetic acid were added into the reaction mixture which was further stirred for 20-24 h at RT. Progress of the reaction was monitored by TLC and/or LCMS. For incomplete reactions 15 another portion of formalin solution (1.8 mL/mmol) and sodium cyanoborohydride or sodium borohydride (2.0 eq.) maybe added and stirring continued for another 20-24 h. After completion of the reaction, the reaction mixture was diluted with water and extracted with EtOAc three times. The collected organic layers were washed with saturated sodium bicarbonate solution followed by brine solution. The organic layer was dried over anhydrous Na₂SO₄and concentrated in vacuo to afford the crude product which was purified by Combi-flash chromatography using mixtures of 20% EtOAc in hexane as eluent to afford a compound of formula (XXI) (30-55% yield) as a white solid.

General Procedure 10

To a stirred solution of a compound of formula (XXX) (0.91 mmol, 1.0 eq.) in DCM (5.5 mL/mmol) was added BBr₃ (2.4 mL/mmol, 1M solution in DCM) at 0-5 °C and the

-70resulting reaction mixture was stirred at room temperature for 10-30 min. Completion of the reaction was monitored by TLC and or LCMS. After completion of the reaction, the mixture was diluted with water and extracted with EtOAc. The combined organics ware washed with brine, dried over anhydrous Na₂SO₄ and concentrated in vacuo to a 5 crude product which was purified by column chromatography using mixtures of EtOAc and hexanes to afford a compound of formula (XXIX) (70-80% yield) as a light yellow to off white solid.

General Procedure 11

O OH

To a stirred solution of a compound of formula (XXVffi) (0.13 mmol, 1.0 eq.) in EtOH, MeOH or THF (15 mL/mmol) was added NaBH₄ or LiAlH₄ (3-6 eq.) at 0-5 °C and the whole then stirred at room temperature for 3-6 h. After completion of the reaction (monitored by TLC and/or LCMS), the mixture was quenched by the addition of ammonium chloride/sodium hydroxide solution, extracted with EtOAc and then evaporated under reduced pressure to obtain a crude product which was purified by prep-HPLC to give a compound of formula (XXVI) (40-45% yield) as a white solid.

General Procedure 12

_2O (XL) (XXXIX)

To a stirred solution of a compound of formula (XL) (6.66 mmol, 1.0 eq.) in sulfuric acid (1.5 mL/mmol) was added KNO₃ (1.0 eq.) portionwise at 0-5 °C and stirring was continued for another 30 min. at the same temperature. Progress of the reaction was monitored by TLC and/or LCMS. After completion of the reaction, the reaction mixture 25 was poured into ice-water to give a solid precipitate which was filtered, washed with hexane and then dried in a vacuum oven overnight to give a compound of formula (XXXIX) (90-95% yield) as a light yellow to off-white solid. The product was pure enough to use in the next step without any further purification.

-71General Procedure 13

O

Cyanation (xiii)

To a stirred solution of a compound of formula (XXXVII) (1.74 mmol, 1.0 eq.) in degassed 1,4-dioxane (23 ml/mmol) was added Zn(CN)₂ (5.0 eq.) followed by tetrakistriphenylphosphine palladium(o) (0.1 eq.) under a nitrogen atmosphere at RT. The resulting reaction mixture was heated at 100-105 °C in a sealed tube for 10-12 h. The progress of the reaction was monitored by TLC and/or LCMS. After completion of the reaction, the mixture was diluted with water and extracted with EtOAc. The combined organics were washed with a brine solution, dried over anhydrous Na₂SO₄and concentrated in vacuo to afford the crude product which was purified by column chromatography to give a compound of formula (XXXVI) (55-60% yield) as an off white solid.

General Procedure 14

O Sonogashira

R_x JL χ3 _Nq₂ coupling

O |1^ _zR¹° (xiv) _t

ΒΓ^Χ^Ν'Πγ⁰^ =—\

R⁹ O ^X_P (XXXVII) ^{K ϋ} (XLVI)

To a stirred solution of a compound of formula (XXXVII) (0.72 mmol, 1.0 eq.) and an appropriate terminal alkyne (XLVI; where X is NH or O and P is any protecting group) 20 (2.0 eq.) in degassed 1,4-dioxane, THF, NMP, DMF, DMA or MeCN (8.4 mL/mmol) was added a mild base such as TEA or DIPEA (4.0 eq.) followed by Cui (0.05 eq.) and tetrakistriphenylphosphine palladium(o), Pd(dba)₂ or Pd₂(PPh₃)₂Cl₂ (0.04 eq.) under an inert atmosphere. The resulting reaction mixture was heated at 70-80 °C for 10-15 h. The progress of the reaction was monitored by TLC and or LCMS and after completion 25 of the reaction the mixture was diluted with EtOAc and filtered through a bed of celite.

The filtrate was washed with water and then a saturated brine solution. The organic layers ware dried over anhydrous Na₂SO₄, passed through a cotton bed and then concentrated in vacuo to afford the crude product which was purified by Combi-flash

-72.chromatography using mixtures of 20% EtOAc in hexane as eluent to afford a compound of formula (XXXVI) (80-95% yield) as an off white to light yellow solid.

General Procedure 15

To a stirred solution of a compound of formula (XXXII) (0.12 mmol, 1.0 eq.) in EDO or DCM (16.5 mL/mmol) was added TFA (3.3 mL/mmol) dropwise with cooling. The reaction mixture was stirred for 1-2 h at room temperature. After completion of the reaction (monitored by TLC and/or LCMS), the reaction mixture was evaporated to dryness and the residue further evaporated in vacuo from solutions with acetonitrile and then 1,4-dioxane. The resulting sticky reaction mixture was dissolved in 1,4dioxane (16.5 mL/mmol) and HC1 added (4M solution in 1,4-dioxane, 16.5 mmol/mL) dropwise with cooling. The neutralized mixture was concentrated in vacuo to give a crude product which was purified by trituration with diethyl ether to afford a white solid that was again suspended in hexane (16.5 mL/mmol) with stirring and filtered to give a compound of formula (XXXI) (35-40% yield) as a white solid. The above procedure is applicable when the protecting group was BOC.

Examples

Nuclear magnetic resonance (NMR) spectra were in all cases consistent with the proposed structures. Characteristic chemical shifts (δ) are given in parts-per-million downfield from tetramethylsilane (for Ή-NMR) and up-field from trichloro-fluoromethane (for ¹⁹F NMR) using conventional abbreviations for designation of major peaks: e.g. s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The following abbreviations have been used for common solvents: CDC1₃, deuterochloroform; de-DMSO, deuterodimethylsulphoxide; and CD₃OD, deuteromethanol.

Mass spectra, MS (m/z), were recorded using electrospray ionisation (ESI). Where relevant and unless otherwise stated the m/z data provided are for isotopes ¹⁹F, 35CI, ⁷⁹Br and ¹²?I.

-73All chemicals, reagents and solvents were purchased from commercial sources and used without further purification. All reactions were performed under an atmosphere of nitrogen unless otherwise noted.

Flash column chromatography was carried out using pre-packed silica gel cartridges in a Combi-Flash platform. Prep-HPLC purification was carried out according to the General purification and analytical methods described above. Thin layer chromatography (TLC) was carried out on Merck silica gel 60 plates (5729). All final compounds were >95% pure as judged by the LCMS or UPLC analysis methods described in the General purification and analytical methods above unless otherwise stated.

Example 1: 4.-(2-chloro-6-fluorobenzyD-i.2-dimethyl-3-oxo-N-(2.4..6trifkiorobenzvl)-i.2.2.4.-tetrahvdrobenzol ell 1.2.4. ltriazine-6-carboxamide

¹

Example 1 was prepared according to the methods described in General Procedures 1-7, and the methods described below.

Preparation 1: Methyl i.2-dimethyl-3-oxo-i.2.3.4-tetrahydrobenzoreHT.2.4ltriazine-620 carboxylate

Step 1: tert-Butyl 2-(4-fmethoxycarbonyl)-2-nitrophenyl')hydrazinecarboxylate

O

To a stirred solution of methyl 4-fluoro-3-nitrobenzoate (3.0 g, 15.07 mmol) in DMSO (25 ml) was added tert-butyl hydrazinecarboxylate (2.19 g, 16.58 mmol). The resulting

-74mixture was stirred at room temperature for 3 h. The course of the reaction was monitored by TLC and/or LCMS and after completion of the reaction the mixture was diluted with water and extracted with EtOAc. The collected organic layer was washed with brine solution and dried over anhydrous Na₂SO₄, filtered and concentrated under 5 reduced pressure to give the title compound tert-butyl-2-(4-(methoxycarbonyl)-2nitrophenyl)hydrazinecarboxylate (4.69 g, 100% yield, purity >95%) as a yellow solid. The product was used in the next step without any further purification. LCMS m/z: 256.05 {M+H (-56)}.

Step 2: tert-Butyl 2-(4-(methoxycarbonyl')-2-nitrophenyl')-i.2-dimethylhydrazine carboxylate

O

To a stirred solution of tert-butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)hydrazinecarboxylate (Step 1) (4.69 g, 15.07 mmol) in DMF (20 mL) was added NaH (0.76 g, 31.65 mmol, 60% suspension in oil) at 0-5 °C under an inert atmosphere and allowed to stir for 20-30 min., then Mel (2.06 mL, 33.16 mmol) was added dropwise with cooling and the whole was then stirred at room temperature for 2 h. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction the mixture was quenched with a saturated solution of NH₄C1, diluted with water and extracted with MTBE. The combined organics were washed with brine twice, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give the title compound tert-butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-i,2-dimethylhydrazine carboxylate (4.0 g, 78% yield, purity >95%) as a yellow semi solid. The product was used in the next step without any further purification. LCMS m/z: 340.14 (M+H),

240.14 {M+H (-100)}.

Step 3: tert-Butyl-2-(2-amino-4-(methoxycarbonyl)phenyl')-i.2-dimethylhydrazine carboxylate

O

-75To a stirred solution of tert-butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-i,2dimethylhydrazine carboxylate (Step 2) (4.0 g, 12.86 mmol) in EtOAc (120 mL) was added Pd-C (50% w/w in water) under an inert atmosphere. The reaction mixture was stirred under a H₂ gas balloon pressure for 3 h at room temperature. The course of the 5 reaction was monitored by TLC and/or LCMS and after completion, the reaction mass was diluted with EtOAc and filtered carefully through a celite bed. The collected organics ware dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give the title compound tert-butyl-2-(2-amino-4(methoxycarbonyl)phenyl)-i,2-dimethylhydrazine carboxylate (3.2 g, 88% yield, purity 10 >90%) as a yellow semi solid. The product was used in the next step without any further purification. LCMS m/z: 310.17 (M+H).

Step 4: Methyl-i.2-dimethyl-3-oxo-i.2.3.4-tetrahydrobenzoreiri.2.4.1triazine-6carboxylate

In a 250 mL two neck round bottom flask fitted with a condenser was placed tert-butyl-

2-(2-amino-4-(methoxycarbonyl)phenyl)-i,2-dimethylhydrazine carboxylate (Step 3) (3.2 g, 10.35 mmol) and THF (100 mL). To this solution was added LiHMDS (21 mL, 20.701 mmol, 1.3 M solution in THF) dropwise at 0-5 °C. After the addition was complete, the reaction mixture was allowed to warm slowly to room temperature. Finally, the reaction mass was heated at 40 °C for 1 h. The progress of the reaction was monitored by TLC and/or LCMS. After completion, the THF was evaporated from the reaction mixture and the residue diluted with EtOAc and poured onto a saturated solution of NH₄C1 and allowed to stir for 30 min. The resulting mixture was diluted with water and extracted with EtOAc. The combined organics were washed with brine solution twice. The collected organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give the crude compound which was purified by Combi-flash chromatography using 30-40% EtOAc /hexane mixture to give the title compound methyl i,2-dimethyl-3-oxo-i,2,3,4-tetrahydrobenzo[e][i,2,4] triazine-6-carboxylate (2.2 g, 90% yield, purity >95%) as a white solid. LCMS m/z: 236.10 (M+H).

-7β-

Preparation 2: Methyl-4-i2-chloro-6-fluorobenzyl')-i.2-dimethyl-3-oxo-i.2.3.4tetrahydrobenzorel Γ i,2.4ltriazine-6-carboxylate

n'ⁿ

ΙΟ

To a stirred solution of methyl-i,2-dimethyl-3-oxo-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxylate (Preparation 1) (0.045 g, 0.19 mmol) in DMF (1.5 mL) was added NaH (4.82 mg, 0.20 mmol, 60% suspension in oil) at 0-5 °C under an inert atmosphere and the whole allowed to stir for 20-30 min., before 2-chloro-6fluorobenzyl bromide (0.02 mL, 0.21 mmol) was added dropwise with cooling and the reaction mass was then stirred at room temperature for 2 h. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction the mixture was quenched with a saturated solution of NH₄C1, diluted with water and extracted with EtOAc. The combined organics were washed with brine twice, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give the title compound methyl-4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4-tetrahydrobenzo[e] [i,2,4]triazine-6 carboxylate (0.07 g, 97% yield, purity >95%) as an off white solid. The product was used in the next step without any further purification. LCMS m/z: 378.09 (M+H).

Preparation 3: 4-i2-chloro-6-fluorobenzyl')-i.2-dimethyl-3-oxo-i.2.3.420 tetrahydrobenzo reHT.2.41triazine-6-carboxylic acid

To a stirred solution of methyl-4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6 carboxylate (Preparation 2) (0.07 g, 0.18 mmol) in a mixture of solvents THF:H₂0:Me0H (2:1:1 mL) was added Li0H.H₂0 (0.08 g, 1.91 mmol) and the whole was allowed to stir at room temperature for 4 h. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction, the

-77reaction mixture was diluted with water and washed with EtOAc. The aqueous layer was acidified with HC1 (1N) to pH 1-2 and then extracted with EtOAc. The combined organics ware dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to afford the title compound 4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,45 tetrahydrobenzo [e][i,2,4]-triazine-6-carboxylic acid (0.06 g, 89% yield, purity >95%) as a white solid. The product was used in the next step without any further purification.

Preparation 4: 4-(2-chloro-6-fluorobenzyl')-i.2-dimethyl-3-oxo-N-(2.4.6trifluorobenzvl)-i.2..q.4-tetrahvdrobenzoreiri.2.4ltriazine-6-carboxamide (Example 1)

To a stirred solution of 4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine-6-carboxylic acid (Preparation 3) (0.06 g, 0.16 mmol) in DMF (1.5 mL) was added HBTU (0.079 g, 0.21 mmol) followed by TEA (0.042 g, 0.41 mmol) and the whole was allowed to stir for 15-20 min. before adding (2,4,6-trifluorophenyl)methanamine (0.026 g, 0.17 mmol). The mixture was allowed to stir at RT for 4 h. The course of the reaction was monitored by TLC and/or LCMS and after completion, the reaction mixture was diluted with EtOAc and then washed with water, a saturated solution of K₂CO₃,1N HC1 and finally brine solution. The organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to give 20 the crude product which was purified by prep-HPLC to afford the title compound 4-(2chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4 tetrahydrobenzo[e]- [i,2,4]triazine-6-carboxamide (0.042 g, 42% yield, purity >98%) as a light yellow solid. LCMS m/z: 507.26 [M+H]; Ή NMR (500 MHz, DMSO-de): δ

2.78 (s, 3H), 3.04 (s, 3H), 4.45 (d, J = 4.9 Hz, 2H), 5.17 (s, 2H), 7.07 (d, J = 8.1 Hz, 1H), 25 7.18-7.23 (m, 3H), 7.30-7.38 (m, 2H), 7.42-7.43 (dd, Λ = 1.4 Hz, J₂ = 8.05 Hz, 1H), 7.56 (d, J = 1.3 Hz, 1H), 8.79 (t, J = 5.05 Hz, 1H).

Examples 2-14

Examples 2-14 were prepared according to the above methods used to make Example 1 and those methods described in General Procedures 1-8 using the product of

Preparation 1 and the appropriate amines and functionalised alkyl halides. Purification was as stated in the aforementioned methods.

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
2	_F ο U _FjX^aUi^f 1	4-(2-fluoro-6methoxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.69 (s, 3H), 3-05 (s, 3H), 3.80 (s, 3H), 4-45 (d, J = 4.6 Hz, 2H), 5.11 (s, 2H), 6.73 (t, J = 9.3 Hz, 1H), 6.83 (d, J = 8.35 Hz, 1H), 7.01 (d, J = 8.05 Hz, 1H), 7.187.27 (m, 3H), 7.37 (d, J = 7.1 Hz, 1H), 7.60 (s, 1H), 8.74 (t, J = 4.85 Hz, 1H).	503-27
3	_{F 0} ,ά/αχ 1	4-(2-fluoro-3methylbenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.23 (s, 3H), 2.81 (s, 3H), 3-07 (s, 3H), 4-41 (d, J = 4.95 Hz, 2H), 5.10 (s, 2H), 6.93-6.96 (m, 1H), 7.00 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 8.1 Hz, 1H), 7.17 (t, J = 8.25 Hz, 3H), 7.32 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 8.84 (t,J=5Hz, 1H).	487.24
4	JUL ^H UL X 1	4-(2-fluoro-6hydroxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2-73 (s, 3H), 3-04 (s, 3H), 4.43 (d, J = 4.7 Hz, 2H), 5.06 (s, 2H), 6.54 (t, J = 8.95 Hz, 1H), 6.64 (d, J = 8.05 Hz, 1H), 7.00-7.08 (m, 2H), 7.20 (t, J = 8.5 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H),	489-15

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
			7.65 (s, 1H), 8.75 (bs, 1H).
5	F 0 .ΔγΧχγ 1	4-(3,5- difluorobenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.85 (s, 3H), 3-10 (s, 3H), 4.40 (d, J = 4.9 Hz, 2H), 5.09 (s, 2H), 6.96 (d, J = 6.45 Hz, 2H), 7.10-7.17 (m, 4H), 7.23 (d, J = 1.0Hz, 1H), 7.487.48 (dd, Jr = 1.25 Hz, J₂= 8.1 Hz, 1H), 8.83 (t, J = 4.95 Hz, 1H).	491.28
6	f\-0 ^H \ / N	N-(benzofuran2-ylmethyl)-4(2-chloro-6fluorobenzyl)i,2-dimethyl-3oxo-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.8ο (s, 3H), 3.05 (s, 3H), 4-63 (d, J = 5-55 Hz, 2H), 5.19 (s, 2H), 6.74 (s, 1H), 7.10 (d, J = 8.1 Hz, 1H), 7.20-7.37 (m, 5H), 7-51-7-55 (m, 2H), 7.60 (d, Λ = 7.15 Hz, 1H), 7.65 (d, J = 1.35 Hz, 1H), 9.05 (t, J = 5.6 Hz, 1H).	493-24
7	^f>^n J- h F 0 .ΔγΑχχ⁰1	4-((3- fluoropyridin-4yl)methyl)-i,2dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.85 (s, 3H), 3.08 (s, 3H), 4.40 (d, J = 4.9 Hz, 2H), 5.17 (s, 2H), 6.12- 7.18 (m, 4H), 7.25 (s, 1H), 7.49 (d, J = 8.15 Hz, 1H), 8.34 (d, J = 4.75 Hz, 1H), 8.58 (s, 1H), 8.84 (t, Jt = 4.9 Hz, 1H).	474-24

- 8ο -

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
8	V/? ΑΧ ΙΧΕ 1	4-(2-chloro-6fluorobenzyl)1,2-dimethyl-N((5-methylfuran2-yl)methyl)-3oxo-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSO-d₆): δ 2.23 (s, 3H), 2.79 (s, 3H), 3-04 (s, 3H), 4-39 (d, J = 5.6 Hz, 2H), 5.19 (s, 2H), 6.00 (d, J = 1.95 Hz, 1H), 7.12 (d, J = 2.95 Hz, 1H), 7.08 (d, J = 6.48 Hz, 1H), 7.187.21 (m, 1H), 7-31-7-36 (m, 2H), 7.47-7.49 (dd, Ji = 1.5 Hz, J₂ = 8.1 Hz, 1H), 7.63 (d, J = 1.4 Hz, 1H), 8.84 (t, Jr = 5-6 Hz, 1H).	457-21
9	, ο /Α.	4-(2-chloro-6fluoro-3methoxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.77 (s, 3H), 3-04 (s, 3H), 3-81 (s, 3H), 4-44 (d, J = 4-8 Hz, 2H), 5-16 (s, 2H), 7.10-7.05 (m, 2H), 7-23-7-15 (m, 3H), 7.41 (d, J = 7.7 Hz, 1H), 7.55 (s, 1H), 8.79 (s, 1H).	537-21
10	Cl^^xJDMe Μ Μ Α ΛΑ ^Η ΙΑ A \ί^-_Ν A 1	4-(2-chloro-6fluoro-4methoxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine	(500 MHz; DMSO-d₆): δ 2.70 (s, 3H), 3.04 (s, 3H), 3-74 (s, 3H), 4-45 (d, J =4-8 Hz, 2H), 5.08 (s, 2H), 6.70 (d, J = 9.9 Hz,2H), 7.06 (d, J=8.i Hz, 1H), 7.22 (t, J = 8.4 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.54 (s, 1H),	521.22

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
		-6-carboxamide	8.82 (s, 1H).
11	XT F 0 \|\ A/W 1	4-(2,6-difluoro- 4hydroxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.69 (s, 3H), 3-03 (s, 3H), 4-45 (d, J = 4-7 Hz, 2H), 5.04 (s, 2H), 6.41 (d, J = 9.8 Hz, 2H), 7.05 (d, J = 8 Hz, 1H), 7.21 (t, J = 8.5 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.52 (s, 1H), 8.81 (t, J = 4.7 Hz, 1H), 10.4 (s, 1H).	507-15
12	^F'Y^i?xi<^O'^s/^OH F 0 [X A^nVyⁿy°^f JLxsX. ^H IXL. .N. 1	4-(2,6-difluoro- 4-(2hydroxyethoxy)b enzyl)-i,2dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.70 (s, 3H), 3.03 (s, 3H), 3.65 (d, J = 5.2 Hz, 2H), 3.96 (s, 2H), 4.45 (d, J = 4.2 Hz, 2H), 4.89 (t, J = 5.3 Hz, 1H), 5.08 (s, 2H), 6.69 (d, J = 10 Hz, 2H), 7.06 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 8.4 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H), 7.54 (s, 1H), 8.82 (bs, 1H).	551-16
13	Fn^^O^xx^OH X J F 0 [Χ¹ Τ^ΑγΑ ΛΑ ^H LA n F^ZXsS^F Χ^ιγ'Χ 1	4-(2,6-difluoro- 4-(3- hydroxypropoxy) benzyl)-i,2dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine	(500 MHz; DMSO-d₆): δ i.8o (t, J = 5.9 Hz, 2H), 2.70 (s, 3H), 3.04 (s, 3H), 3.50 (d, J = 5.7 Hz, 2H), 4.00 (t, J = 12.0 Hz, 2H), 4.45 (d, J = 4.3 Hz, 2H), 4.55 (t, J = 9.5 Hz, 1H), 5.08 (s, 2H), 6.68 (d, J = 10 Hz, 2H), 7.05 (d, J = 8.1 Hz, 1H),	565-24

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
		-6-carboxamide	7.22 (t, J = 8.5 Hz, 2H), 7.42 (d, J = 11.1 Hz, 1H), 7.54 (s, 1H), 8.81 (s, 1H).
14	F 0 /VOm 1	4-(2-chloro-6fluoro-3hydroxybenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.78 (s, 3H), 3.04 (s, 3H), 4.43 (d, J = 4.4 Hz, 2H), 5.13 (s, 2H), 6.896.87 (m, 1H), 6.99 (t, J = 9.3 Hz, 1H), 7.05 (d, J = 7.9 Hz, 1H), 7.21 (t, J = 8.4 Hz, 2H), 7.41 (d, J = 8.05 Hz, 1H), 7.54 (s, 1H), 8.77 (s, 1H).	523-13

Example is: 4.-(2-Chloro-6-fluorobenzyD-i.2-dim ethyl-3-oxo-N-(2.4..6Lrifluorobenzvl)-i.2.2.4.-LeLrahvdroauinoxaline-6-carboxamide

Example 15 was prepared according to the methods described in General Procedures 13 and 8, and the methods described below.

Preparation 5: Methyl-i.2-dimethyl-B-oxo-i.2.B.4-tetrahydroquinoxaline-6-carboxylate

-83Step 1: Methyl-4-iii-methoxy-i-oxopropan-2-yl')amino')-3-nitrobenzoate

To a stirred solution of methyl-4-fluoro-3-nitrobenzoate (0.75 g, 3.77 mmol) in THF (10 mL) was added NaHCOs (0.63 g, 7.54 mmol) followed by methyl 2-aminopropanoate (P-79 g, 5.66 mmol) at RT and the whole reaction mixture was stirred at RT for 20 h.

The progress of the reaction was monitored by TLC and/or LCMS and after completion the reaction mixture was diluted with EtOAc and washed with cold water followed by brine solution. The organic layer was concentrated under reduced pressure to give the title compound methyl-4-((i-methoxy-i-oxopropan-2-yl)amino)-3-nitrobenzoate (0.95 g, 89% yield) as a yellow solid. The product was used in the next step without any further purification.

Step 2: Methyl-2-methyl-3-oxo-i.2.3.4-tetrahydroquinoxaline-6-carboxylate

H

To a stirred solution of methyl-4-((i-methoxy-i-oxopropan-2-yl)amino)-3nitrobenzoate (Step 1) (0.3 g, 1.06 mmol) in glacial acetic acid (4.0 mL) was added Fe powder (0.30 g, 5.31 mmol) at RT. The reaction mixture was heated at 80 °C for 14 h and after completion (monitored by TLC and or LCMS) the reaction mixture was poured into ice cold water and extracted with DCM. The organic layers were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to give the cyclized title compound methyl-2-methyl-3-oxo-i,2,3,4-tetrahydroquinoxaline-6-carboxylate (0.23 g, 98% yield, purity 99%) as a yellow solid. The product was used in the next step without any further purification. LCMS m/z: 221.06 [M+H].

-84Preparation 6: Methyl-4-i2-chloro-6-fluorobenzyl)-2-methyl-3-oxo-i.2.3.4tetrahydroquinoxaline-6-carboxylate

H

To a stirred solution of methyl-2-methyl-3-oxo-i,2,3,4-tetrahydroquinoxaline-65 carboxylate (Preparation 5) (0.2 g, 0.91 mmol) in dry DMF (5.0 mL) was added K₂CO₃(0.25 g, 1.82 mmol) followed by 2-chloro-6-fluorobenzyl bromide (0.22 g, 1.0 mmol) at RT. The whole reaction mixture was stirred at RT for 12 h and after completion (monitored by TLC and or LCMS) the reaction mixture was diluted with cold water and extracted with MTBE (x3). The combined organics were washed with brine solution, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the crude product which was purified by Combi-flash chromatography using 15% EtOAc /hexane mixture to give the title compound methyl-4-(2-chloro-6-fluorobenzyl)-2methyl-3-oxo-i,2,3,4-tetrahydroquinoxaline-6-carboxylate (0.12 g, 36% yield, purity 99.5%) as a white solid. LCMS m/z: 363.07 [M+H].

Preparation 7: Methyl-4-f2-chloro-6-fluorobenzyl')-i.2-dimethyl-.?-oxo-i.2..?.4tetrahydroauinoxaline-6-carboxylate

To a stirred solution of methyl-4-(2-chloro-6-fluorobenzyl)-2-methyl-3-oxo-i,2,3,420 tetrahydroquinoxaline-6-carboxylate (Preparation 6) (0.1 g, 0.28 mmol) in MeOH (1.0 mL) was added formalin solution (0.1 mL) and the whole was further stirred at RT for 2 h. Sodium cyanoborohydride (0.1 g, 0.55 mmol) and 1-2 drop of acetic acid ware added and the mixture was stirred at RT for 24 h. Completion of the reaction was monitored by TLC and/or LCMS, which showed around 40% reaction completion. A further portion of formalin solution (0.05 mL) and sodium cyanoborohydride (0.1 g, 0.55 mmol) were added and the reaction mixture was stirred for another 24 h at RT. After

-85completion of the reaction, the reaction mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed subsequently with a saturated solution of sodium bicarbonate followed by brine. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford a crude compound which was purified by Combi-flash chromatography using 20% EtOAc/hexane mixture as eluent to afford the title compound methyl-4-(2-chloro-6fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4-tetrahydroquinoxaline-6-carboxylate (0.035 g, 34% yield) as a white solid. LCMS m/z: 377.1 [M+H].

Preparation 8: 4-(2-Chloro-6-fluorobenzyl')-i.2-dimethyl-3-oxo-N-(2.4.6trifkiorobenzvl)-i,2,.q,4-tetrahvdrociiiinoxaline-6-carboxamide (Example 15) Ck

To a stirred solution of 4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydroquinoxaline-6-carboxylic acid (synthesized from the product of Preparation 7 15 according to the method of Preparation 3) (0.025 g, 0.068 mmol) in DCM (1.5 mL) was added TEA (0.024 mL, 0.17 mmol) followed by HBTU (0.031 g, 0.083 mmol) at 0 °C and the whole was allowed to stir for 5-10 min at the same temperature. 2,4, 6Trifluorobenzyl amine (0.01 mL, 0.075 mmol) was then added and the reaction mixture was brought to RT and stirred for 3 h. Completion of the reaction was monitored by 20 TLC and LCMS. After completion of the reaction, the mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed with a saturated solution of sodium bicarbonate, HC1 (1N) and finally by brine. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford crude compound which was purified by Combi-flash using 35% EtOAc/hexane 25 mixture as eluent to afford the title compound 4-(2-chloro-6-fluorobenzyl)-i,2dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4-tetrahydroquinoxaline-6carboxamide (0.01 g, 29% yield, purity 98%) as a white solid. LCMS m/z: 506.13 [M+H]; 1H NMR (400 MHz, DMSO-d6): δ 1.ΟΟ-Ο.98 (t, J = 6.76 Hz, 3H), 2.83 (s, 3H), 4.12-4.07 (q, J = 6.72 Hz, 1H ), 4.46-4.31 (m, 2H), 5.16-5.12 (d, J = 16.04 Hz, 1H), 30 5.44-5.40 (d, J = 15.92 Hz, 1H), 6.69-6.67 (d, J = 15.92 Hz, 1H), 7.18-7.07 (m, 3H),

7.32-7.26 (m, 2H), 8.53-8.50 (t, J = 5.12 Hz, 1H).

-86Example 16: 2-allyl-4-(2-chloro-6-fluorobenzyl)-i-methyl-.3-oxo-N-(2,4.6trifluorobenzyO-i.2.3.4-tetrahydrobenzoreHT.2.4ltriazine-6-carboxamide

Example 16 was prepared according to the methods described in General Procedures i-

7, and the methods described below.

Preparation Q: Methyl-2-allyl-i-methyl-3-oxo-i.2.3.4-tetrahydrobenzore1IT.2.4l io triazine-6-carboxylate

Step 1: tert-Butyl-i-allyl-2-(4-fmethoxycarbonyl')-2-nitrophenyl) hydrazinecarboxylate

O

To a stirred solution of tert-butyl-2-(4-(methoxycarbonyl)-2nitrophenyl)hydrazinecarboxylate (Preparation 1, Step 1) (1.0 g, 3.21 mmol) in DMF (15 mL) was added Cs₂CO₃ (1.25 g, 3.85 mmol) at 0 °C followed by dropwise addition of allyl bromide (0.30 mL, 3.53 mmol) and the reaction mixture was then stirred at RT for 1 h. Progress of the reaction was monitored by TLC and/or LCMS. After completion of 20 the reaction, the mixture was diluted with water and extracted with EtOAc and then washed with brine. The organic layer was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain a crude product which was purified by Combi-flash eluting with 15% EtOAc in hexane mixture to afford the title compound tert-butyl-i-allyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-hydrazinecarboxylate (1.0 g, 25 89% yield) as a yellow viscous oil. LCMS m/z: 256.05 [M+H(-56)].

-S7Step 2: terf-Butyl-i-allyl-2-(4-imethoxycarbonyl)-2-nitrophenyl')-2methylhydrazinecarboxylate

O

To a stirred solution of tert-butyl-i-allyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)5 hydrazinecarboxylate (Step 1) (1.1 g, 3.13 mmol) in DMF (20 mL) was added NaH (0.25 g, 6.26 mmol) at 0 °C followed by dropwise addition of methyl iodide (0.391 mL, 6.26 mmol) and the reaction mixture was heated at 60 °C for 1 h. Progress of the reaction was monitored by TLC and LC. After completion, the reaction mixture was quenched with saturated NH4CI solution, extracted with EtOAc and the organics washed with brine. The collected organic layer was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford the title compound tert-butyl-i-allyl-2(4-(methoxycarbonyl)-2-nitrophenyl)-2-methylhydrazinecarboxylate (1.1 g, yield 95% and purity 98%) as a yellow oil. LCMS m/z: 366.12 [M+H].

Step 3: tert-Butyl-i-allyl-2-(2-amino-4-(methoxycarbonyDphenyD-2methylhydrazinecarboxylate

O

To a stirred solution of tert-butyl-i-allyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-2methylhydrazinecarboxylate (Step 2) (1.3 g, 3.55 mmol) in a mixture of acetonitrile and

H₂0 (36 mL, 2:1) was added sodium hydrosulfite (4.95 g, 28.46 mmol), tetrabutyl ammonium hydrosulfate (0.6 g, 1.77 mmol) and potassium carbonate (2.94 g, 21.34 mmol) at RT and the reaction mixture stirred for 1 h. Completion of the reaction was monitored by TLC and LCMS. After completion of the reaction solvents were evaporated to give an oily liquid which was dissolved in 1N HC1 and extracted with

EtOAc. The organics were washed with a saturated brine solution, dried over anhydrous Na₂SO₄ and concentrated in vacuo to afford the title compound tert-butyl-iallyl-2-(2-amino-4-(methoxycarbonyl)phenyl)-2-methylhydrazinecarboxylate (1.0 g, 84% yield, purity 98%) as a yellowish solid. The product was used in the next step without any further purification. LCMS m/z: 336.2 [M+H].

-88Step 4: Methyl-2-allyl-i-methyl-3-oxo-i.2.3.4-tetrahydrobenzoreiri.2.4l -triazine-6carboxylate

To a stirred solution of tert-butyl-i-allyl-2-(2-amino-4-(methoxycarbonyl)phenyl)-25 methylhydrazinecarboxylate (Step 3) (1.0 g, 2.98 mmol) in dryTHF was added

LiHMDS solution (5 mL, 5.96 mmol, 1.3 M) at 0 °C and the reaction mixture was stirred for 5 min. at the same temperature before heating at 60 °C for 15 min. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was quenched with a saturated solution of NH₄C1 and extracted with EtOAc.

The combined organics ware washed with brine solution, dried over Na₂SO₄ and concentrated under reduced pressure to give the title compound (0.5 g, 64% yield, purity 94%) as a brown solid. The product was used in the next step without any further purification. LCMS m/z: 262.08 [M+H].

Preparation 10: 2-allyl-4-(2-chloro-6-fluorobenzyl')-i-methyl-3-oxo-N-(2.4.6trifluorobenzyl')-i.2.3.4-tetrahydrobenzoreiri.2.4ltriazine-6-carboxamide (Example 16)

2-allyl-4-(2-chloro-6-fluorobenzyl)-i-methyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,420 tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de (Example 16) was prepared from methyl 2-allyl-i-methyl-3-oxo-i,2,3,4tetrahydrobenzofe] [1,2,4] -triazine-6-carboxylate (Preparation 9) according to methods described in Preparations 2, 3 and 4 and General Procedures 1-3. LCMS m/z: 533.14 [M+H]; Ή NMR (500 MHz, DMSO-d6): δ 2.8ο (s, 3H), 4.07 (d, J = 5.15 Hz, 2H), 4.45 (d, J = 4.75 Hz, 2H), 5.06 (s, 1H), 5.09 (d, J = 7.25 Hz, 1H), 7.17 (s, 2H), 5-75-5-82 (m,

1H), 7.05 (d, J = 8.05 Hz, 1H), 719-7.23 (m, 3H), 7.31-7.38 (m, 2H), 7.43 (d, J = 8.0 Hz,

1H), 7.59 (bs, 1H), 8.79 (t, J = 4.8 Hz, 1H).

-89Example 17: 4-(2-chloro-6-fluorobenzyl)-i-methyl-.3-oxo-2-propyl-N-(2,4.6trifluorobenzyl)-i,2,.3.4-tetrahydrobenzoreiri.2,4ltriazine-6-carboxamide

Example 17 was prepared according to the methods described in General Procedures 15 3, and the methods described below.

Preparation 11: Methyl-i-methyl-3-oxo-2-propyl-i.2.3.4-tetrahydrobenzoreiri.2.4ltriazine-6-carboxylate

To a stirred solution of methyl 2-allyl-i-methyl-3-oxo-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxylate (Preparation 9) (0.2 g, 0.77 mmol) in methanol (4 mL) was added Pd/C (10%) at RT under an inert atmosphere and the reaction mixture was stirred under H₂ gas balloon pressure at RT for 2 h. The progress of the reaction was monitored by TLC and LCMS, showing that the starting material had all been consumed. The reaction mixture was filtered through a celite pad and concentrated under reduced pressure to afford the title compound methyl-i-methyl-3oxo-2-propyl-i,2,3,4-tetrahydrobenzo-[e][i,2,4]triazine-6-carboxylate (0.2 g, 99% yield, purity 98%) as a greenish solid. The product was used in the next step without any further purification. LCMS m/z: 264.1 [M+H].

Preparation 12: 4-f2-Chloro-6-fluorobenzyl')-i-methyl-.q-oxo-2-propyl-N-(2.4.6trifluorobenzyl')-i.2..q.4-tetrahydrobenzoreiri.2.4ltriazine-6-carboxamide (Example 17)

-904-(2-Chloro-6-fluorobenzyl)-i-methyl-3-oxo-2-propyl-N-(2,4,6-trifluorobenzyl)i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide (Example 17) was prepared from methyl-i-methyl-3-oxo-2-propyl-i,2,3,4-tetrahydrobenzo-[e][i,2,4]triazine-6carboxylate (Preparation 11) according to methods described in Preparations 2, 3 and 4 5 and General Procedures 1-3. LCMS m/z: 535.17 [M+H]; Ή NMR (500 MHz, DMSOd₆): δ 0.73 (t, J = 7-35 Hz, 3H), 1-49-1-53 (m, 2H), 2.77 (s, 3H), 3.38 (d, J = 6.7 Hz, 2H), 4.44 (d, J = 4.85 Hz, 2H), 5.16 (s, 2H), 7.08 (d, J = 8.05 Hz, 1H), 7.17-7.22 (m, 3H), 7-30-7-37 (m, 2H), 7.42 (d, J = 8.05 Hz, 1H), 7.56 (s, 1H), 8.80 (t, J = 4.9 Hz, 1H).

Example 18: i-Allyl-4-(2-chloro-6-fluorobenzyl)-2-methyl-3-oxo-N-(2,4.6Lrifluorobenzvl)-i.2.rt.4.-tetrahvdrobenzol ell 1.2.4. ltriazine-6-carboxamide

Example 18 was prepared according to the methods described in General Procedures 1-

7, and the methods described below.

Preparation 13: Methyl-i-allyl-2-methyl-.2-oxo-i.2.2.4-tetrahydrobenzore][i.2.4] triazine-6-carboxylate

Step 1: tert-Butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl')-i-methylhydrazine carboxylate

To a stirred solution of tert-butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)hydrazine carboxylate (Preparation 1, Step 1) (1.5 g, 4.81 mmol) in DMF (20 mL) was added

-91Cs₂CO₃ (3.14 g, 9.63 mmol) at 0-5 °C followed by dropwise addition of methyl iodide (0.351 mL, 5.78 mmol) and the reaction mixture was stirred at RT for 1 h. Completion of the reaction was monitored by TLC and LC. After completion the reaction mixture was diluted with water and extracted with EtOAc followed by a brine wash. The collected organic layer was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain a crude material which was purified by Combi-flash eluting with 15% EtOAc/hexane as eluent to afford the title compound (1.5 g, 95.5% yield and purity >82%) as a brown solid. LCMS m/z: 324.09 [M-H].

Step 2: tert-Butyl 2-allyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-i-methylhydrazine carboxylate

O

To a stirred solution of tert-butyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-imethylhydrazinecarboxylate (Step 1) (0.6 g, 1.84 mmol) in DMF (10 mL) was added

NaH (0.15 g, 3.68 mmol, 60% suspension in mineral oil) at 0-5 °C followed by dropwise addition of allyl bromide (0.31 mL, 3.68 mmol) and the reaction mixture was heated at 60 °C for 1 h. After completion of the reaction the reaction mixture was quenched with a saturated solution of NH₄C1 and extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain a crude compound which was purified by Combi-flash eluting with 20% EtOAc/hexane as eluent to afford the title compound (0.35 g, 52% yield and purity 85%) as a yellow viscous oil. LCMS m/z: 266.02 [M+H (-100)].

Step 3: tert-Butyl-2-allyl-2-(2-amino-4-(methoxycarbonyl)phenyl)-i-methylhydrazine carboxylate

O

To a stirred solution of tert-butyl-2-allyl-2-(4-(methoxycarbonyl)-2-nitrophenyl)-imethylhydrazinecarboxylate (Step 2) (0.4 g, 1.09 mmol) in MeCN:H₂0 (12 mL, 2:1) was

-92added sodium hydrosulfite (1.52 g, 8.75 mmol), tetrabutyl ammonium hydrosulfate (0.185 g, 0.54 mmol) and potassium carbonate (0.91 g, 6.56 mmol) at RT and the reaction mixture was stirred at RT for 1 h. Completion of the reaction was monitored by TLC and LC. After completion of the reaction the solvents were evaporated under reduced pressure to give an oily liquid which was dissolved in 1N HC1 and extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous Na₂SO₄ and concentrated in vacuo to afford the title compound (0.35 g, 68% yield and purity >85%) as a yellowish solid. LCMS m/z: 336.17 [M+H].

Step 4: tert-Butyl-2-allyl-2-(2-((ethoxycarbonyl')amino')-4-(methoxycarbonyl')phenyl')i-methylhydrazinecarboxylate

To a stirred solution of tert-butyl-2-allyl-2-(2-amino-4-(methoxycarbonyl)phenyl)-imethylhydrazine carboxylate (Step 3) (0.35 g, 1.04 mmol) in DCE (5 mL) at 0-5 °C was 15 added pyridine (0.185 mL, 2.29 mmol) and ethyl chloroformate (0.119 mL, 1.25 mmol) and the whole stirred at RT for 1 h. Completion of the reaction was monitored by TLC and LC. After complete consumption of the starting materials, the reaction mixture was quenched with 1N HC1 solution and extracted with DCM. The combined organics were washed with brine, dried over anhydrous Na₂SO₄ and concentrated in vacuo to afford a 20 crude oil which was purified by Combi-flash eluting with 20% EtOAc/hexane as eluent to afford the title compound (0.35 g, 82% yield and purity 98%) as a white solid. LCMS m/z: 408.24 [M+H].

Step 5: Methyl-4-ii-allyl-2-methylhydrazinyl')-3-iiethoxycarbonyl)amino')benzoate hydrochloride

0^0

-93To a stirred solution of tert-butyl-2-allyl-2-(2-((ethoxycarbonyl)amino)-4(methoxycarbonyl)phenyl)-i-methylhydrazinecarboxylate (Step 4) (0.5 g, 1.41 mmol) in

1,4-dioxane (10 mL) at 0-5 °C was added 4M HC1 in dioxane (5 mL) and the whole was stirred at RT overnight. Completion of the reaction was monitored by TLC and LC and after completion the solvents were evaporated under reduced pressure to give a crude residue which was azeotropically distilled with MeCN to afford the title compound (0.2 g, 53% yield and purity >90%) as a yellow solid. LCMS m/z: 308.15 [M+H].

Step 6: Methyl-i-allyl-2-methyl-3-oxo-i.2.3.4-tetrahydrobenzoreHT.2.41triazine-610 carboxylate

O

To a stirred solution of methyl-4-(i-allyl-2-methylhydrazinyl)-3-((ethoxycarbonyl)amino)benzoate hydrochloride (Step 5) (0.2 g, 0.65 mmol) in methanol (4 mL) and water (2 mL) was added K₂CO₃ (0.178 g, 1.29 mmol) and the reaction mixture was stirred at RT for 2 h. Completion of the reaction was monitored by TLC and LC and after completion the solvents were evaporated under reduced pressure to give an oil which was diluted with water and extracted with EtOAc. The combined organics were washed with brine, dried over Na₂SO₄ and concentrated under reduced pressure to give the title compound (0.12 g, 70.5% yield and purity 97%) as a grey white solid. LCMS m/z: 262.06 [M+H].

Preparation 14: i-Allyl-4-(2-chloro-6-fluorobenzyl')-2-methyl-.3-oxo-N-(2.4.6trifluorobenzyl)-i.2..3.4-tetrahydrobenzore]ri.2.4]triazine-6-carboxamide (Example 18)

-94Example 18 was prepared from methyl-i-allyl-2-methyl-3-oxo-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxylate (Preparation 13) according to the methods described in Preparations 2,3 and 4 and General Procedures 1-3. LCMS m/z: 533-17 [M+H]; Ή NMR (500 MHz, DMSO-d₆): δ 3-05 (s, 3H), 3-63 (d, J = 6.85 Hz,

2H), 4.45 (d, J = 4.85 Hz, 2H), 4.97 (d, J = 17.05 Hz, 1H), 5.05 (d, J = 10.15 Hz, 1H),

5.15 (s, 2H), 5-85-5-93 (m, 1H), 7.02 (d, J = 8.05 Hz, 1H), 7.21 (t, J = 8.75 Hz, 3H), 7.327.39 (m, 2H), 7.40-7.42 (dd, Λ = 1.0 Hz, J₂ = 8.05 Hz, 1H), 7.60 (bs, 1H), 8.81 (t, J = 5.05 Hz, 1H).

Examples 19-26

Examples 19-26 were prepared according to the above method used to make Example 1 and Preparations 2-4 using substrates described in General Procedures 4 and 5 and the methods described in General Procedures 1-9 using the appropriate reagents. Purification was as stated in the aforementioned methods.

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
19	1 1 OH	4-(2-chloro-6fluorobenzyl)-2(3hydroxypropyl)i-methyl-3-oxo- N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 1.24 (s, 2H), 1.64-1.67 (m, 2H), 2.77 (s, 3H), 3.32 (t, J = 6.45 Hz, 1H), 3.48 (t, J = 6.65 Hz, 2H), 4-45 (d, J = 4.65 Hz, 2H), 5.16 (s, 2H), 7.08 (d, J = 8.1 Hz, 1H), 7.18-7.23 (m, 3H), 7-31-7-38 (m, 2H), 7.42 (d, J = 8.25 Hz, 1H), 7-57 (s, 1H), 8.79 (bs, 1H).	551-18
20	1 1 ^OH	4-(2-chloro-6fluorobenzyl)-2(2hydroxypropyl)i-methyl-3-oxo-	(500 MHz; DMSO-d₆): δ 0.95 (d, J = 6.15 Hz, 3H), 2.78 (s, 3H), 3.22- 3.26 (dd, Λ = 6.15 Hz, J₂ = 13.75 Hz, 1H),	551-18

Exa mple	Structure	IUPAC Name	II-NMR	LCMS [M+H]
		N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	3-41-3-45 (dd, Λ = 6.05 Hz, J₂ = 13.7 Hz, 1H), 3-77-3-81 (m, 1H), 4.45 (d, J = 4-7 Hz, 2H), 4.59 (d, J = 5.2 Hz, 1H), 5.11 (q, J = 15.4 1H), 5.21 (d, J = 15.3 Hz, 1H), 7.06 (d, J = 8.05 Hz, 1H), 7.18-7.23 (m, 3H), 7.30-7.38 (m, 2H), 7.42 (d, J = 8.05 Hz, 1H), 7.58 (s, 1H), 8.79 (t, J = 4.85 Hz, 1H).
21	T}	6-(2-chloro-6fluorobenzyl)-5oxo-N-(2,4,6trifluorobenzyl)- 2,3,5,6tetrahydro-iHbenzo[e]pyrazol 0[l,2a][i,2,4]triazine8-carboxamide	(500 MHz; DMSO-d₆): δ 2.19-2.22 (m, 2H), 3.18 (m, 2H), 3.50 (t, J = 7.3 Hz, 2H), 4.41 (d, J = 4.45 Hz, 2H), 5.13 (s, 2H), 6.72 (d, J = 8.15 Hz, 1H), 7.14-7.22 (m, 3H), 7.28-7.35 (m, 3H), 7-40 (d, J = 7-55 Hz, 1H), 8.64 (bs, 1H).	519-14
22	F 0 OH	4-(2-chloro-6fluorobenzyl)-i- (3- hydroxypropyl)- 2-methyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo	(500 MHz; DMSO-d₆): δ 1-53 (t, J = 6.8 Hz, 2H), 3.02 (t, J = 7.30 Hz, 2H), 3.05 (s, 3H), 3-43 (d, J = 5-4 Hz, 2H), 4.45-4.49 (m, 3H), 5-15 (s, 2H), 7-03 (d, J = 8.0 Hz, 1H), 7.17-7.23 (m, 3H),	551-11

Exa mple	Structure	IUPAC Name	II-NMR	LCMS [M+H]
		[e][i,2,4]triazine -6-carboxamide	7.30-7.36 (m, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.61 (s, 1H), 8.81 (bs, 1H).
23	OH	4-(2-chloro-6fluorobenzyl)-i- (2- hydroxypropyl)- 2-methyl-3-oxo- N-(2,4,6trifluorobenzyl)1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ i.o6 (d, J = 6.1 Hz, 3H), 2.80-2.91 (m, 2H), 3.06 (s, 3H), 3.633.65 (m, 1H), 4.45 (d, J = 4.35 Hz, 2H), 4.76 (d, J = 4.4 Hz, 1H), 5.115.19 (q, J = 15-35 2H), 7.09 (d, J = 8.05 Hz, 1H), 7-18-7-23 (m, 3H), 7.31-7.38 (m, 2H), 7.43 (d, J = 8.1 Hz, 1H), 7.60 (s, 1H), 8.80 (t, J = 4.4 Hz, 1H).	551-H
24	rQ Mx 1 1 CN	4-(2-chloro-6fluorobenzyl)-2(2-cyanoethyl)i-methyl-3-oxo- N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.79 (s, 5H), 3-71 (t, J = 6.1 Hz, 2H), 4.45 (d, J = 4.7 Hz, 2H), 5.18 (s, 2H), 7.11 (d, J =8.1 Hz, 1H), 7-29-7-19 (m, 3H), 7.38-7.31 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 8.82 (t, J = 4.8 Hz, 1H).	546.13
25	^FX1 F 0 ^ΖχΌ^Ζ	2- allyl-4-(2- chloro-6-fluoro- 3- methoxybenzyl)i-methyl-3-oxoN-(2,4,6-	(500 MHz; DMSO-d₆): δ 2.78 (s, 3H), 3.81 (s, 3H), 4.07 (d, J = 3.2 Hz, 2H), 4-44 (d, J = 2.7 Hz, 2H), 5.09-5.06 (m, 2H), 5.17 (s, 2H),	563· 15

Exa mple	Structure	IUPAC Name	II-NMR	LCMS [M+H]
		trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	5.81-5.76 (m, 1H), 7.117.04 (m, 2H), 7.23-7.16 (m, 3H), 7.42 (d, J = 7.9 Hz, 1H), 7.57 (s, 1H), 8.79 (s, 1H).
26	F 0 OH 1	2- allyl-4-(2- chloro-6-fluoro- 3- hydroxybenzyl)i-methyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz; DMSO-d₆): δ 2.79 (s, 3H), 4.07 (d, J = 4.6 Hz, 2H), 4.44 (d, J = 4-6 Hz, 2H), 5.09-5.06 (m, 2H), 5.14 (s, 2H), 5.81-5.76 (m, 1H), 6.90-6.87 (m, 1H), 7.05-6.98 (m, 2H), 7.21 (t, J = 8.5 Hz, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.57 (s, 1H), 8.78 (s, 1H), 10.19 (s, 1H).	549-17

Example 27: N-(Benzofuran-2-ylmethyl)-4-(2-chloro-6-fluorobenzyl)-i,2dimethyl-.3-oxo-i,2,.3.4-tetrahydropyridor.3.2-eiri.2,4ltriazine

Preparation is: Methyl-i.2-dimethyl-B-oxo-i.2.B.4-tetrahydropyridorB.2el Γ i.2.41triazine-6-carboxylate

-98Step 1: Methyl-6-(2-(tert-butoxycarbonyllhydrazinyll-s-nitronicotinate

O

To a stirred solution of methyl-6-chloro-5-nitronicotinate (1.0 g, 4.63 mmol) in anhydrous DMSO (20 mL) was added tert-butyl-hydrazinecarboxylate (0.67 g, 5.07 mmol) and whole reaction mixture was stirred at RT for 14 h. After completion of the reaction (monitored by TLC and LCMS) the reaction mass was quenched with water and extracted with EtOAc. The combined organics were washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give the title compound methyl-6-(2-(tert-butoxycarbonyl)hydrazinyl)-5-nitronicotinate (1.2 g, 83% yield, purity 100%) as a yellow solid. The product was used in the next step without any further purification. LCMS m/z: 313.10 [M+H].

Step 2: Methyl-6-f2-ftert-butoxycarbonyl')-i.2-dimethylhydrazinyl')-.s-nitronicotinate

O

To a stirred solution of methyl-6-(2-(tert-butoxycarbonyl)hydrazinyl)-5-nitronicotinate (Step 1) (0.82 g, 2.63 mmol) in anhydrous DMF (15 mL) under a N₂ atmosphere was added Mel (0.76 g, 5.35 mmol) followed by NaH (0.221 g, 5.5 mmol, 60% suspension in oil) portionwise at 0-5 °C. The resulting mixture was stirred at the same temperature for 4 h. The course of the reaction was monitored by TLC and/or LCMS. After completion of the reaction the mixture was quenched with a saturated solution of NH₄C1, diluted with water and extracted with MTBE. The combined organics were washed with brine twice, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give a crude product which was purified by column chromatography using silica gel with 60% EtOAc/hexane mixture as eluent to afford the title compound methyl-6-(2-(teit-butoxycarbonyl)-i,2-dimethylhydrazinyl)-5nitronicotinate (0.86 g, 96.6% yield, purity 89%) as a yellow solid. LCMS m/z: 341.11 [M+H],

-99Step 3: Methyl-5-amino-6-(2-(tert-butoxycarbonyl')-i.2-dimethylhydrazinyl')ni cotinate

O

To a stirred solution of 6-(2-(tert-butoxycarbonyl)-i,2-dimethylhydrazinyl)-5nitronicotinate (Step 2) (0.86 g, 2.53 mmol) in anhydrous EtOAc was added Pd/C (10%, 0.027 g, 0.26 mmol) and the mixture was flushed twice with N₂ gas followed by

H₂. After purging the mixture was stirred at RT under H₂ balloon pressure for 3 h. After completion of the reaction, the mixture was filtered through a bed of celite carefully and the celite bed was washed with EtOAc 3 times under a N₂ atmosphere. The combined filtrate was evaporated under reduced pressure to obtain the title compound methyl-510 amino-6-(2-(tert-butoxycarbonyl)-i,2-dimethylhydrazinyl)nicotinate (0.78 g, 96% yield, purity >97%) as a gummy solid. The product was used in the next step without any further purification. LCMS m/z: 311.08 [M+H].

Step 4: Methyl-i.2-dimethyl-3-oxo-i.2.3.4-tetrahydropyridolA.2-eHT.2.4.1triazine-615 carboxylate

To a stirred solution of methyl-5-amino-6-(2-(tert-butoxycarbonyl)-i,2dimethylhydrazinyl)nicotinate (Step 3) (0.6 g, 1.93 mmol) in anhydrous THF (20 mL) was added LiHMDS (0.64 g, 3.82 mmol, 1.3 M sol. in THF) dropwise at 0 °C under an argon atmosphere. After complete addition, the reaction mixture was warmed to RT and then heated at 40-45 °C for 1 h. The reaction was monitored by TLC and LCMS and after completion of the reaction; the mixture was quenched by the addition of a saturated solution of NH₄C1 and extracted with EtOAc. The combined organics ware washed with a saturated brine solution, dried over anhydrous Na₂SO₄ and the solvents evaporated under reduced pressure to obtain a crude product which was purified by column chromatography over silica gel using a 40% EtOAc/hexane mixture as eluent to afford the title compound methyl-i,2-dimethyl-3-oxo-i,2,3,4-tetrahydropyrido[3,2-

e][i,2,4]triazine-6-carboxylate (0.4 g, 85% yield, purity 100%) as a white solid. LCMS m/z: 237.08 [M+H].

- 100 Preparation 16: lV-(Benzofuran-2-ylmethyl')-4-(2-chloro-6-fluorobenzyl)-i.2-dimethyl-

3-oxo-i.2.3.4.-tetrahydropyridor3.2-e1IT.2.4.1triazine-6-carboxamide (Example 27)

Cl

To a stirred solution of 4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydropyrido[3,2-e][i,2,4]triazine-6-carboxylic acid (synthesized from the product of Preparation 13 according to methods described in Preparations 2 and 3) (0.05 g, 0.14 mmol in DMF (2.0 mL) was added TEA (0.042 g, 0.42 mmol) under a N₂ atmosphere followed by HATU (0.063 g? 0.17 mmol) and the reaction mixture was stirred for 5 minutes. Benzofuran-2-ylmethanamine (0.023 g, 0.14 mmol) was added to the reaction and the whole was stirred at RT for 1 h. After completion of the reaction, it was quenched with water and extracted with EtOAc. The combined organic layers were washed with a saturated brine solution, dried over Na₂SO₄ and evaporated under reduced pressure to obtain a crude compound which was purified by prep-HPLC to afford the title compound (Example 27) JV-(benzofuran-2-ylmethyl)-4-(2-chloro-6fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4-tetrahydropyrido-[3,2-e][i,2,4]triazine-6carboxamide (0.045 g, yield, purity >99%) as an off white solid. LCMS m/z: 494.11 [M+H]; Ή NMR (500 MHz, DMSO-d₆): δ 1.28 (d, J = 6.6 Hz, 3H), 2.97 (s, 3H), 3.34 (m, 2H), 4.42-4.52 (m, 2H), 4.57-4.61 (m, 1H), 4.92 (d, J = 15.75 Hz, 1H), 5.49 (d, J =

15.7 Hz, 1H), 7.16-7.24 (m, 3H), 6.30-7.37 (m, 2H), 7.76 (s, 1H), 8.52 (s, 1H), 9.01 (bs,

1H).

Examples 28-36

Examples 28-36 were prepared according to the methods described for the synthesis of

Example 1 and the procedures described in General Procedures 1-15 starting from an appropriately substituted phenyl/pyridine and using the appropriate benzyl halides and amines.

- 101 -

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
28	. , 1	4-(2-chloro-6fluorobenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydropyrido [3,2e][i,2,4]triazine6-carboxamide	(500 MHz, DMSOd₆): δ 2.97 (s, 3H), 3.08 (s, 3H), 4-45 (d, J = 4.7 Hz, 2H), 5.12 (s, 2H), 7.19-7.24 (m, 3H), 7.30-7.38 (m, 2H), 7.73 (s, 1H), 8.34 (s, 1H), 8.92 (bs, 1H).	508.10
29	Tj AJaX 1	4-(2-chloro-6fluorobenzyl)-7hydroxy-1,2dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSOd₆): δ 2.76 (s, 3H), 2.98 (s, 3H), 4.52 (d, J = 4.6 Hz, 2H), 5.08 (s, 2H), 6.56 (s, 2H), 7.17 (t, J = 9.2 Hz, 1H), 7.25 (t, J = 8.55 Hz, 2H), 7.297.30 (m, 1H), 7.327.37 (m, 1H), 7.57 (s, 1H), 9.01 (bs, 1H).	523-11
30	F θ X⁵ r it ^N τ r JI ^H JU- A k^OH 1	4-(2-chloro-6fluorobenzyl)-7(3hydroxypropyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSOd₆): δ 1.52-1.58 (m, 2H), 2.53-2.55 (m, 2H), 2.75 (s, 3H), 3.05 (s, 3H), 3-253.29 (q, J = 6.1 Hz, 2H), 4.39-4.44 (m, 3H), 5.12 (S, 2H), 6.87 (s, 1H), 6.94 (s, 1H), 7.16 (t, J = 9.5 Hz, 1H), 7.23 (t, J = 8.5 Hz, 2H), 7.30 (d,	565· 15

- 102 -

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
			J = 8 Hz, 1H), 7.347-39 (m, 1H), 8.69 (t, J =5.15 Hz, 1H).
31	Tj ΑδαΤ 1	4-(2-chloro-6fluorobenzyl)-7cyano-1,2dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSOd₆): δ 2.83 (s, 3H), 3.06 (s, 3H), 4.47 (d, J = 5.1 Hz, 2H), 5.18 (s, 2H), 7.19 (t, J = 9.5 Hz, 1H), 7.27 (t, J = 8.55 Hz, 2H), 7.31-7.40 (m, 3H), 7.58 (s, 1H), 9.05 (t, J = 5.2 Hz, 1H).	532.14
32	Tj F 0 [ T if n >1 τ ΛΑ ^H ΛΛ X 1 1 nh₂hci	7-(3- aminopropyl)-4(2-chloro-6fluorobenzyl)i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide hydrochloride	(500 MHz, DMSOd₆): δ 1.73-1.79 (m, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.67-2.68 (m, 2H), 2.77 (s, 3H), 3.06 (s, 3H), 4.42 (d, J = 5.0 Hz, 2H), 5.13 (s, 2H), 6.97 (d, J = 14-95 Hz, 2H), 7-16 (t, J = 9.15 Hz, 1H), 7.247.31 (m, 3H), 7-347-39 (m, 1H), 7.80 (bs, 3H), 8.77 (t, J = 5.2 Hz, 1H).	56414
33	, . x> Α'ύαΤ 1	4-(2-chloro-6fluorobenzyl)-7methoxy-1,2dimethyl-3-oxoN-(2,4,6-	(500 MHz, DMSOd₆): δ 2.81 (s, 3H), 3.04 (s, 3H), 3.80 (s, 3H), 4-47 (d, J = 5.45 Hz, 2H), 5.12	537-13

-103-

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
		trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(s, 2H), 6.83 (s, 1H), 7.16-7.22 (m, 3H), 7.29-7.37 (m, 2H), 7.42 (s, 1H), 8.48 (t, J= 5.5 Hz, 1H).
34	^Cl\^x Tj F O \| T ΛΑ ^H ΛΛ X r ¹ OH	4-(2-chloro-6fluorobenzyl)-7(3-hydroxyprop- l-yn-l-yl)-l,2- dimethyl-3-oxo- N-(2,4,6- trifluorobenzyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	(500 MHz, DMSOd₆): δ 2.78 (s, 3H), 3.05 (s, 3H), 4-14 (d, J = 5.5 Hz, 2H), 4.42 (d, J = 5.05 Hz, 2H), 5.15 (s, 2H), 5-25 (t, J = 5.8 Hz, 1H), 7.06 (s, 1H), 7.11 (s, 1H), 7.17-7.25 (m, 3H), 7.32 (d, J = 8.05 Hz, 1H), 7-35-7-38 (m, 1H), 8.65 (t, J = 5.15 Hz, 1H).	561.12
35	^Cl\^\ Tj iW? F F O N V^H1 0	2-((4-(2-chloro- 6- fluorobenzyl)i,2-dimethyl-3oxo-6-((2,4,6trifluorobenzyl)c arbamoyl)- 1,2,3,4- tetrahydrobenzo [e][i,2,4]triazin- 7- yl)oxy)acetic acid	(500 MHz, DMSOd₆): δ 2.82 (s, 3H), 3.04 (s, 3H), 4-50 (d, J = 4.95 Hz, 2H), 4.77 (s, 2H), 5.15 (s, 2H), 6.84 (s, 1H), 7.16-7.21 (m, 3H), 7-30-7-37 (m, 2H), 7.61 (s, 1H), 8.93 (bs, 1H), 13.33 (bs, 1H).	581.10
36	JO F 0 \| 1 Ate* k^OH 1	4-(2-chloro-6fluorobenzyl)-7- (2- hydroxyethoxy)-	(500 MHz, DMSOde): δ 2.80-2.85 (m, 3H), 3-03-3-07 (m, 3H), 3.62-3.64 (m,	567-17

-104 -

Exa mple	Structure	IUPAC Name	Ή-NMR	LCMS [M+H]
		i,2-dimethyl-3oxo-N-(2,4,6trifluorobenzyl)1,2,3,4tetrahydrobenzo [e][i,2,4]triazine -6-carboxamide	2H), 4.05-4.08 (m, 2H), 4.46-4.49 (m, 2H), 4.88-4.93 (m, 1H), 5-13-5-16 (m, 2H), 6.86-6.90 (m, 1H), 7.18-7.20 (m, 3H), 7-31-7-34 (m, 2H), 7.52-7.56 (m, 1H), 8.65-8.68 (m, 1H).

Biological Assays

Stable cell line generation

a) Stable STING expressing cells - Stable HEK293T STING-expressing cell lines were generated using plasmids purchased from Invivogen, CA, USA, that contain STING cDNA cloned into the pUNO-1 vector under hEFi-HTLV promoter and containing the Blasticidin selection cassette. The plasmids hSTING(R232), hSTING(H232), hSTING(HAQ) were directly procured from Invivogen while hSTING (AQ) and hSTING (Q) were derived from hSTING(HAQ) and hSTING (R232) plasmids respectively by using a PCR based site directed mutagenesis method. These vectors were individually transfected into HEK293T cells using Lipofectamine (Invitrogen) and transfected cells were selected under Blasticidin selection. These transfected cells were further subjected to clonal selection using the limiting dilution method to obtain clonally pure populations of HEK cells transfected with each of the above mentioned human STING variants. Only those clones were selected in which ligand independent activation of STING was minimal.

b) Stable Luciferase reporter gene expressing cells - Stable HEK293T Luciferase reporter gene expressing cell lines were generated using pCDNA4 plasmids under an IRF-inducible promoter. This promoter is comprised of five tandem interferon-stimulated response elements (ISRE) fused to an ISG54 minimal promoter. This vector was transfected into HEK293T cells using Lipofectamine (Invitrogen) and transfected cells were selected under Zeocin selection. These

-105transfected cells were further subjected to clonal selection using the limiting dilution method to obtain clonally pure populations of HEK cells transfected the Luciferase reporter construct. Only those clones were selected in which ligand independent induction of luciferase was minimal. .

Luciferase Assay x io⁵ clonally selected HEK293T-hSTING-Luciferase cells were seeded in 384-well plates in growth medium and stimulated with novel compounds. After 2ohr of stimulation supernatant were removed and secretary reporter gene activity were measured using the Quanti-Luc detection system (Invivogen) on a Spectramax 13X luminometer.

In the tables below, EC₅₀ value ranges for exemplary compounds are given. The EC₅₀ranges are indicated as “A” for values less than or equal to 1 pM, “B” for values greater than 1 pM and less than or equal to 10 pM, and “C” for values greater than 10 pM.

All compounds were first tested in a primary screen to obtain a ‘fold-induction’ over baseline levels of protein activity. Only those compounds that had a fold induction >1 have been included in the table and all are considered ‘active’.

Results

Example	Human activity	Example	Human activity
1	A	21	B
2	A	22	C
3	A	23	C
4	A	24	B
5	A	25	A
6	A	26	A
7	A	27	B
8	A	28	B
9	A	29	B
10	A	30	B
11	A	31	C
12	A	32	C

- ιο6 -

Example	Human activity	Example	Human activity
13	A	33	C
14	A	34	C
15	A	35	C
16	A	36	C
17	A
18	A
19	B
20	B

Conclusion

The inventors have synthesised a large number of compounds which fall within the general formula (I). They have shown that these compounds activate the STING 5 protein, and so could be used to treat a number of diseases, including cancer.

Claims

Claims

1. A compound of formula (I):

R⁹

5 CD or a pharmaceutically acceptable salt or prodrug thereof, wherein:

XGs CR¹ orN;

X²is CR²orN;

X3is CR3orN;

io Z is NR¹⁰ or CR^1OR^U;

Q is C=0, S=0, S0₂, C=S or CR⁴Rs;

L is optionally substituted Ci-Ce alkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₃-C6 cycloalkyl,, optionally substituted C₂-C& alkynyl, C=0, S=0, S0₂, -CH₂C(0)-, -CH₂C0NH-, or -C0NH-;

15 Y is an optionally substituted Ci-Ce alkyl, an optionally substituted C₂-C6 alkenyl, an optionally substituted C₂-G> alkynyl, an optionally substituted C₃-Ce cycloalkyl, or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle;

R¹, R² and R³ are each independently selected from the group consisting of H, halogen, CN, hydroxyl, COOH, CONR¹²R¹³, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce alkyl, 20 optionally substituted Ci-Ce alkylsulfonyl, optionally substituted mono or bicyclic C₃-Ce cycloalkyl, optionally substituted C₂-G> alkenyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl group, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic 3 to 8 25 membered heterocycle, optionally substituted aryloxy, optionally substituted heteroaryloxy, and optionally substituted heterocyclyloxy;

R4 and Rs are each independently selected from the group consisting of H, halogen, optionally substituted G-G> alkyl and optionally substituted C₃-Ce cycloalkyl; or R⁴ and Rs together with the atom to which they are attached form a spirocyclic ring;

30 R⁶ is a mono or bicyclic optionally substituted C₅-Go aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted G_?,-G cycloalkyl or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle;

- ιο8 R7 is H, optionally substituted Ci-Ce alkyl, optionally substituted sulfonyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C₃-C6 cycloalkyl, optionally substituted C₂-C6 alkenyl and optionally substituted C₂-C& alkynyl;

R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally

5 substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic C₃-Ce cycloalkyl or an optionally substituted mono or bicyclic 3 to 8 membered heterocycle; R⁹ is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C₂-C& alkenyl, H, hydroxyl, CONR¹²R¹³, sulfonyl, NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce thioalkyl, optionally substituted Ci-Ce alkylsulfonyl,

10 optionally substituted C₃-Ce cycloalkyl,, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted heterocycle, optionally substituted aryloxy, and an optionally substituted heteroaryloxy;

15 when Z is NR¹⁰ then R¹⁰ is selected from the group consisting of optionally substituted Ci-Ce alkyl, H, hydroxyl, optionally substituted Ci-Ce thioalkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C₃-Ce cycloalkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted

20 C5-C10 aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted heterocycle, optionally substituted aryloxy, and an optionally substituted heteroaryloxy;

when Z is CR¹⁰Rⁿ then R¹⁰ and R¹¹ are each independently selected from the group consisting of optionally substituted Ci-Ce alkyl, H, hydroxyl, CONR¹²R¹³, sulfonyl,

25 NR¹²R¹³, NHCOR¹², optionally substituted Ci-Ce thioalkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted C₃-Ce cycloalkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₂-C& alkynyl, optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl, mono or bicyclic optionally substituted C₅C10 aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl,

30 optionally substituted heterocycle, optionally substituted aryloxy, an optionally substituted heteroaryl oxy, CN, halogen, azido and C0₂H; or R¹⁰ and R¹¹ together with the atoms to which they are attached can combine to form an optionally substituted spirocyclic ring;

or, when Z is NR¹⁰ or CR¹⁰Rⁿ, then R⁹ andR¹⁰ together with the atoms to which they are

35 attached can combine to form an optionally substituted fused ring; and

-109 R¹² and R¹³ are each independently selected from the group consisting of H, halogen, CN, hydroxyl, COOH, C0NH₂, NH₂, NHCOH, optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkylsulfonyl, optionally substituted mono or bicyclic C₃-C6 cycloalkyl, optionally substituted C₂-C& alkenyl, optionally substituted C₂-C& alkynyl,

5 optionally substituted Ci-Ce alkoxy, optionally substituted Ci-Ce alkoxycarbonyl group, mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl, optionally substituted mono or bicyclic 3 to 8 membered heterocycle, optionally substituted aryloxy, optionally substituted heteroaryloxy, and optionally substituted heterocyclyloxy.
2. A compound according to claim 1, wherein Z is CR^1OR^U.
3. A compound according to claim 2, wherein R¹⁰ and R¹¹ are independently an optionally substituted Ci-Ce alkyl, H, optionally substituted C₂-C& alkenyl or a C₃-Ce

15 cycloalkyl or R⁹ and R¹⁰ together with the N atoms to which they are attached combine to form an optionally substituted fused ring.
4. A compound according to claim 1, wherein Z is NR¹⁰.

20 5. A compound according to claim 4, wherein R¹⁰ is an optionally substituted Ci-Ce alkyl, H, an optionally substituted C₂-C& alkenyl or a C₃-Ce cycloalkyl or R⁹andR¹⁰together with the N atoms to which they are attached combine to form an optionally substituted fused ring.

25 6. A compound according to any one of claims 2 to 5, wherein R⁹ and R¹⁰ together with the N atoms to which they are attached form a five-membered ring.

7. A compound according to any preceding claim, wherein Q is C=0 or CR⁴R⁵.

30 8. A compound according to claim 7, wherein Q is C=0.

9. A compound according to any preceding claim, wherein X¹ is CR¹, X² is CR² and X³ is CR³.

- 110 -

10. A compound according to any preceding claim, wherein R¹, R² and R³ are each independently selected from the group consisting of H, halogen, and optionally substituted Ci-Ce alkyl.
5 11. A compound according to any preceding claim, wherein L is optionally substituted Ci-Ce alkyl, -CH₂C(0)- or -CH₂C0NH-.

12. A compound according to any preceding claim, wherein R⁶ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl or a mono or bicyclic optionally substituted 5 to 10

10 membered heteroaryl.

13. A compound according to claim 12, wherein R⁶ comprises between 1 and 5 substituents, and the or each substituent is independently selected from the list consisting of halogen, optionally substituted Ci-Ce alkyl, CN, optionally substituted Ci-

15 C₆ alkoxy, azido, CONR¹²R¹³, OH, C00R¹², 0C(0)R¹², NR¹²R¹³, 0C(0)0R¹², OC(O)NR¹²R¹³ and 0P(0)(0H)₂.

14. A compound according to any preceding claim, wherein R⁷ is preferably H or an optionally substituted Ci-Ce alkyl.

15. A compound according to any preceding claim, wherein Y is an optionally substituted Ci-Ce alkyl.

16. A compound according to any preceding claim, wherein R⁸ is a mono or bicyclic 25 optionally substituted C₅-Ci₀ aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl.

17. A compound according to any preceding claim, wherein R⁹ is an optionally substituted Ci-Ce alkyl, H, optionally substituted C₂-Co alkenyl or a C₃-Ce cycloalkyl.

18. A compound according to any preceding claim, wherein:

X¹ is CR¹;

X² is CR²;

X3 is CR3;

35 Z is NR¹⁰ or CR^1OR^U;

Q is C=0 or CR⁴R⁵;

- Ill L is an optionally substituted C1-C3 alkyl;

Y is an optionally substituted Ci-Ce alkyl;

R¹, R² and R³ are each independently selected from the group consisting of H, halogen,

CN, optionally substituted Ci-Ce alkyl, and optionally substituted mono and bicyclic C₃5 Ce cycloalkyl;

R4 and R⁵ are each independently selected from the group consisting of H and Ci-Ce alkyl;

R⁶ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl or a mono or bicyclic optionally substituted 5 to 10 membered heteroaryl;

10 R⁷ is H;

R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl, mono or bicyclic optionally substituted 5 to 10 membered heteroaryl or an optionally substituted mono or bicyclic C₃-Ce cycloalkyl; and

R⁹, R¹⁰ and R¹¹ are each independently selected from the group consisting of optionally 15 substituted Ci-Ce alkyl, H, hydroxyl, CONR¹²R¹³, sulfonyl, NR¹²R¹³, NHCOR¹², optionally substituted C₃-Ce cycloalkyl, optionally substituted C2-C
6 alkenyl, and optionally substituted Ci-Ce alkoxy, or R⁹ and R¹⁰ together with the N atoms to which they are attached can combine to form an optionally substituted fused ring.

20 19. A compound according to claim 18, wherein:

X¹ is CH;

X² is CH;

X³ is CH;

Z is NR¹⁰;

25 Q is C=O;

L is a Ci-C₃ alkyl;

Y is a Ci-C₃ alkyl;

R⁶ is a mono or bicyclic substituted C₅-Ci₀ aryl or a mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl are

30 substituted with one or more substituents selected from a halogen, Ci-C₃ alkoxy, OH, NH₂ and/or Ci-C₃ alkyl;

R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl or mono or bicyclic optionally substituted 5 to 10 membered heteroaryl; and

R⁹ and R¹⁰ are each independently a Ci-Ce alkyl, H, optionally substituted C₂-Ce alkenyl 35 or a C₃-Ce cycloalkyl, even more preferably a Ci-C₃ alkyl or a C₃-Ce cycloalkyl.

- 112 -

20. A compound according to either claim 18 or claim 19, wherein:

Z is NR¹⁰;

Q is CO;

L is a C1-C2 alkyl;

5 Y is a C1-C2 alkyl;

R⁶ is a mono or bicyclic C₅-Ci₀ aryl substituted with one or more substituents selected from a halogen, C1-C3 alkoxy, OH, NH₂ and/or C1-C3 alkyl;

R⁸ is a mono or bicyclic optionally substituted C₅-Ci₀ aryl or mono or bicyclic optionally substituted 5 to 10 membered heteroaryl; and

10 R⁹ and R¹⁰ are each independently selected from the group consisting of Ci-Ce alkyl, C₂Ce alkenyl and H.

21. A compound according to claim 20, wherein:

X¹ is CH;

15 X² is CH;

X3 is CH;

L is -CH₂-;

Y is -CH₂-;

R⁶ is a phenyl or pyridinyl substituted with one or more substituents selected from a

20 halogen, C1-C3 alkoxy, OH, NH₂ and/or C1-C3 alkyl;

R⁸ is a mono or bicyclic substituted C₅-Ci₀ aryl or mono or bicyclic substituted 5 to 10 membered heteroaryl and the C₅-Ci₀ aryl or 5 to 10 membered heteroaryl is substituted with one or more substituents selected from Ci-Ce alkyl, halogen, OH, Ci-Ce alkoxy, CONR¹²R¹³, CN and/or azido; and

25 R⁹ and R¹⁰ are each independently a C1-C3 alkyl or a C₂-C₄ alkenyl.

22. A compound according to claim 21, wherein R⁸ is a phenyl substituted with at least one halogen.

30 23. A compound according to claim 1, wherein the compound is:

4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de;

4-(2-fluoro-6-methoxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de;

35 4-(2-fluoro-3-methylbenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de;

-1134-(2-fluoro-6-hydroxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de;

4-(3,5-difluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

5 N-(benzofuran-2-ylmethyl)-4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-((3-fluoropyridin-4-yl)methyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-N-((5-methylfuran-2-yl)methyl)-3-oxo10 i,2,3,4-tetrahydrobenzo[e] [i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluoro-3-methoxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluoro-3-methoxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

15 4-(2,6-difluoro-4-hydroxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2,6-difluoro-4-(2-hydroxyethoxy)benzyl)-i,2-dimethyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2,6-difluoro-4-(3-hydroxypropoxy)benzyl)-i,2-dimethyl-3-oxo-N-(2,4,620 trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluoro-3-hydroxybenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-Chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydroquinoxaline-6-carboxamide;

25 2-allyl-4-(2-chloro-6-fluorobenzyl)-i-methyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-i-methyl-3-oxo-2-propyl-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

i-Allyl-4-(2-chloro-6-fluorobenzyl)-2-methyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,430 tetrahydrobenzo [e][i,2,4]triazine-6-carboxami de;

4-(2-chloro-6-fluorobenzyl)-2-(3-hydroxypropyl)-i-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-2-(2-hydroxypropyl)-i-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

35 6-(2-chloro-6-fluorobenzyl)-5-oxo-N-(2,4,6-trifluorobenzyl)-2,3,5,6-tetrahydro-iHbenzo[e]pyrazolo[i,2-a][i,2,4]triazine-8-carboxamide;

-1144-(2-chloro-6-fluorobenzyl)-i-(3-hydroxypropyl)-2-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-i-(2-hydroxypropyl)-2-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

5 4-(2-chloro-6-fluorobenzyl)-2-(2-cyanoethyl)-i-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

2-allyl-4-(2-chloro-6-fluoro-3-methoxybenzyl)-i-methyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

2-allyl-4-(2-chloro-6-fluoro-3-hydroxybenzyl)-i-methyl-3-oxo-N-(2,4,610 trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

N-(benzofuran-2-ylmethyl)-4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-i,2,3,4tetrahydropyrido[3,2-e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)-i,2,3,4tetrahydropyrido[3,2-e][i,2,4]triazine-6-carboxamide;

15 4-(2-chloro-6-fluorobenzyl)-7-hydroxy-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)- i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-7-(3-hydroxypropyl)-i,2-dimethyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide; 4-(2-chloro-6-fluorobenzyl)-7-cyano-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)20 i,2,3,4-tetrahydrobenzo[e] [i,2,4]triazine-6-carboxamide;
7-(3-aminopropyl)-4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide hydrochloride;

4-(2-chloro-6-fluorobenzyl)-7-methoxy-i,2-dimethyl-3-oxo-N-(2,4,6-trifluorobenzyl)25 1,2,3,4-tetrahydrobenzofe] [i,2,4]triazine-6-carboxamide;

4-(2-chloro-6-fluorobenzyl)-7-(3-hydroxyprop-i-yn-i-yl)-i,2-dimethyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide;

2-((4-(2-chloro-6-fluorobenzyl)-i,2-dimethyl-3-oxo-6-((2,4,6trifluorobenzyl)carbamoyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazin-7-yl)oxy)acetic

30 acid; or

4-(2-chloro-6-fluorobenzyl)-7-(2-hydroxyethoxy)-i,2-dimethyl-3-oxo-N-(2,4,6trifluorobenzyl)-i,2,3,4-tetrahydrobenzo[e][i,2,4]triazine-6-carboxamide.

24. A pharmaceutical composition comprising a compound according to any one of

35 claims 1 to 23 or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

-11525- A compound according to any one of claims 1 to 23, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, or a pharmaceutical composition according to claim 24, for use in therapy.

26. A compound according to any one of claims 1 to 23, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, or a pharmaceutical composition according to claim 24, for use in modulating the Stimulator of Interferon Genes (STING) protein.

2.7. A compound or composition for use according to claim 26, wherein the compound or composition is for use in activating the STING protein.

28. A compound according to any one of claims 1 to 23, or a pharmaceutically

15 acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, or a pharmaceutical composition according to claim 24, for use in treating, ameliorating or preventing a disease selected from cancer, bacterial infection, viral infection, parasitic infection, fungal infection, immune-mediated disorder, central nervous system disease, peripheral nervous system disease, neurodegenerative disease, mood disorder, sleep 20 disorder, cerebrovascular disease, peripheral artery disease or cardiovascular disease.

29. A compound or composition for use according to claim 28, wherein the disease is cancer.

25 30. A compound or composition for use according to claim 29, wherein the cancer is selected from the group consisting of colorectal cancer, aero-digestive squamous cancer, lung cancer, brain cancer, liver cancer, stomach cancer, sarcoma, leukaemia, lymphoma, multiple myeloma, ovarian cancer, uterine cancer, breast cancer, melanoma, prostate cancer, bladder cancer, pancreatic carcinoma or renal carcinoma.

31. A compound for use according to any one of claims 26 to 30, wherein the compound is for use with a second therapeutic agent, optionally wherein the second therapeutic agent comprises an antiviral agent, an anti-inflammation agent, conventional chemotherapy, an anti-cancer vaccine and/or hormonal therapy.

-116 -

32. A compound for use according to claim 31, wherein the second therapeutic agent comprises a B7 costimulatory molecule, interleukin-2, interferon-g, GM-CSF, a CTLA-4 antagonist (such as Ipilimumab and tremilimumab), an IDO inhibitor or IDO/TDO inhibitor (such as Epacadostat and GDC-0919), a PD-1 inhibitor (such as

5 Nivolumab, Pembrolizumab, Pidilizumab, AMP-224, and MDX-1106), a PD-Li inhibitor (such as Durvalumab, Avelumab and Atezolizumab), an OX-40 ligand, a LAG3 inhibitor, a CD40 ligand, a 41BB/CD137 ligand, a CD27 ligand, Bacille CalmetteGuerin (BCG), liposomes, alum, Freund’s complete or incomplete adjuvant, a TLR agonist (such as Poly I:C, MPL, LPS, bacterial flagellin, imiquimod, resiquimod,
10 loxoribine and a CpG dinucleotide) and/or detoxified endotoxins.

33. A process for making the composition of claim 24, the process comprising contacting a therapeutically effective amount of a compound according to any one of claims 1 to 23, or a pharmaceutically acceptable salt, solvate, tautomeric form or
15 polymorphic form thereof, and a pharmaceutically acceptable vehicle.

34. A compound of the formula (II), (III), (IV) or (V):

Formula (II)

Formula (III) Formula (IV) Formula (V) wherein, X¹, X², X3, Q, L, R⁶, R⁷, R⁸, R⁹ and Z are as defined in any one of claims 1 to 22;

and

R is H or a Ci-Ce alkyl, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or

25 polymorphic form thereof.

35. A compound according to claim 34, wherein the compound is selected from:

-117-

-118-

36. A conjugate of formula (VI):

(VI)

ΙΟ wherein C is a compound as defined in any one of claims 1 to 23; L¹ is a linker;

T is a targeting moiety; and a is an integer between 1 and 10.