WO2023092175A1

WO2023092175A1 - Novel p2x7 receptor antagonists

Info

Publication number: WO2023092175A1
Application number: PCT/AU2022/051400
Authority: WO
Inventors: Michael Kassiou; James O'BRIEN-BROWN; Jayson GILCHRIST; Gemma Figtree
Original assignee: The University Of Sydney
Priority date: 2021-11-24
Filing date: 2022-11-23
Publication date: 2023-06-01

Abstract

The invention relates generally to a compound of Formula (I), wherein R1 is a bicyclic fused heterocyclic moiety and R2 is H or F. The compound of Formula (I) or a pharmaceutically acceptable salt thereof has potential medical use in the treatment or prevention of conditions such as stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer s disease; Huntington s disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction.

Description

NOVEL P2X7 RECEPTOR ANTAGONISTS

Related Application

[001] This application claims convention priority to Australian provisional patent application 2021903786, filed on 24 November 2021.

Field of the Invention

[002] The present invention relates to a series of novel heterocyclic adamantyl and F3- adamantyl cyanoguanidine P2X7 receptor antagonists of Formula (I), wherein Ri is preferably a bicyclic aromatic heterocyclic moiety such as indazolyl, benzimidazolyl, benzotriazolyl, indolyl, benzoxazolyl, benzoxadiazolyl and benzothiazolyl; and wherein R2 is H or F:

(i).

[003] Other forms of the invention relate to synthesis and medical uses of such compounds.

[004] Although the present invention will be described hereinafter with reference to its preferred embodiment, it will be appreciated by those of skill in the art that the spirit and scope of the invention may be embodied in many other forms.

Background of the Invention

[005] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[006] The P2X7 receptor (P2X7R) is a purinergic receptor which has been implicated in numerous central nervous system (CNS) disorders due to its central role in the inflammatory response. Activation of the P2X7R releases pro-inflammatory cytokines such as interleukin 1β, which have been shown to underlie the pathogenesis of many neurodegenerative disorders. Accordingly, P2X7R antagonist compounds may have utility in the treatment of neurodegenerative conditions associated with several progressive CNS disorders, including stroke, ALS, MS and Alzheimer's disease.

[007] P2X7 signalling has also been heavily implicated in atherosclerosis and atherothrombosis through in vitro and in vivo experiments, with blockage of receptor- mediated signalling mitigating atherosclerosis, hypertension and diabetic retinopathy, as well as dilated cardiomyopathy and arrhythmia post myocardial infarction.

[008] Finally, recent studies have shown that the P2X7R also plays a role in cardiovascular diseases, with a special focus on ischaemic injury.

[009] International patent publication WO 99/02145, to Cambridge Neuroscience, Inc., broadly teaches the use of adamantyl guanidines as antibiotics. More particularly taught is a method for treating an infection in a mammal, comprising administering to a mammal suffering from or susceptible to an infection an effective amount of an aminoglycoside antibiotic and a compound that is an N,N' -disubstituted guanidine, N,N,N'-trisubstituted guanidine, N,N,N',N' -tetrasubstituted guanidine, or an N,N -disubstituted guanidine.

[010] United States patent publication US 2005/049312, to Rotta Research Uaboratorium SpA, teaches adamantane derivatives with neuroprotective, antidepressant and anti- ischaemic activities, and process for preparing them. The moiety representing Ri of the present invention is a linear, branched or cyclic alkyl group containing from 1 to 3 carbon atoms and a simple amino group or an amino group substituted with a nitro group. The reference further teaches that the compounds are non-competitive receptor antagonists for the NMDA receptor and may have a favourable use either for treating CNS diseases, for instance Alzheimer's disease, senile dementia, cerebral ischaemia and depression, or for treating neuropathic peripheral forms, these pathologies possibly being correlated at least partially with dysfunction of the glutamatergic system.

[011] United States patent publication US 2005/171195, to Abbott Uaboratories, teaches P2X7 antagonists for treating neuropathic pain. The moiety corresponding to Ri of the present invention can be aryl, arylalkyl, heterocycle or heterocyclealkyl. The reference teaches more generally that antagonists of the P2X7 receptor are useful in the treatment of neuropathic pain states.

[012] International patent publication WO 2014/005129, to Abbvie, Inc., teaches substituted cyanoguanidines as oral anti-virals. More particularly, the reference teaches cyanoguanidine compounds that are useful for inhibiting respiratory syncytial virus (RSV) infection and/or replication

[013] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[014] The development of a P2X7R inhibitor capable of reducing central and peripheral proinflammatory signalling has been a significant goal of many pharmaceutical and academic groups. CNS-penetrant P2X7R antagonists have yet to proceed past clinical trials. In the case of cardiovascular diseases, there is no available drug for P2X7R modulation. [015] Accordingly, the present invention comprises a series of 5,6-fused heterocyclic adamantyl and F₃ -adamantyl cyanoguanidines, developed with the aim of refining the pharmacophore for P2X7R antagonism.

[016] The adamantyl indazoles 302 and 303 (IC₅₀ = 18.6 ± 0.5 nM and 22.2 ± 6 nM, respectively) and the adamantyl benzoxazole 13 (IC₅₀ = 22.1 ± 1.2 nM) have immediately demonstrated potent inhibition in vitro. Structure-activity relationship data from this series has identified several requirements for potent P2X7R inhibition.

[017] The inventive series of compounds represents a potentially-significant advancement in the development of a drug-like P2X7R antagonist.

[018] Although the invention will be described with reference to specific examples it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Definitions

[019] In describing and defining the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[021] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[022] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When the phrase "consists of' (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase "consisting essentially of' limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter.

[023] With respect to the terms "comprising", "consisting of' and "consisting essentially of", where one of these three terms are used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of "comprising" may be replaced by "consisting of' or, alternatively, by "consisting essentially of'.

[024] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about", having regard to normal tolerances in the art. The examples are not intended to limit the scope of the invention. In what follows, or where otherwise indicated, "%" will mean "weight %", "ratio" will mean "weight ratio" and "parts" will mean "weight parts".

[025] The term "substantially" as used herein shall mean comprising more than 50%, where relevant, unless otherwise indicated.

[026] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[027] The terms "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[028] It must also be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[029] The person skilled in the art would appreciate that the embodiments described herein are exemplary only and that the electrical characteristics of the present application may be configured in a variety of alternative arrangements without departing from the spirit or the scope of the invention.

[030] Although example embodiments of the disclosed technology are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosed technology be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosed technology is capable of other embodiments and of being practised or carried out in various ways.

[031] The term "heteroaryl," as used herein, means an aromatic monocyclic ring or an aromatic bicyclic ring. The aromatic monocyclic rings are five or six membered rings containing 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, and S. The nitrogen heteroatoms can be optionally quatemised or oxidized to the N- oxide. The nitrogen containing rings can be optionally N-protected. The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. The aromatic bicyclic rings are composed of an aromatic monocyclic ring fused to a phenyl group. Alternatively, aromatic bicyclic rings are composed of an aromatic monocyclic ring fused to another aromatic monocyclic ring. The aromatic monocyclic rings and the aromatic bicyclic rings are connected to the parent molecular moiety through a carbon or nitrogen atom. Representative examples of heteroaryl include, but are not limited to, benzothienyl, benzoxadiazolyl, cinnolinyl, dibenzofuranyl, furopyridinyl, furyl, imidazolyl, indazolyl, indolyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridinium N-oxide, pyrrolyl, quinolinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienopyridinyl, thienyl, triazolyl, and triazinyl.

[032] The heteroaryl groups of the present invention are substituted with 0, 1, 2, 3, or 4 substituents independently selected from alkenyl, -OR_a, alkylOR_a, -C(O)OR_a, alkyl, - C(O)R_a, -OC(O)R_a, -SR_a, alkynyl, -C(O)O- cyano, formyl, haloalkoxy, haloalkyl, halogen, hydroxy, hydroxyalkyl, mercapto, nitro, -NR_cR_c, and (NR_c R_d)carbonyl, wherein R_c and R_d are independently selected from hydrogen, alkyl, -C(O)R_a, formyl, aryl and arylalkyl. Representative examples include, but are not limited to, 3-cyanopyridin-2-yl, 5- hydroxypyridin-2-yl, and 3-methylpyridin-2-yl.

[033] The term "heterocycle" as used herein, refers to a monocyclic or bicyclic, nonaromatic, saturated or partially unsaturated ring system. Monocyclic ring systems are exemplified by any 4-membered ring containing a heteroatom independently selected from oxygen, nitrogen and sulfur; or a 5-, 6-, 7-, or 8-membered ring containing one, two or three heteroatoms wherein the heteroatoms are independently selected from nitrogen, oxygen and sulfur. The 5 -membered ring has 0 or 1 double bond. The 6-memebered ring has 0, 1 or 2 double bonds. The 7- or 8-membered ring has 0, 1, 2 or 3 double bonds.

[034] Representative examples of monocyclic ring systems include, but are not limited to azetidinyl, azepanyl, azepinyl, diazepinyl, dioxolanyl, dioxanyl, dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 3- oxo-morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, 2-oxo-oxazolinyl, oxazolidinyl, piperazinyl, piperidyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydropyridyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, 1,4-diazepanyl and trithianyl.

[035] Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to a phenyl group, a monocyclic cycloalkenyl group, as defined herein, a monocyclic cycloalkyl group, as defined herein, or an additional monocyclic heterocycle group, as defined herein.

[036] Representative examples of bicyclic ring systems include but are not limited to, benzodioxinyl, benzopyranyl, benzothiopyranyl, 2,3-dihydroindolyl, indolizinyl, pyranopyridinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiopyranopyridinyl, 2-oxo- 1,3-benzoxazolyl, 3-oxo-benzoxazinyl, 3-azabicyclo[3.2.0]heptyl, 3,6- diazabicyclo[3.2.0]heptyl, octahydrocyclopenta[c]pyrrolyl, hexahydro-lH-furo[3,4- c]pyrrolyl, and octahydropyrrolo[3,4-c]pyrrolyl. The monocyclic or bicyclic ring systems as defined herein may have two of the non-adjacent carbon atoms connected by a heteroatom selected from nitrogen, oxygen or sulfur, or an alkylene bridge between one and three additional carbon atoms.

[037] Representative examples of monocyclic or bicyclic ring systems that contain such connection between two non-adjacent carbon atoms include, but not limited to, 2- azabicyclo[2.2.2]octyl, 2-oxa-5-azabicyclo[2.2.2]octyl, 2,5-diazabicyclo[2.2.2]octyl, 2- azabicyclo[2.2.1]heptyl, 2-oxa-5-azabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.1]heptyl, 2- azabicyclo[2.1.1]hexyl, 5-azabicyclo[2.1.1]hexyl, 3 -azabicyclo [3. l. l]heptyl, 6-oxa-3- azabicyclo[3.1.1]heptyl, 8-azabicyclo[3.2.1]octyl, 3-oxa-8-azabicyclo[3.2.1]octyl, 1,4- diazabicyclo [3.2 ,2]nonyl, 1 ,4-diazatricyclo [4.3.1. 1^3,8 | undecyl . 3,10- diazabicyclo[4.3.1]decyl, or 8 -oxa-3 -azabicyclo [3.2.1] octyl, octahydro- 1H-4, 7- methanoisoindolyl, and octahydro- lH-4,7-epoxyisoindolyl.

[038] The heterocycle groups of this invention, including the representative examples listed above, can be optionally substituted with 1, 2, or 3 substituents independently selected from alkenyl, -OR_a, -C(O)OR_a, -alkylC(O)OR_a, alkyl, -COR_a, -OC(O)R_a, alkylOC(O)R_a, - S(O)R_a, -S(O)2R_a, -S(O)₂NR_a R_b, alkynyl, -C(O)NR_aR_b>, cyano, halo, haloalkyl, haloalkoxy, nitro, -NR_aR_b, and (NR_aR_b)alkyl, wherein R_a and R_b are independently selected from the group consisting of hydrogen, alkyl, aryl, haloalkyl and arylalkyl; furyl, imidazolyl, isothiazolyl, isoxazolyl, naphthyl, oxadiazolyl, oxazolyl, phenyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrrolyl, tetrazinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazinyl, triazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzothienyl, benzoxadiazolyl, benzoxazolyl, benzofuranyl, cinnolinyl, indolyl, naphthyridinyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, and quinolinyl wherein said furyl, imidazolyl, isothiazolyl, isoxazolyl, naphthyl, oxadiazolyl, oxazolyl, phenyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrrolyl, tetrazinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazinyl, triazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzothienyl, benzoxadiazolyl, benzoxazolyl, benzofuranyl, cinnolinyl, indolyl, naphthyridinyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, and quinolinyl may be substituted with 1 or 2 substituents independently selected from alkenyl, - OR_a, -C(O)OR_a, alkylC(O)OR_a, alkyl, -COR_a, -OC(O)R_a, alkylOC(O)R_a, -S(O)R_a, -SO₂R_a, - SO₂NR_aR_b, alkynyl, -C(O)R_b , cyano, halo, haloalkyl, haloalkoxy, nitro, -NR_aR_b, and (NR_aR_b)alkyl, wherein R_a and R_b are independently selected from the group consisting of hydrogen, alkyl, aryl, haloalkyl and arylalkyl. The heterocycle groups of this invention are connected to the parent molecular moiety through any substitutable carbon or nitrogen atom in the groups. The nitrogen heteroatom may or may not be quatemised and may or may not be oxidized to the N-oxide. In addition, the nitrogen containing heterocyclic rings may or may not be N-protected.

Summary of the Invention

[039] Applicant has surprisingly discovered a series of 24 heterocyclic adamantyl and F3- adamantyl cyanoguanidines which display potent antagonistic properties at the P2X7 receptor. These new molecules possess physiochemical properties suitable for in vivo studies and represent exciting lead structures for the development of a therapy for pathologies characterised by inflammation. The 24 compounds are listed in Tables 1 and 2.

Table 1. 5,6-fused heterocyclic adamantyl and F^-adamantyl cyanoguanidine series; preferred Ri moieties

Table 2. 5,6-fused heterocyclic adamantyl and F₃-adamantyl cyanoguanidine series; compounds of the invention

[040] According to a first aspect of the present invention there is provided a compound of Formula (I):

wherein Ri is a bicyclic fused heterocyclic moiety; and wherein R2 is H or F. [041] In an embodiment, the Ri moiety is a 5,6-fused heterocyclic moiety.

[042] In an embodiment, the the Ri moiety is selected from the group consisting of: benzothienyl, benzoxadiazolyl, cinnolinyl, dibenzofuranyl, furopyridinyl, furyl, imidazolyl, indazolyl, indolyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridinium N-oxide, pyrrolyl, quinolinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienopyridinyl, thienyl, triazolyl, triazinyl and benzothiazolyl.

[043] In an embodiment, the Ri moiety is selected from the group consisting of: indazolyl, benzimidazolyl, benzotriazolyl, indolyl, benzoxazolyl, benzoxadiazolyl and benzothiazolyl. [044] In an embodiment, the Ri moiety is selected from the group consisting of: 4-NH-l- Me-indazolyl; 4-NH-l-Me-benzimidazolyl; 4-NH-2-Me-indazolyl; 7-NH-2-Me-indazolyl; 7-NH-l -Me -indazolyl; 7 -NH-1 -Me -benzimidazolyl; 7-NH-l -Me -benzotriazolyl; 7-NH-l - Me-indolyl; 6-NH-l-Me-indazolyl; 6-NH-l-Me-benzimidazolyl; 6-NH-2-Me-indazolyl; 5- NH-2-Me-indazolyl; 5-NH-l-Me-indazolyl; 5-NH-l-Me-benzimidazolyl; 4-NH- benzoxazolyl; 5-NH-benzoxazolyl; 6-NH-benzoxazolyl; 7-NH-benzoxazolyl; 4-NH- benzoxadiazolyl; 4-NH-benzodioxolyl; 5-NH-benzodioxolyl; and 7-NH-benzothiazolyl. [045] In an embodiment, the compound is selected from the group consisting of:

[046] In an embodiment, the Ri moiety is selected from the group consisting of: 7-NH- benzoxazolyl; 4-NH-l-Me-indazolyl; 6-NH-l-Me-indazolyl; 7-NH-l-Me-indazolyl; 5-NH- 2-Me-indazolyl; 6-NH-l-Me-benzimidazolyl; and 7-NH-benzothiazolyl.

[047] In an embodiment, the compound is selected from the group consisting of:

[048] In an embodiment, Ri is selected from the group consisting of: 7-NH-benzoxazolyl;

7-NH-l-Me-indazolyl; and 4-NH-2-Me-indazolyl.

[049] In an embodiment, the compound is selected from the group consisting of:

[050] In an embodiment, Ri is 7-NH-benzoxazolyl and the compound is:

[051] In an embodiment, Ri is 7-NH-l-Me-indazolyl and the compound is:

[052] In an embodiment, Ri is 4-NH-2-Me-indazolyl and the compound is:

[053] According to a second aspect of the present invention there is provided a compound, being l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7-yl)-2-cyanoguanidine:

[054] According to a third aspect of the present invention there is provided a compound, being 1 -(adamantan- 1 -ylmethyl)-2-cyano-3 -( 1 -methyl- lH-indazol-7 -yl)guanidine :

[055] According to a fourth aspect of the present invention there is provided a compound, being l-((adamantan-l-yl)methyl)-2-cyano-3-(2-methyl-2H-indazol-4-yl)guanidine:

[056] According to a fifth aspect of the present invention there is provided a compound, being (E)-l-(benzo[d]thiazol-7-yl)-2-cyano-3-(((3s,5s,7s)-3,5,7-trifluoroadamantan-l- yl)methyl)guanidine :

[057] In an embodiment of the first, second, third, fourth or fifth aspects, the compound is a P2X7 receptor antagonist.

[058] According to a sixth aspect of the present invention there is provided a method for the treatment of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction, the method comprising administering to a patient in need to such treatment and effective amount of a compound as defined according to the first, second, third, fourth or fifth aspects of the invention, or a pharmaceutically acceptable salt thereof.

[059] According to a seventh aspect of the present invention there is provided use of a compound as defined according to the first, second, third or fourth aspects of the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction.

[060] According to an eighth aspect of the present invention there is provided a compound as defined according to the first, second, third, fourth or fifth aspects of the invention, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction.

[061] According to a ninth aspect of the present invention there is provided a method for the synthesis of a compound according to the first, second, third, fourth or fifth aspects of the invention, the method comprising the steps of:

[062] a) forming adamant- 1-ylmethylamine hydrochloride wherein R2 is H or ((3s, 5s, 7s)- 3,5,7-trifluoroadamantan-l-yl)methanamine hydrochloride wherein R2 is F;

[063] b) forming a cyanocarbamimidate by reduction of an aryl amine and subsequent reaction with diphenyl A-cyanocarbonimidate; and

[064] c) coupling the adamant- 1-ylmethylamine hydrochloride or ((3s,5s,7s)-3,5,7- trifluoroadamantan-l-yl)methanamine hydrochloride to the cyanocarbamimidate.

[065] According to an eighth aspect of the present invention there is provided a method for the synthesis of l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7-yl)-2-cyanoguanidine

the method comprising the steps of:

[066] a) reacting 2-chloro-l,3-dinitrobenzene under basic conditions to form 2,6- dinitrophenol;

[067] b) aminating the 2,6-dinitrophenol to form 2-amino-6-nitrophenol;

[068] c) treating the 2-amino-6-nitrophenol with trimethyl orthoformate to give 7- nitrobenzo [d] oxazole ;

[069] d) reducing the 7-nitrobenzo[d]oxazole to benzo[d]oxazol-7-amine;

[070] e) reacting the benzo[d]oxazol-7-amine with diphenyl cyanocarbonimidate to give phenyl N-(benzo[d]oxazol-7-yl)-N'-cyanocarbamimidate; and

[071] f) coupling the phenyl N-(benzo[d]oxazol-7-yl)-N'-cyanocarbamimidate with 1- adamantylmethylamine to give l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7-yl)-2- cyanoguanidine.

[072] In an embodiment the method is substantially as herein described with reference to Scheme 1, below.

Scheme 1. Synthesis of l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7-yl)-2- cyanoguanidine 13

(continued next page) H₂, Pd/C EtOAc, 18 h, quant.

[073] According to a ninth aspect of the present invention there is provided a method for the synthesis of l-(adamantan-l-ylmethyl)-2-cyano-3-(1-methyl-lH-indazol-7-yl)guanidine

the method comprising the steps of:

[074] a) methylating 7 -nitroindazole to give 1-methyl-7-nitro-lH-indazole;

[075] b) reducing the 1-methyl-7-nitro-lH-indazole to provide 1 -methyl- lH-indazol-7- amine;

[076] c) treating the 1-methyl-lH-indazol-7-amine with diphenyl N-cyanocarbonimidate to provide phenyl N'-cyano-N-(1-methyl- lH-indazol-7-yl)carbamimi date; and

[077] d) coupling the phenyl N'-cyano-N-(1-methyl-lH-indazol-7-yl)carbamimidate with 1-adamantyhnethylamine to give l-(adamantan-l-yhnethyl)-2-cyano-3-(1-methyl-lH- indazol -7 -yl)guanidine .

[078] In an embodiment the method is substantially as herein described with reference to Scheme 2, below. Scheme 2. Synthesis of l-(adamantan-l-ylmethyl)-2-cyano-3-(1-methyl-lH-indazol-7- yl)guanidine 302

302

[079] According to a tenth aspect of the present invention there is provided a method for the synthesis of l-((adamantan-l-yl)methyl)-2-cyano-3 -(2 -methyl -2H-indazol-4- yl)guanidine

the method comprising the steps of:

[080] a) preparing 2-methyl-4-nitro-2H-indazole by methylating 4 -nitroindazole;

[081] b) reducing the 2-methyl-4-nitro-2H-indazole to provide 2-methyl-2H-indazol-4- amine;

[082] c) reacting the 2-methyl-2H-indazol-4-amine with diphenyl N-cyanocarbonimidate to provide phenyl N'-cyano-N-(2-methyl-2H-indazol-4-yl)carbamimidate; and

[083] d) coupling the phenyl N'-cyano-N-(2-methyl-2H-indazol-4-yl)carbamimidate with 1-adamantyhnethylamine to provide l-((Adamantan-l-yl)methyl)-2-cyano-3-(2-methyl-2H- indazol -4-yl)guanidine.

[084] In an embodiment the method is substantially as herein described with reference to Scheme 3, below. Scheme 3. Synthesis of 1 -((adamantan- 1 -yl)methyl)-2-cyano-3-(2-methyl-2H-indazol-4- yl)guanidine 302

Description of a Preferred Embodiment

Assay Method: THP-1 cell line

[085] Tamm -Horsfall protein 1 (THP-1) cells are a human monocytic leukaemia cell line derived from a 1 year old male infant, and used extensively as a model of monocyte and macrophage function. Cells were cultured in suspension in T 175 flasks (Coming), using RPMI 1640 media (with ATCC modification; Gibco) supplemented with 10% heat- inactivated foetal bovine serum (FBS; Gibco). THP-1 cells were separated from old media by centrifugation (125 g, 5 min) and a proportion of cell pellet resuspended back into a T175 flask with fresh media.

Dye uptake assay with THP-1 cells

[086] THP-1 cells were harvested from culture by centrifugation (125 g, 5 min) and resuspended in RPMI 1640 media (Gibco) supplemented with 5% FBS (Gibco), 100 ng ml- ¹ lipopolysaccharide (LPS; from E. coli strain 0111:B4, Sigma-Aldrich), and 10 ng mL^-1 recombinant human interferon gamma (IFN-γ; R&D Systems). Cells were immediately seeded onto 96-well black-walled, CellBIND plates (Coming) at a density of 1.5 x 10⁵ cells per well and incubated for 24 h (37 °C, 5% CO2) to allow differentiation into macrophages. Supernatant was removed from each well, and cells were washed once with 150 μL of warm modified Hanks' Balanced Salt Solution containing low Ca²⁺ (HBSS; KC1 5.37 mM, KH2PO4 0.44 mM, NaCl 136.89 mM, Na₂HPO₄ 0.34 mM, glucose 5.55 mM, NaHCO₃ 4.17 mM, CaCl₂0. 1 mM, pH 7.4, 37 °C). Cells were treated with 100 pL of warmed HBSS buffer (as above, pH 7.4, 37 °C) containing: (i) YO-PRO-1® iodide (1 μM; Life Technologies), (ii) 100 μM BzATP (Sigma- Aldrich) or vehicle control (0.4% ultra-pure water) (v/v), and (iii) test compound (1 nM - 10 μM) or vehicle control (0.1% DMSO) (v/v). Fluorescence in each well was immediately recorded every 30 s for 1.5 h at 37 °C using a BMG POLARstar Omega (λ_eX = 485-12, λ_em = 520). Fluorescence values recorded at 1 h were used to determine the IC₅₀ in GraphPad Prism 7.0 (GraphPad software, USA) by using a 4- paramater sigmoidal dose-response (inhibition) fit. Each test compound was assayed in duplicates and repeated a minimum of three times, with the calculated IC₅₀ from each assay used to determine the average and standard error of the mean (SEM).

Measurement of IL-1β release by THP-1 cells

[087] Cultured THP-1 cells were harvested by centrifugation (125 g, 5 min), and resuspended in RPMI 1640 media (Gibco) supplemented with 25 ng mL^-1 LPS (E. coll strain 0111:B4, Sigma-Aldrich) and 10 ng mL^-1 IFN-γ (R&D Systems). Cells were seeded onto 96-well black -walled, CellBIND plates (Coming) at a density of 2 x 10⁵ cells per well, and incubated for 3 h (37 °C, 5% CO₂). Supernatant was removed and cells incubated with test compound (0.1 - 10 μM) or vehicle control (0.1% DMSO) (v/v) diluted in RPMI 1640 media for 30 min at 37 °C. Pre-treatments were aspirated, and cells were washed once with 150 pL dPBS buffer (pH 7.4, 37 °C). Cells were treated with (i) 1 mM BzATP (Sigma- Aldrich) or vehicle control (0.4% ultra-pure water) (v/v), and (ii) test compound (0.1 - 10 μM) or vehicle control (0.1% DMSO) (v/v) for 1 h (37 °C, 5% CO2). Supernatant was aspirated and centrifuged at 10,000 g for 10 s. The final supernatant was collected and stored at -80 °C. Supernatant samples were defrosted and assayed for IL-1β with a human IL-1β ELISA kit (R&D Systems) as per the manufacturer's instructions. Absorbance was recorded using a BMG POLARstar Omega microplate reader set to 450 nm. Absorbance readings were basal corrected, and IL-ip concentration in each supernatant was determined by interpolating from a standard curve generated using a sigmoidal dose-response (variable slope) fit in GraphPad Prism 7.0 (GraphPad software, USA). Interpolated values were then expressed as a percentage of the positive control, and analysed using a 4-paramater sigmoidal dose-response (inhibition) fit to determine the IC₅₀ of each test compound.

Samples and standards were assayed in duplicates and repeated a minimum of three repetitions to calculate the average IC₅₀ and standard error of the mean. Table 3. In vitro P2X7R antagonist potency for the heterocyclic adamantyl cyanoguanidines of the invention

[088] Of the compounds tested to date, compounds 13, 299, 301, 302, 303, 304, 309 and JSG B29 were found to be potent inhibitors of the P2X7R in two in vitro assays, with compounds 13, 302 and 303 demonstrating particular potency.

[089] Further data will suggest appropriate lead compounds. Leads will be assessed on their ability to reduce secreted levels of interleukin- 1β in a human monocytic cell line, and in ex vivo peripheral blood mononuclear cells from ST elevation myocardial infarction patients. Mouse models for different cardiovascular disease states are planned and a combination of echocardiography, histology and ELISA will be used to measure endpoints and establish effectiveness of each therapy.

[090] Inhibitors that progress through the first cellular screen will be assessed for cytotoxicity, AMES fluctuations, CYP profiles, and possible liabilities such as hERG, followed by in vivo pharmacokinetic profiling. After demonstration of the efficacy of any lead compound in these models, the Inventors aim to select a candidate to take forward to clinical trials.

[091] As the P2X7R has been implicated in a wide range of disease states for which no good treatment currently exists (e.g., stroke, ALS, MS, Alzheimer's disease, Huntington's disease, atherosclerosis, diabetic retinopathy, dilated cardiomyopathy, ischemic injury and left ventricular hypertrophy post myocardial infarction), development of a small molecule capable of modulating the signaling pathway is of significant commercial interest.

[092] The Inventors are confident that the inventive series of 5,6-fiised heterocyclic analogues have never been explored in any previous P2X7R SAR study. This series has to date revealed valuable pharmacophoric criteria for potent P2X7R inhibition within this scaffold: the optimum position and identity of distal hydrogen bond acceptors. The activity achieved by these adamantyl heterocycles is likely to extend beyond the current cyanoguanidine linker backbone.

[093] A close structural analogue of the inventive compounds, "PKT 100", has recently shown efficacy in pre-clinical models of CVD. Early stage testing of the inventive series shows superior potency to PKT 100, and the breadth of potent assay results increases the chance of identifying a lead with a promising pharmacokinetic profde. Heteroaromatic adamantyl cyanoguanidines have previously been shown to bind non-competitively to P2X7Rs, meaning these ligands do not compete with endogenous ATP. Non-competitive inhibitors are superior drugs as they avoid increased concentrations of endogenous ligand in the extracellular space, which can lead to off-site binding, side-effects, etc.

[094] To the best of the Inventors' knowledge, there are no clinical trials underway investigating inhibitors of the P2X7R for any cardiovascular disease. There is one trial currently underway for Major Depressive Disorder (Inflammation) by Janssen (Phase 2 clinical trial with API JNJ-54175446).

Synthetic Protocols

[095] The synthesis and characterisation of the compounds according to the invention are described below.

General procedure “C” for coupling aniline-like amines with diphenyl N -cyanocarb onimidate [096] A solution of the aryl amine (1.00 eq) in anhydrous tetrahydrofuran (0.1 M substrate concentration) was cooled to 0 °C, and then treated portion-wise with sodium hydride (60% w/w dispersion in mineral oil, 3.0 eq). After stirring at 0 °C for 15 min, the reaction mixture was warmed to room temperature, and then treated portionwise (~0.5 eq every 1 h) with diphenyl N-cyanocarbonimidate (2.0-3.0 eq; effervescence). After stirring at room temperature for 17 h, volatiles were removed under a stream of nitrogen gas, and the reaction mixture was purified by flash chromatography to afford the cyanocarbamimidate product.

General procedure “D "for coupling of aliphatic amines with cyanocarbamimidate intermediates

[097] The aliphatic amine (1.3 eq) was added to a stirred suspension of the cyanocarbamimidate (1.0 eq) in 2-propanol (3 mL) and then heated at reflux for 17-24 h. The resulting precipitate was collected by vacuum filtration, washing the retentate with diethyl ether (3 x 2 mL) to give the title compound as a white solid unless otherwise noted.

General procedure “M” for N-methylation of nitroindazoles

[098] A solution of the nitroindazole (2.0 g, 12 mmol, 1 eq) in tetrahydrofuran (20-40 mL) was added to a flask containing a suspension of sodium hydride (60% w/w dispersion in mineral oil, 0.62 g, 15 mmol, 1.25 eq) in tetrahydrofuran (12 mL) under an argon atmosphere with stirring at 0 °C. The syringe was rinsed with an additional 12 mL of tetrahydrofuran and allowed to warm to room temperature over 30 min. Methyl iodide (0.88 mL, 14 mmol, 1.15 eq) was then added dropwise with rapid stirring, and allowed to stir until the reaction had reached completion. Excess volatiles were removed under nitrogen gas flow, and the crude residue was dissolved in ethyl acetate (300 mL). This was washed with aqueous sodium hydroxide (1 M, 4 x 100 mL), brine (150 mL), dried over MgSCri, and then reduced in vacuo. The crude residue was purified by flash chromatography with hexane/ethyl acetate eluent, to yield a mixture of 1 -methyl- and 2-methyl-nitroindazoles. The 1 -methyl regioisomer was less polar and eluted before the 2-methyl regioisomer for all of the nitroindazoles synthesised. The regioselectivity of the methylation reactions were confirmed by full assignment with HSQC and HMBC for all methylated nitroindazoles.

General procedure “N” for reduction of nitroindazoles

[099] Ethyl acetate (50 mL) was added to a flask containing the nitroindazole (1 g, 6 mmol) under a nitrogen atmosphere. The reaction flask was evacuated and purged with nitrogen three times, and then palladium on carbon (10% w/w, 0.25 g, ~5 mol% Pd) was added to the reaction mixture, rinsing into the flask with an additional 50 mL of ethyl acetate. The reaction mixture was evacuated and purged with hydrogen gas three times and left open to the hydrogen atmosphere, and then stirred at room temperature until the reaction had reached completion. The reaction mixture was filtered through a silica plug, and the eluate was reduced in vacuo to yield the aminoindazole. Due to stability issues with aminoindazoles, the crudely purified products were immediately coupled with diphenyl N- cyanocarbonimidate according to the general procedure C or general procedure O.

[0100] N.B. the 2-methylated aminoindazoles are less thermally stable than the 1- methylated indazoles, and general procedure O forms complex product mixtures when used for these derivatives and as such general procedure C is suggested for these methylated regioisomers. 7 -amino- 1 -methylindazole is not sufficiently nucleophilic to undergo coupling with diphenyl N-cyanocarbonimidate at reflux under neutral conditions, and general procedure C (using sodium hydride) is required to form this derivative. General procedure "O " for coupling of bicyclic heteroaryl amines with diphenyl N- cyanocarbonimidate

[0101] A solution of the aminoindazole (0. 150 g, 1.00 mmol) and diphenyl N- cyanocarbonimidate (0.255 g, 1.05 mmol) in acetonitrile (7.5 mL) was heated at reflux with stirring until the reaction had reached completion. Volatiles were removed under a flow of nitrogen gas, and the crude residue was purified by flash chromatography (0.5-1% v/v MeOH in CH₂Cl₂) to yield the phenyl N'-cyano- N'-(indazolyl)carbamimidatc as a white to off-white solid.

60 - 1 -Adamantane carb oxamide⁶

[0102] 1 -adamantanecarboxylic acid (10.0 g, 55.5 mmol) and 1,1 ' -carbonyldiimidazole (10.8 g, 66.6 mmol) were dissolved in anhydrous THF (~0.3 mmol of acid/mL) and stirred under a nitrogen atmosphere for 1 h. The solution was cooled in an ice bath, and aqueous ammonia (28% w/v, 0.2 mL per mmol of acid) was added. The reaction mixture was allowed to stir for a further 1.25 h, and THF was removed in vacuo. The crude product was dissolved in dichloromethane, washed with aqueous sodium hydroxide (1 M, 3 x 100 mL), aqueous hydrochloric acid (1 M, 2 x 150 mL), and water (2 x 150 mL). The organic fraction was dried over MgSC>4, and dichloromethane was removed by rotary evaporation to yield 62 (9.20 g, 92%) as a white solid, mp 185-186 °C (lit. 185.5-191.7 °C); ¹H NMR(300 MHz, d6 -DMSO): δ 6.92 (1H, br s, NH_a), 6.66 (1H, br s, NH_b), 1.94 (3H, br s, H2), 1.74-1.65 (12H, m, H1+H3) ppm. All spectroscopic data for this compound matched that previously reported in the literature.⁶

64 - Adamant- 1-ylmethylamine hydrochloride⁶

[0103] 62 (4.80 g, 26.8 mmol) in anhydrous THF (0.25 mmol/mL) was treated with lithium aluminium hydride (4.07 g, 107.2 mmol) at 0 °C with stirring, warmed to room temperature over 30 min, and then heated at reflux until the starting material was consumed. The reaction mixture was allowed to cool to room temperature, and then chilled water (0.17 mL/mmol) was added dropwise, followed by aqueous sodium hydroxide (4 M, 0.17 mL/mmol), and additional water (0.5 mL/mmol). THF was added to mobilise the viscous slurry, and after stirring for 30 min, MgSO₄ was added directly to the reaction mixture. The product mixture was filtered through a Celite® plug, washing with dichloromethane, and the filtrate was reduced in vacuo to yield the amine. The amine was dissolved in diethyl ether (1 mL/mmol), and then washed with aqueous sodium hydroxide (1 M, 1 mL/mmol), water (1 mL/mmol), brine (1 mL/mmol), dried over MgSO₄ and filtered. The filtrate was cooled in an ice bath, and then HC1 in dioxane (4 M, 0.4 mL/mmol) was added dropwise with stirring to form a white precipitate. The precipitate was collected by vacuum filtration and washed with diethyl ether to afford 64 (3.04 g, 56%) as a white solid, mp 326-332 °C; ¹H NMR (300 MHz, d₆- DMSO) δ 6.86 (3H, br s, NH3⁺), 2.43 (2H, s, H4), 1.94 (3H, s, H2), 1.68-1.51 (12H, m, H1+H3) ppm; ¹³C NMR (75 MHz, d₆- DMSO): δ 50.0, 38.9, 36.1, 31.7, 27.4 ppm. All spectroscopic data matched that reported in the literature.⁶

Methylation of 4-nitroindazole 315

[0104] General procedure M was followed using 4-nitroindazole 315 (2.01 g, 12.3 mmol) to provide 319 as a pale-yellow solid (1.15 g, 53%), and 323 as a pale-yellow solid (743 mg, 34%; 87% yield overall for 2 regioisomers).

319 - l-Methyl-4-nitro-1H-indazole

[0105] R_f 0.52 (hexane/EtOAc, 1 : 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.57 (s, 1H, H4), 8.12 (d, J= 7.6 Hz, 1H, Hl), 7.76 (d, J= 8.3 Hz, 1H, H3), 7.50 (t, J= 8.0 Hz, 1H, H2), 4.17 (s, 3H, H5) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 141.6, 140.8, 132.7, 125.5, 118.2, 117.1,

116.2, 36.2 ppm. Spectroscopic data matches that reported in the literature.⁷

323 - 2-Methyl-4-nitro-2H-indazole

[0106] R_f0.48 (hexane/EtOAc, 1 : 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.54 (s, 1H), 8.17 (d, J= 7.5 Hz, 1H), 8.06 (d, J= 8.5 Hz, 1H), 7.39 (dd, J= 8.5, 7.6 Hz, 1H), 4.31 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 150.2, 140.7, 126.0, 125.4, 124.5, 120.7, 115.3, 41.0 ppm. Spectroscopic data matches that reported in the literature.⁷

[0107] Method B: Trimethyloxonium tetrafluoroborate (638 mg, 4.31 mmol, 1.39 eq) was added to a partially dissolved suspension of 4-nitroindazole 315 (507 mg, 3.11 mmol, 1.00 eq) in ethyl acetate (10 mL). The reagent was weighed in a vial in the fumehood, and all materials in contact with the methylating agent were placed in a beaker filled with methanol saturated with ammonia. This was stirred at room temperature, quickly becoming a lighter shade of orange and forming a viscous syrup-like mixture. After stirring for an hour, the reaction mixture was more mobile and after stirring at room temperature for 20 h, had become a light pink colour. When stirring was stopped a white precipitate with a yellow supernatant was evident. The reaction mixture was diluted with ethyl acetate (60 mL) and aqueous sodium bicarbonate (50 mL). The phases were separated, and the aqueous fraction was extracted with ethyl acetate (2 x 50 mL). The combined organic fractions were washed with brine (50 mL), dried over MgSO₄, and reduced in vacuo to yield a yellow-orange solid. This was purified by flash chromatography (5% v/v PhMe + 45% v/v hexane in CH₂CI₂) to elute the title compound as a yellow band with significant co-elution with an orange band. Pure fractions were collected and reduced in vacuo to yield the title compound as a paleyellow solid (353 mg, 64%). All spectra for this compound matched the spectra reported immediately above.

Methylation of 5 -nitroindazole 316

[0108] General procedure M was followed using 5 -nitroindazole 316 (2.55 g, 15.6 mmol) to provide 320 (1.19 g, 43%) as a pale-yellow solid, and 324 (1.06 g, 38%; 81% yield overall for 2 regioisomers) as a pale-yellow solid.

320 - l-Methyl-5-nitro-lH-indazole

[0109] R_f0.54 (hexane/EtOAc, 1 : 1 v/v); ¹H NMR (500 MHZ, CDCl₃) δ 8.69 (d, J= 2.1 Hz, 1H), 8.25 (dd, J= 9.2, 2.1 Hz, 1H), 8.17 (d, J= 0.9 Hz, 1H), 7.44 (d, J= 9.1 Hz, 1H), 4.13 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 142.4, 141.6, 135.7, 123.2, 121.5, 119.0, 109.3, 36.1 ppm. Spectroscopic data matches that reported in the literature.¹⁰

324 - 2-Methyl-5-nitro-2H-indazole

[0110] R_f 0.22 (hexane/EtOAc, 1 : 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.70 (d, J = 2.1 Hz,

1H), 8.19 (s, 1H), 8.08 (dd, J= 9.5, 2.2 Hz, 1H), 7.72 (d, J= 9.4 Hz, 1H), 4.28 (s, 3H) ppm;

¹³C NMR (126 MHz, CDCl₃) δ 150.1, 143.2, 127.8, 120.5, 120.4, 119.3, 118.3, 41.1 ppm.

Spectroscopic data matches that reported in the literature.¹⁰

[0111] Method B: To a stirred pale orange suspension of 5-nitro- 1 H -indazole 316 (1.04 g, 6.36 mmol, 1.00 eq) in dichloromethane (35 mL) was added a solution of trifluoromethanesulfonic acid (1.06 g, 7.01 mmol, 1.11 eq) in dichloromethane (15 mL). This mixture was stirred at room temperature for 15 min and then treated with methyl 2,2,2- trichloroacetimidate (2.0 mL, 16 mmol, 2.5 eq). After stirring for ~10 min, the reaction mixture had formed a clear orange-red solution. The reaction mixture was stirred at room temperature for 18 h, and then slowly quenched with saturated aqueous sodium bicarbonate (40 mL). The phases were separated, and the aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organic fractions were then washed with water (2 x 40 mL), aqueous lithium chloride (5% w/v, 2 x 40 mL; to remove 2,2,2- trichloroacetamide byproduct), brine (80 mL), dried over MgSO₄ and reduced in vacuo to yield an orange solid. This was purified by flash chromatography (20% v/v EtOAc in hexane until a dark yellow non UV-active band eluted, then increased to 40% v/v EtOAc in hexane until a pale yellow band (title compound) started to elute just after a pink band, and then increased to 50% v/v EtOAc in hexane until the product completely eluted). The product was isolated as a yellow solid (274 mg, 24%) which exactly matched the spectra reported.

Methylation of 6-nitroindazole 317

[0112] General procedure M was followed using 6-nitroindazole (2.62 g, 16.1 mmol) to provide 320 (1.18 g, 41%) as a pale-yellow solid, and 324 (1.40 g, 49%; 90% overall yield for 2 regioisomers) as a golden-yellow solid.

321 - l-Methyl-6-nitro-lH-indazole

[0113] R_f0.74 (hexane/EtOAc, 1 : 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.40-8.36 (m, 1H), 8.10 (d, J= 0.7 Hz, 1H), 8.01 (dd, J= 8.8, 1.9 Hz, 1H), 7.83 (dd, J = 8.9, 0.6 Hz, 1H), 4.18 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 146.6, 138.8, 133.3, 127.2, 122.0, 115.5, 105.9, 36.2 ppm. Spectroscopic data matches that reported in the literature.¹¹

325 - 2-Methyl-6-nitro-2H-indazole

[0114] R_f0.30 (hexane/EtOAc, 1: 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.72 - 8.61 (m, 1H), 8.03 (s, 1H), 7.89 (dd, J = 9.1, 2.0 Hz, 1H), 7.74 (d, J= 9.1 Hz, 1H), 4.30 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 147.1, 146.7, 124.8, 124.7, 121.4, 115.9, 115.5, 41.2 ppm.

Spectroscopic data matches that reported in the literature.¹¹

Methylation of 7 -nitroindazole 318

[0115] 326 was prepared from 7-nitroindazole 318 (2.43 g, 14.9 mmol) according to the general procedure M to provide the title compound as a yellow solid (2. 10 g, 80%). The 1- methyl-7-nitro- 1 H-indazole 322 was only formed in trace amounts and its alternative synthesis is described below.

326 - 2-Methyl-7-nitro-2H-indazole

[0116] R_f 0.12 (hexane/EtOAc, 1: 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.33 (dd, J= 7.6, 0.8 Hz, 1H), 8.17 (s, 1H), 8.03 (dd, J= 8.2, 0.7 Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 4.35 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 141.0, 137.5, 128.8, 126.3, 125.9, 125.3, 120.3, 41.3 ppm. Spectroscopic data matches that obtained in the literature.⁷ 322 - l-Methyl-7 -nitro- IH-indazole

[0117] Acetone (15 mL) was added to a flask containing 7-nitroindazole 318 (1.01 g, 6.19 mmol) and potassium hydroxide (0.503 g, 8.96 mmol) under nitrogen and allowed to stir for 30 min at room temperature. The reaction mixture was then cooled to 0 °C, and methyl iodide (390 pL, 6.26 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature with stirring for 17 h, and volatiles were removed under a flow of nitrogen gas. The crude residue was dissolved in ethyl acetate (100 mL), washed with water (3 x 50 mL), brine (75 mL), dried over MgSO₄ and reduced in vacuo. The resulting residue was purified by flash chromatography (5% v/v EtOAc in hexane) to provide the title compound as a bright yellow solid (0.477 g, 43%). R_f0.78 (hexane/EtOAc, 1: 1 v/v); ¹H NMR (500 MHz, CDCl₃) δ 8.14 (s, 1H), 8.11 (dd, J = 7.8, 0.9 Hz, 1H), 8.02 (dd, J= 7.9, 1.1 Hz, 1H), 7.22 (t, J= 7.8 Hz, 1H), 4.25 (s, 3H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 135.4, 134.2, 131.3, 129.1, 128.1, 124.8, 119.9, 41.1 ppm. Spectroscopic data matches that obtained in the literature.⁷

S10 - 1 -Methyl- lH-indazol-4-amine

[0118] S10 was prepared from 319 (1.05 g, 5.92 mmol) according to the general procedure N to yield the product as a white crystalline solid (0.829 g, 95%) which rapidly discolours in air and was used immediately in the subsequent coupling step without characterisation.

333 - Phenyl N'-cyano-N-(l-methyl-lH-indazol-4-yl)carbamimidate

[0119] 333 was prepared from S10 (150 mg, 1.02 mmol) and diphenyl N- cyanocarbonimidate (255 mg, 1.07 mmol, 1.05 eq) according to the general procedure O to yield the title compound as a white solid (254 mg, 86%). mp 185-188 °C; Rf 0.15 (1.5% v/v MeOH in CH₂C1₂); IR (diamond cell): 3136, 3034, 2192, 1640, 1607, 1579, 1427, 1245, 1196 cm’¹; ¹H NMR (400 MHz, DMSO-d₆): δ 10.92 (1H, s, -NH), 8.21 (1H, s, H7), 7.71 (1H, br s, H5 or H6), 7.66 (1H, d, J= 8.6 Hz, H5 or H6), 7.45 (2H, t, J= 7.9 Hz, H2), 7.38- 7.22 (4H, m, Hl + H3 + H4), 3.83 (3H, s) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.7, 145.6, 141.7, 134.4, 130.7, 129.8, 126.1, 120.7 (br), 119.2, 118.9 (br), 113.5 (br), 106.4 (br), 30.7 ppm (one aromatic carbon unresolved due to extensive broadening of some resonances); LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C16H13N5O [M+Na]⁺: 314.1018, found: 314.1018.

[0120] 299 was prepared from 333 (100 mg, 0.342 mmol, 1.00 eq), 64 HC1 (72 mg, 0.36 mmol, 1.1 eq), and triethylamine (0.15 mL, 1.1 mmol, 3.0 eq) according to general procedure D to provide the title compound (106 mg, 85%) as a white solid, mp 222-223 °C; IR (diamond cell): 3442, 3348, 3186, 2971, 2888, 2846, 2177, 1595, 1564, 950 cn^-1: ¹H NMR (500 MHz, DMSO-d₆): δ 9.26 (1H, br s), 7.99 (1H, s), 7.42 (1H, d, J= 8.4 Hz), 7.36 (1H, t, J= 7.9 Hz), 7.11 (1H, br s), 6.95 (1H, d, J= 7.3 Hz), 4.04 (3H, s), 3.01 (2H, d, J = 4.8 Hz), 1.95 (3H, br s), 1.68 (3H, d, J= 11.9 Hz), 1.61 (3H, d, J= 11.6 Hz), 1.48 (6H, s) ppm; ¹³C NMR (126 MHZ, DMSO-d₆): δ 158.6, 140.8, 130.6, 126.5, 118.8, 116.9, 114.2, 106.3, 52.7, 36.4, 35.5, 34.3, 27.6 ppm (one aromatic carbon unresolved); LRMS (+ESI) m/z-. 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2117; Anal. Calc, for C₂₁H₂₆N₆: C, 69.58; H, 7.23; N, 23.19. Found: C, 69.61; H, 7.24; N, 23.16.

Sil - l-Methyl-lH-indazol-5-amine

[0121] Sil was prepared from 320 (1.06 g, 5.98 mmol) according to the general procedure N to yield the product as a light pink solid (0.845 g, 96%) which was taken through to the next step without further characterisation.

334 - Phenyl N’-cyano-N-(l -methyl- lH-indazol-5-yl)carbamimidate

[0122] The title compound 334 was prepared from Sil (149 mg, 1.01 mmol) according to the general procedure O to yield a white solid (250 mg, 85%). mp 175-177 °C; R_f0.17 (1.5% v/v MeOH in CH₂CI₂); IR (diamond cell): 3176, 3050, 2935, 2198, 1649, 1452, 1420 cm’¹; ¹H NMR (500 MHz, CDCl₃) δ 9.26 (br s, 1H, -NH), 7.97 (s, 1H), 7.74 (s, 1H), 7.46 - 7.33 (m, 4H), 7.28 (d, J = 7.4 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 4.06 (s, 3H) ppm; ¹³C NMR

(126 MHz, CDCl₃) δ 151.3, 138.5, 133.1, 129.8, 128.5, 126.9, 124.1, 123.8, 121.5, 116.5, 109.7, 35.9 ppm; LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C16H13N5O [M+Na]⁺: 314.1018, found: 314.1015.

[0123] The title compound was prepared from 334 (151 mg, 0.520 mmol, 1.00 eq), 64 HC1 (111 mg, 0.550 mmol, 1.05 eq), and triethylamine (0.22 mL, 1.6 mmol, 3.0 eq) according to the general procedure D, and purified by flash chromatography (0.5-1.0% v/v MeOH in CH₂CI₂) to yield 300 (88 mg, 47%) as a white solid. This was recrystallised from methanol to give the title compound (66 mg, 35%) as a white crystalline solid, mp 247-248 °C; IR (diamond cell): 3243, 3199, 2898, 2843, 2165, 1565 cm’¹; ¹H NMR (500 MHz, DMSO-d₆): δ 8.93 (1H, br s), 8.03 (1H, s), 7.64 (1H, d, J= 8.8 Hz), 7.57 (1H, d, J= 1.2 Hz), 7.25 (1H, dd, J= 8.9, 1.6 Hz), 6.73 (1H, br s), 4.04 (3H, s), 2.95 (2H, d, J= 5.9 Hz), 1.95 (3H, br s), 1.68 (3H, d, J= 11.7 Hz), 1.61 (3H, d, J= 11.6 Hz), 1.46 (6H, s) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 159.2, 137.8, 132.3, 130.2, 124.6, 123.6, 117.4, 116.3, 110.0, 52.3, 36.5, 35.4, 34.4, 27.6 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2115; Anal. Calcd for C₂₁H₂₆N₆: C, 69.58; H, 7.23; N, 23.19. Found: C, 69.60; H, 7.26; N, 23.18.

S12 - 1 -Methyl- lH-indazol-6-amine

321 S12

[0124] The title compound S12 was prepared from 321 (156 mg, 0.881 mmol) according to the general procedure N to give a white solid which was used immediately in the subsequent coupling step without further characterisation.

335 - Phenyl N’-cyano-N-(l-methyl-lH-indazol-6-yl)carbamimidate

[0125] The title compound 335 was prepared from S12 according to the general procedure O to give a white solid (249 mg, 97% over 2 steps), mp 173-175 °C; R_f0.27 (1.5% v/v MeOH in CH₂CI₂); IR (diamond cell): 3120, 2920, 2203, 1639, 1613, 1421, 1200 cm’¹; ¹H NMR (500 MHz, CDCl₃) δ 9.02 (1H, br s, -NH), 7.97 (1H, d, J= 0.8 Hz, H7), 7.71 (1H, d, J= 8.6 Hz, H5), 7.48 (1H, br s, H6), 7.46-7.41 (2H, m, H5), 7.32 (1H, t, J= 7.4 Hz, Hl), 7.21-7.09 (3H, m, H3 + H4), 4.05 (3H, s, H8) ppm; ¹³C NMR (126 MHz, CDCh): 8 151.1, 140.0, 133.8, 133.0, 130.0, 127.1, 122.5, 122.0, 121.5, 117.2, 103.4, 35.8 ppm (2 aromatic carbons unresolved); LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C16H13N5O [M+Na]⁺: 314.1018, found: 314.1014.

[0126] The title compound was prepared from 335 (96 mg, 0.330 mmol, 1.00 eq), 64 HC1 (70 mg, 0.35 mmol, 1.1 eq), and triethylamine (0.14 mL, 1.0 mmol, 3.0 eq) according to the general procedure D, and purified by flash chromatography (0.5-1.0% v/v MeOH in CH₂CI₂) to yield 301 (73 mg, 61%) as a white solid. This was recrystallised from ethyl acetate to give the title compound (54 mg, 45%) as a white crystalline solid, mp 217-218 °C; IR (diamond cell): 3309, 3177, 2905, 2893, 2174, 1598, 1583, 1560 cm”¹; ¹H NMR (500 MHz, DMSO-d₆): δ 9.14 (1H, br s, -NH), 7.99 (1H, s, H8), 7.71 (1H, d, J= 8.6 Hz, H6), 7.45 (1H, br s, H7), 7.07 (1H, br t, J= 6.2 Hz, -NH), 7.01 (1H, dd, J= 8.5, 1.4 Hz, H5), 3.99 (3H, s, H9), 2.99 (2H, d, J= 6.0 Hz, H4), 1.96 (3H, s, H2), 1.72-1.58 (6H, m, Hl), 1.49 (6H, d, J= 3.0 Hz, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 158.7, 139.9, 136.1, 132.3, 121.2, 120.9, 117.7, 117.2, 103.4, 52.5, 39.7, 36.5, 35.2, 34.4, 27.6 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2114; Anal. Calcd for C₂₁H₂₆N₆ C, 69.58; H, 7.23; N, 23.19. Found: C, 69.59; H, 7.24; N, 23.20.

S13 - l-Methyl-lH-indazol-7-amine

[0127] S13 was prepared from 322 (200 mg, 1.13 mmol, 1.00 eq) according to the general procedure N to provide the title compound (170 mg, >quant.) as a white crystalline solid with black impurities which was taken through to the next step immediately below.

[0128] 336 was prepared from S13 (170 mg, 1.16 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 144 mg, 3.6 mmol, 3.1 eq), and diphenyl N- cyanocarbonimidate (858 mg, 3.60 mmol, 3.12 eq) in anhydrous tetrahydrofuran (10 mL) according to general procedure C, and then purified by flash chromatography (5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to yield a white solid. This was recrystallised from 2- propanol to yield the title compound (222 mg, 66%) as a white solid with a similar texture to paper fibres, mp 195-196 °C; R_ƒ= 0.18 (2.5% v/v MeOH in CH₂CI₂); IR (diamond cell): 3147, 2190, 1637, 1426, 1247, 1202 cm^-1: ¹H NMR (400 MHz, DMSO-d₆): δ 10.92 (1H, br s, -NH), 8.12 (1H, s, H7), 7.78 (1H, d, J= 7.8 Hz), 7.58-7.21 (5H, m), 7.17 (1H, t, J= 7.7 Hz, H5), 4.23 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.1, 135.4, 132.8, 129.8 (br), 126.5, 126.5, 126.1 (br), 121.4 (br), 121.2 (br), 120.7, 119.2 (br), 39.9 ppm (2 aromatic carbons unresolved; large broadening of ¹³C resonances); LRMS (+ESI) m/z-. 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z-. Calc, for C16H13N5O [M+Na]⁺: 314.1018, found: 314.1014.

[0129] 302 was prepared from 64 (61 mg, 0.369 mmol, 1.08 eq) and 336 (100 mg, 0.343 mmol, 1.00 eq) according to general procedure D to provide the title compound (78 mg, 63%) as a white solid, mp 236-237 °C; R_ƒ= 0.12 (2.5% v/v MeOH in CH₂CI₂); IR (diamond cell): 2915, 2889, 2840, 2175, 2151, 1997, 1598, 1564 cm-¹; ¹H NMR (400 MHz, DMSO- d₆): δ 9.08 (1H, br s, -NH), 8.08 (1H, s, H8), 7.74 (1H, dd, J= 7.4, 1.3 Hz, H7), 7.15 (2H, m, H5+H6), 6.64 (1H, br s, -NH), 4.10 (3H, s, H9), 2.93 (2H, d, J= 5.9 Hz, H4), 1.92 (3H, s, H2), 1.71-1.51 (6H, m, H3), 1.41 (6H, s, Hl) ppm (assignments of H5 and H7 were assisted by ³J_C8-H7 HMBC correlation); ¹³C NMR (101 MHz, DMSO-d₆): δ 160.5, 137.0, 133.1, 127.4, 126.7, 121.4, 121.2, 121.0, 117.8, 52.8, 40.2, 38.1, 36.9, 34.8, 28.1 ppm; LRMS (+ESI) m/z-. 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2113; HPLC: 98.2% (λ_maxi = 230 nm); 99.5% (λ_maX2 = 300 nm), RT: 27.7 min.

S14 - 2-Methyl-2H-indazol-4-amine

323 S14

[0130] S14 was prepared according to general procedure N using 323 (245 mg, 1.38 mmol, 1.00 eq) and palladium on carbon (Pd/C, 10% w/w, 70 mg, 5 mol% Pd) in EtOAc (25 mL). The reaction mixture was fdtered through a basic alumina plug, eluting with an additional 100 mL of ethyl acetate to yield a colourless filtrate. The filtrate was reduced in vacuo to give a white solid which quickly discoloured in the presence of air, and this was further purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂), eluting rapidly. This was reduced in vacuo to yield the title compound as a white solid (178 mg, 87%) which was used immediately in the subsequent coupling reaction.

34% (recryst.)

[0131] 337 was prepared according to the general procedure C using S14 (178 mg, 1.20 mmol, 1.00 e), sodium hydride (60% w/w dispersion in mineral oil, 146 mg, 3.7 mmol, 3.0 eq), and diphenyl N-cyanocarbonimidate (582 mg, 2.44 mmol, 2.03 eq; ~0.5 eq every 20 min, effervescence) in tetrahydrofuran (10 mL) at room temperature for 17 h. Volatiles were removed under a stream of nitrogen gas, and the crude residue was eluted through a silica plug with 10% MeOH sat. w/ NH₃ in CH₂CI₂. Despite the polarity of the eluent, the product moved very slowly on silica, so for the subsequent purification the column was run using a basic alumina stationary phase. Even with 0.5% v/v MeOH in CH₂CI₂ eluent, the compound eluted from the first fraction and with no separation. To avoid silica retention issues, the crude product was recrystallised from EtOAc, forming a white crystalline solid. This was collected by vacuum filtration, washing with diethyl ether (3 x 2 mL) to yield the title compound as an off-white solid (119 mg, 34%). mp 178-180 °C; Rf= 0.25 (2.5% v/v MeOH sat. W/ NH₃ in CH₂CI₂; stains orange with vanillin); *HNMR (400 MHz, DMSO-d₆): δ 11.04 (1H, s, -NH), 8.54 (1H, s, H7), 7.54 (1H, dt, J= 8.6, 0.7 Hz, H6), 7.48-7.41 (2H, m, H2), 7.33-7.24 (3H, m, H1+H3), 7.26 (1H, dd, J= 8.6, 7.2 Hz, H5), 7.09 (1H, dd, J= 7.1, 0.5 Hz, H6), 4.20 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.5, 149.0, 129.8, 127.9, 126.2, 125.2, 123.6, 120.9, 118.6, 116.8, 115.7, 113.3, 40.1 ppm; LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₆H₁₃N_5O [M+Na]⁺: 314.1018, found: 314.1013.

[0132] 303 was prepared according to general procedure D using 64 (77 mg, 0.47 mmol, 1.4 eq) and 337 (94.5 mg, 0.324 mmol, 1.00 eq) in 2-propanol (3 mL) at reflux for 20 h. The reaction mixture was cooled to room temperature, and the precipitate was collected by vacuum filtration, washing with 2-propanol ( 1 mL) and then diethyl ether (3 x 2 mL) to yield a white solid (85 mg, 72%). mp 226-227 °C; R_ƒ= 0.21 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; stains white with vanillin); IR (diamond cell): 3253, 3077, 2889, 2845, 2175, 1586, 1352 cm’¹; ¹H NMR (400 MHz, DMSO-d₆): δ 9.11 (1H, br s, -NH), 8.28 (1H, s, H8), 7.42 (1H, d, J= 8.6 Hz), 7.21 (1H, dd, J= 8.5, 7.3 Hz, H6), 6.85 (2H, d, J= 7.1 Hz, H5), 4.17 (3H, s, H9), 2.98 (2H, d, J= 6.2 Hz, H4), 1.95 (3H, br s, H2), 1.74-1.54 (6H, m, Hl), 1.46 (6H, br s, H3) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 158.6, 149.2, 129.9, 125.5, 123.4, 118.4, 117.1, 115.2, 114.1, 52.6, 40.0, 39.7, 36.5, 34.3, 27.7 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2112; HPLC: 99.5% (λ_maxi = 236 nm); 99.6% (λ_max2 = 305 nm), RT: 26.4 min.

SI 5 - 2-Methyl-2H-indazol-5-amine

324 63% S15

[0133] S15 was prepared using 324 (222 mg, 1.25 mmol, 1.00 eq) according to the general procedure N. This was purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to elute the compound as a colourless fraction just before a deep blue-coloured band, which was reduced in vacuo to yield a white solid (116 mg, 63%) which rapidly discolours to yellow in air. This was used in the next step immediately without further characterisation.

338 - Phenyl N’-cyano-N-(2-methyl-2H-indazol-5-yl)carbamimidate

[0134] 338 was prepared according to the general procedure C using S15 (116 mg, 0.788 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 98 mg, 2.5 mmol, 3. 1 eq), and diphenyl N-cyanocarbonimidate (379 mg, 1.59 mmol, 2.02 eq). Volatiles were removed under a stream of nitrogen gas, and the crude residue was eluted through a silica plug with 10% MeOH sat. w/ NH₃ in CH₂CI₂. The filtrate was reduced in vacuo and then purified by flash chromatography (1% v/v MeOH sat. w/ NH₃) to give the title compound (210 mg, 91%) as a fluffy white solid.

[0135] Mp 189 °C (decomp); R_ƒ= 0.25 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; ninhydrin stain); IR (diamond cell): 3148, 3021, 2189, 1643, 1427, 1212, 1192 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.79 (1H, br s, -NH), 8.36 (1H, s, H7), 7.79 (1H, br s, H5), 7.62 (1H, dt, J= 9.2, 0.9 Hz, H4), 7.50-7.40 (2H, m, H2), 7.34-7.25 (4H, m, H1+H3+H6), 4.16 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.7 (br), 146.4, 129.8 (br), 129.1, 126.1, 125.1, 123.7 (br), 121.0, 120.6 (br), 117.3, 115.1 (br), 113.5 (br), 40.1 ppm (one aromatic carbon unresolved; extremely broad resonances for some ¹³C peaks which may obscure signals); LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z'. Calc, for C₁₆H₁₃N₅₀ [M+Na]⁺: 314.1018, found: 314.1013.

[0136] 304 was prepared according to general procedure D using 64 (63 mg, 0.38 mmol, 1.1 eq) and 338 (99 mg, 0.34 mmol, 1.0 eq) in 2-propanol (3 mL) at reflux for 20 h. The reaction mixture was cooled to room temperature, and the precipitate was collected by vacuum filtration, washing with 2-propanol (1 mL) and then diethyl ether (3 x 2 mL) to yield a white solid (94 mg, 76%). This compound was very poorly soluble in most organic solvents, and NMR analysis was conducted by dissolving 304 in d₄ -trifluoroacctic acid, mp 241-242 °C; R_ƒ= 0.05 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; vanillin stain); IR (diamond cell): 3127, 2898, 2843, 2340, 2171, 1597, 1581, 793 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.87 (1H, br s), 8.30 (1H, br s), 7.57 (1H, br s), 7.50 (1H, br s), 7.09 (1H, br s), 6.71 (1H, br s), 4. 15 (3H, br s), 2.95 (2H, br s), 1.95 (3H, br s), 1.78-1.53 (6H, m), 1.46 (6H, br s) ppm; ¹H NMR (400 MHz, TFA-tfi): 8 8.78 (1H, s, H8), 8.12 (1H, d, J= 1.5 Hz, H7), 7.93 (1H, d, J= 92 Hz, H6), 7.74 (1H, dd, J= 92, 1.7 Hz, H5), 4.47 (3H, s, H9), 3.21 (2H, s, H4), 2.02 (3H, br s, H2), 1.80 (3H, m, Hl), 1.71-1.53 (9H, m, H1+H3) ppm; ¹³C NMR (101 MHz, TFA- d₄): 5 57.0, 41.8, 41.7, 38.0, 35.4, 30.1 ppm; LRMS (+ESI) m/z-. 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₆H₁₃N₅₀ [M+Na]⁺: 314.1018, found: 314.1013;

HPLC: 96.5% (λ_maxi = 215 nm), RT: 25.1 min. Due to the low solubility of 304 in all deuterated solvents sans TFA-d₄ , only ¹ H NMR spectra could be obtained for 304 in DMSO-d₆, with extensive broadening of all signals. Complex ¹ H NMR spectra in TFA-d₄ arose due to tautomerism/configurational isomerism and potential product instability, which also prevented complete ¹³C NMR spectra from being obtained.

S16 - 2-Methyl-2H-indazol-6-amine

[0137] S16 was prepared according to general procedure N using 325 (508 mg, 2.87 mmol, 1.00 eq) and palladium on carbon (Pd/C, 10% w/w, 125 mg, 4mol% Pd) in EtOAc (40 mL). The reaction mixture was filtered through a basic alumina plug, eluting with an additional 100 mL of ethyl acetate to yield a golden coloured filtrate. This was reduced in vacuo to yield an orange oil, which under a flow of nitrogen overnight formed an orange solid. This was recrystallised from EtOAc (~8 mL) to yield the title compound (187 mg, 44%) as a white crystalline solid which was used in the next step immediately without further characterisation.

339 - Phenyl N’-cyano-N-(2-methyl-2H-indazol-6-yl)carbamimidate

90%

[0138] 339 was prepared according to the general procedure C using S16 (187 mg, 1.27 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 153 mg, 3.8 mmol, 3.0 eq), and diphenyl /V-cyanocarbonimidate (673 mg, 2.82 mmol, 2.22 eq; ~0.5 eq every 20 min, effervescence) in tetrahydrofuran (10 mL) at room temperature for 17 h. Volatiles were removed under a stream of nitrogen gas, and the crude residue was eluted through a silica plug with 10% MeOH sat. w/ NH₃ in CH₂CI₂. The filtrate was reduced in vacuo to yield a light brown-coloured oil which was purified by flash chromatography (1% v/v MeOH sat. w/ NH₃) to give the title compound as a white solid (334 mg, 90%) which existed as a mixture of tautomers in a 10: 1 ratio, mp 160-162 °C; R_ƒ= 0.08 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; stains white with vanillin, while S16 with same Rf stains yellow); IR (diamond cell): 3340, 3161, 2971, 2194, 1618, 1420, 1392, 948 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.88 (1H, br s, -NH), 8.33 (1H, s, H7), 7.70 (2H, m, H5+H6), 7.49-7.39 (2H, m, H2), 7.34-7.26 (3H, m, H1+H3), 7.14 (1H, dd, J= 8.8, 1.8 Hz, H4), 4.15 (3H, s, H8) ppm; ¹³C NMR (101 MHZ, DMSO-d₆): δ 161.7, 159.6, 152.6, 151.7, 147.8, 147.7, 133.4, 133.3, 129.8, 126.2, 126.1, 124.8, 124.7, 120.9, 120.7, 120.6, 119.8, 119.7, 119.5, 119.0, 118.9, 113.4, 111.0, 110.8, 56.3, 40.0 ppm; LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₆H₁₃N₅₀ [M+Na]⁺: 314.1018, found: 314.1013.

[0139] 305 was prepared according to general procedure D using 64 (63 mg, 0.381 mmol, 1.1 eq) and 339 (104 mg, 0.357 mmol, 1.00 eq) in 2-propanol (3 mL) at reflux for 20 h. The reaction mixture was cooled to room temperature, and the precipitate was collected by vacuum filtration, washing with 2-propanol ( 1 mL) and then diethyl ether (3 x 2 mL) to yield the title compound (73 mg, 57%) as a white solid, mp 210-211 °C; IR (diamond cell): 3420, 3214, 2894, 2843, 2167, 1585, 1366, 1157, I I 14 cm^-1: ¹H NMR (400 MHz. DMSO-d ₆): δ 9.00 (1H, br s, -NH), 8.29 (1H, s, H8), 7.66 (1H, d, J= 8.8 Hz, H6), 7.38 (1H, s, H7), 6.96 (1H, br t, J= 6.2 Hz, -NH), 6.92 (1H, dd, J= 8.9, 1.7 Hz, H5), 4.14 (3H, s, H9), 2.98 (2H, d, J = 6.3 Hz, H4), 1.95 (3H, br s, H2), 1.73-1.54 (6H, m, Hl), 1.48 (6H, br s, H3) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 158.8, 148.2, 135.0, 124.6, 120.9, 119.3, 119.3, 117.3, 109.6, 52.4, 39.9, 39.7, 36.5, 34.4, 27.7 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2111;

HPLC: 99.9% (X_max1 = 240 nm); 99.8% (X_max2 = 292 nm), RT: 26.0 min. S17 - 2-Methyl-2H-indazol-7 -amine

[0140] S17 was prepared from 326 (442 mg, 2.49 mmol, 1.00 eq) according to general procedure N, yielding the title compound (367 mg, quant.) as a pale pink oil which was used immediately in the following step without further characterisation.

90%

[0141] 340 was prepared according to the general procedure C using S17 (367 mg, 2.49 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 300 mg, 7.5 mmol, 3.0 eq), and diphenyl N-cyanocarbonimidate (1.19 g, 4.99 mmol, 2.00 eq) in tetrahydrofuran (20 mL). This was purified by flash chromatography (1% v/v MeOH sat. w/ NH₃, eluting as a pale salmon -pink band on silica after a yellow band had eluted). 340 was collected as a pale pink to off-white solid (651 mg, 90%). This compound discolours to a blue-green colour overtime with no apparent degradation observed via NMR analysis, mp 177 °C (decomp); R_ƒ= 0. 17 (1% v/v MeOH sat. w/ NH₃ in CH₂CI₂; stains white with ninhydrin, while S 17 stains green); IR (diamond cell): 3136, 2921, 2852, 2190, 1631, 1436, 1201, 950, 742, 695 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.91 (1H, br s, -NH), 8.41 (1H, s, H7), 7.66 (1H, d, J= 8.3 Hz, H6), 7.46-7.39 (2H, m, H2), 7.31-7.22 (4H, m, H1+H3+H4), 7.06 (1H, dd, J = 8.2, 7.2 Hz, H5), 4.22 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 161.1, 151.6, 143.9, 129.6, 126.1, 125.5, 124.5, 122.9, 121.3, 121.2, 120.7, 119.8, 113.8, 40.1 ppm;

LRMS (+ESI) m/z-. 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z'. Calc, for C16H13N5O [M+Na]⁺: 314.1018, found: 314.1012.

[0142] 306 was prepared according to general procedure D using 64 (139 mg, 0.841 mmol, 1.18 eq) and 340 (208 mg, 0.714 mmol, 1.00 eq) in 2-propanol (5 mL), providing the title compound as a white solid (196 mg, 76%). mp 214-215 °C; R_ƒ= 0.38 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; stains white with vanillin, while 339 stains yellow); IR (diamond cell): 3380, 3217, 3124, 2900, 2841, 2176, 1598, 1572, 1357, 748 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 8.94 (1H, br s, -NH), 8.39 (1H, s, H8), 7.50 (1H, dd, J= 8.3, 0.7 Hz, H7), 7.21 (1H, dd, J= 12, 0.7 Hz, H4), 7.01 (1H, dd, J= 8.3, 7.3 Hz, H6), 7.01 (1H, br s, -NH), 4.18 (3H, s, H9), 3.04 (2H, d, J= 5.4 Hz, H4), 1.94 (3H, s, H2), 1.72-1.54 (6H, m, Hl), 1.52- 1.44 (6H, m, H3) ppm (the NH and H6 resonances at 7.01 ppm overlap); ¹³C NMR (101 MHz, DMSO-d₆): δ 158.0, 143.1, 126.1, 125.5, 122.7, 121.2, 118.0, 117.2, 116.8, 52.8, 40.0, 39.5, 36.4, 33.9, 27.6 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2111.

349 - l-Methyl-4-nitrobenzimidazole

[0143] Aqueous hydrochloric acid (32% w/w, 1.0 mL, 11 mmol, 6.7 eq) was added to a solution of 3-nitro-l,2-phenylenediamine 348 (253 mg, 1.65 mmol, 1.00 eq) in ethanol (3 mL). After the addition of aqueous formaldehyde (37% w/w, 0.30 mL, 4.0 mmol, 2.4 eq), the reaction mixture was heated at reflux for 3 h after which the reaction had gone to completion. Volatiles were removed under a stream of nitrogen gas to yield a pale red residue which was dissolved in aqueous sodium hydroxide (1 M, 50 mL), and then extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO₄ and reduced in vacuo. The residue was purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to elute the title compound as a pale salmon -pink band (eluting just before, and minorly co-e luting with a yellow band; coeluted fractions discarded). The combined clean fractions were reduced in vacuo to yield the title compound as an off-white, crystalline solid (182 mg, 62%). Reaction was repeated with same mass of starting material and columned with 0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to isolate 349 as an off-white crystalline solid (199 mg, 68%). mp 165-167 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 8.49 (1H, s, H4), 8.05 (2H, d, J= 8.1 Hz, H1+H3), 7.46 (1H, t, J = 8.0 Hz, H2), 3.94 (3H, s, H5) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 148.1, 138.5, 137.3, 136.3, 121.6, 118.3, 117.5, 31.2 ppm. Spectroscopic data matches that reported in the literature.¹²

350 - l-Methyl-4-aminobenzimidazole

349 97% 350

[0144] 350 was prepared from 349 (145 mg, 0.818 mmol, 1.00 eq) according to the general procedure N, providing the title compound (119 mg, 97%) as a white, crystalline solid which rapidly discolours to yellow and brown in air. This compound was used immediately in the subsequent coupling step without further characterisation.

352 - N-methyl-2, 6-dinitroaniline ¹³

351 quant. 352

[0145] A solution of aqueous methylamine (40% w/w, 1.3 , 1 m5 L mmol, 3.1 eq) was added dropwise to a pale yellow, partially dissolved solution of 2-chloro- 1,3 -dinitrobenzene (975 mg, 4.81 mmol, 1.00 eq) in methanol (10 ). m ALfter 3 h at room temperature, the reaction had gone to completion and volatiles were removed under a stream of nitrogen gas. The resulting yellow residue was partitioned between sodium bicarbonate (50 ) an mdL ethyl acetate (50 mL), the phases were separated, and the aqueous fraction was extracted again with ethyl acetate (2 x 50 m).L The combined organics were washed with water (2 x 50 mb), brine (100 m)L, dried over MgSO₄ and reduced in vacuo to yield the title compound 352 as a yellow-orange solid (945 mg, quant.). ¹ H NMR (400 MHz, CDCl₃): δ 8.47 (1H, br s, -NH), 8.16 (2H, d, J= 8.2 Hz, Hl), 6.74 (1H, t, J= 8.2 Hz, H2), 2.88 (3H, s, H3) ppm;

¹³C NMR (400 MHz, CDCl₃): δ 141.1, 137.7, 132.3, 113.9, 32.8 ppm. Spectroscopic data matches that reported in the literature.¹⁴ 353 - N²-methylbenzene-l ,2,3-tricimine^Xi

[0146] 353 was prepared from 352 (292 mg, 1.48 mmol, 1.00 eq) according to the general procedure N, obtained the title compound (203 mg, quant.) as a brown oil which was used immediately in the subsequent cyclisation reaction without further characterisation.

354 - N-(l-methyl-lH-benzo[d]imidazol-7-yl)formamide^Xi

[0147] A brown solution of 353 (181 mg, 1.32 mmol, 1.00 eq) in formic acid (1 ) wa ms L heated at reflux for 2 h after which the starting material had been consumed. Formic acid was removed under a stream of nitrogen gas overnight, and the brown residue was purified by flash chromatography (hexane/chloroform/methanol, 9:9: 1 v/v), eluting as a colourless eluate immediately preceding a yellow band. Pure fractions were combined and reduced in vacuo to yield the title compound as a white solid (175 mg, 76%). Compound appears as a mixture of rotainers in ~1.0:0.6 ratio; mp 166—167 °C; R_ƒ= 0.19 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂, then CHCI₃/hexane/MeOH, 9:9:2 v/v); IR (diamond cell): 3222, 3191, 3100, 1642, 1505, 1274, 1187, 739 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.11 (0.6H, br s, - NH), 10.00 (1H, br s, -NH), 8.39 (1H, s, -CHO), 8.36 (0.6H, br s, -CHO), 8.13 (0.6H, s, H4B), 8.11 (1H, s, H4A), 7.57 (0.6H, dd, J= 8.1, 0.8 Hz, H3_B), 7.54 (1H, dd, J= 7.8, 1.2 Hz, H3_A), 7.22-7.14 (1.6H, m, H2_A+H2_B), 7.11 (1H, dd, J= 7.6, 1.1 Hz, H1_A), 7.05 (0.6H, dd, J = 7.6, 0.7 Hz, H1_B), 3.99 (1.8H, s, H5_B), 3.92 (3H, s, H5A) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 164.6, 161.9, 146.4, 146.2, 146.0, 145.9, 138.1, 130.0, 123.1, 122.3, 121.8, 121.7, 121.4, 120.3, 118.6, 118.4, 33.6, 32.9 ppm.

355 - 1 -Methyl-7-aminobenzimidazole¹⁵

[0148] Hydrochloric acid (32% w/w, 1.5 mL, 15 mmol, 18 eq) was added to a dispersion of 354 (150 mg, 0.856 mmol, 1.00 eq) in water (1.5 mL) to form a pink solution, and the reaction was heated at 90 °C for 3 h after which the reaction had gone to completion. After cooling to room temperature, the reaction mixture was basified with ammonium hydroxide (28% w/w, 3 mL) to a pH of 12, and diluted with aqueous sodium hydroxide (1 M, 20 mL). This was extracted with dichloromethane (3 x 15mL), and the combined organic phases were washed with brine (25 mL), dried over MgSO₄ and reduced in vacuo to yield the title compound 355 (95 mg, 75%) as an off-white solid which was used immediately in the subsequent coupling reaction following NMR analysis. R_ƒ= 0.20 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂); ¹H NMR (400 MHz, CDCl₃): δ 7.64 (1H, s, H4), 7.27 (1H, dd, J= 8.1, 0.8 Hz, H3), 7.03 (1H, dd, J= 8.1, 7.6 Hz, H2), 6.55 (1H, dd, J= 7.5, 0.7 Hz, Hl), 4.07 (3H, s, H5), 3.68 (2H, br s, -NH2) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 146.1, 144.2, 132.8, 124.9, 123.0, 112.5, 110.8, 33.8 ppm.

358 - Phenyl N’-cyano-N-(l-methyl-3a, 7a-dihydro-lH-benzo [d]imidazol-4- yl)carbamimidate

[0149] 358 was prepared from 350 (101 mg, 0.686 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 300 mg, 7.5 mmol, 3.0 eq), and diphenyl N- cyanocarbonimidate (327 mg, 1.37 mmol, 2.00 eq) according to the general procedure C. The resulting oily brown residue was crudely purified by flash chromatography (10% v/v MeOH sat. w/ NH₃ in CH₂CI₂), and then re-purified by flash chromatography (1-2% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to afford the title compound (180 mg, 90%) as a white solid, mp 175-176 °C; Rf= 0.30 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; stains white with vanillin); ¹H NMR (400 MHz, DMSO-d₆): δ 10.88 (1H, br s, -NH), 8.26 (1H, s, H7), 7.53 (1H, dd, J= 8.0, 0.8 Hz, H6), 7.44-7.38 (2H, m, H2), 7.30 (1H, t, J= 7.7 Hz, H5), 7.28- 7.21 (4H, m, H1+H3+H6), 3.86 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 161.0 (br), 151.6, 144.7, 138.5, 135.5, 129.6, 126.7, 126.1, 122.2, 121.2, 117.9, 113.8 (br), 109.4, 30.9 ppm; LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%). 307 - l-((Adamantan-l-yl)methyl)-2-cyano-3-(l-methyl-lH-benzo[d]imidazol-4- yl)guanidine

[0150] The title compound was prepared from 358 (101 mg, 0.686 mmol, 1.00 eq) and 64 (63 mg, 0.38 mmol, 1. 1 eq) according to general procedure D to afford 307 (76 mg, 69%) as a white solid which was collected by vacuum filtration. This was recrystallised from absolute ethanol to yield 307 as a white crystalline solid (63 mg, 57%). N.B. The product's Rf was identical to the starting material in a wide range of solvent systems, TLC analysis with hexane/chloroform/methanol (9:9:2 v/v), eluted three times can help differentiate starting material and product spots, mp 253-254 °C; Rf= 0.45 (CHCE/hexane/MeOH, 9:9:2 v/v, eluted 3 times); IR (diamond cell): 3455, 3197, 2893, 2176, 1596, 1573, 1262, 745 cm- ¹H NMR (400 MHz, DMSO-d₆): δ 9.00 (1H, br s, -NH), 8.25 (1H, s, H8), 7.86 (1H, br s, - NH), 7.38 (1H, dd, J= 7.5, 1.3 Hz, H7), 7.25 (1H, t, J= 7.8 Hz, H6), 7.22 (1H, dd, J= 7.7, 1.3 Hz, H5), 3.86 (3H, s, H9), 3.04 (2H, d, J= 5.8 Hz, H4), 1.94 (3H, s, H2), 1.67 (3H, d, J = 12.0 Hz, H3), 1.59 (3H, d, J= 11.7 Hz, H3), 1.54-1.44 (6H, m, Hl) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 158.2, 144.1, 136.2, 136.0, 129.1, 123.3, 117.4, 115.4, 107.3, 53.6, 39.9, 36.9, 34.2, 31.4, 28.1 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2112.

84%

[0151] The title compound was prepared from 356 (203 mg, 1.38 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 118 mg, 3.0 mmol, 2.1 eq), and diphenyl /V- cyanocarbonimidate (657 mg, 2.76 mmol, 2.00 eq) according to the general procedure C. The resulting brown oily residue was crudely purified by flash chromatography (5% v/v MeOH sat. w/ NH₃ in CH₂CI₂), and then re-purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ + 1 % PhMe in CH₂CI₂), eluting as a white band (product) preceding a yellow band (impurity). The combined product fractions were reduced in vacuo to yield the title compound (338 mg, 84%) as a white solid, mp 212-213 °C; Rf= 0.24 (CHCh/hexane/MeOH, 9:9:2 v/v); ¹H NMR (400 MHz, DMSO-d₆): δ 10.84 (1H, s), 8.22 (1H, s), 7.76 (1H, d, J= 1.6 Hz), 7.58 (1H, dd, J= 8.6, 0.4 Hz), 7.48-7.40 (2H, m), 7.37 (1H, dd, J= 8.6, 2.0 Hz), 7.34-7.24 (2H, m), 3.83 (3H, s) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.7, 145.7, 143.2, 133.1, 130.1, 129.7, 126.1, 120.9, 119.8, 115.5, 113.5, 110.1, 30.8 ppm (one aromatic carbon unresolved); LRMS (+ESI) m/z: 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₆H₁₃N₅₀ [M+Na]⁺: 314.1018, found: 314.1013.

308 - l-((Adamantan-l-yl)methyl)-2-cyano-3-(l-methyl-lH-benzo[d]imidazol-5- yl)guanidine

[0152] The title compound was prepared from 359 (105 mg, 0.360 mmol, 1.00 eq) and 64 (65 mg, 0.39 mmol, 1.1 eq) according to the general procedure D, isolating the title compound as a white solid which was collected by vacuum fdtration (62 mg, 47%). mp 241-242 °C; R_ƒ= 0.36 (CHCh/hexane/MeOH, 9:9:2 v/v); IR (diamond cell): 3346, 2900, 2846, 2164, 1597, 1572, 1507, 1313 cm^-1: ¹H NMR (400 MHz. DMSO-d₆): δ 8.92 (1H, br s, -NH), 8.19 (1H, s, H8), 7.56 (1H, d, J= 8.6 Hz, H6), 7.48 (1H, d, J= 1.7 Hz, H7), 7.14 (1H, dd, J= 8.6, 1.9 Hz, H5), 6.66 (1H, brt, J= 5.6 Hz, -NH), 3.84 (3H, s, H9), 2.94 (2H, d, J= 6.3 Hz, H4), 1.95 (3H, br s, H2), 1.72-1.56 (6H, m, Hl), 1.51-1.41 (6H, m, H3) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 159.2, 145.4, 143.5, 132.6, 131.4, 120.2, 117.5, 115.6, 110.3, 52.3, 39.7, 36.5, 34.4, 30.8, 27.6 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2112.

360 - Phenyl N’-cyano-N-(l-methyl-lH-benzo[d]imidazol-6-yl)carbamimidate

92%

[0153] 360 was prepared from 1-methyl-6-aminobenzimidazole 357 (203 mg, 1.38 mmol, I.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 113 mg, 2.8 mmol, 2.1 eq), and diphenyl /V-cyanocarbonimidate (657 mg, 2.76 mmol, 2.00 eq) according to the general procedure C. The resulting brown oily residue was purified crudely by flash chromatography (5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) and then re-purified by flash chromatography (1% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to afford the title compound 360 (370 mg, 92%) as a white solid, mp 101 °C (decomp); Rf= 0.27 (CHCE/hexane/MeOH, 9:9:2 v/v); IR (diamond cell): 3322, 2970, 2186, 1618, 1417, 948, 690 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ

I I.16 (1H, s, -NH), 8.24 (1H, d, J= 0.8 Hz, H7), 7.57 (1H, d, J= 8.5 Hz, H5), 7.47-7.39 (3H, m, H2 + H6), 7.35-7.25 (3H, m, Hl + H3), 7.21 (1H, d, J= 7.3 Hz, H4), 4.05 (3H, s) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 160.0, 151.4, 140.6, 130.8, 129.7, 128.3, 127.6, 126.2, 126.1, 121.0, 119.4, 116.3, 113.3, 108.4, 101.4, 96.1, 35.5 ppm (additional resonances due to isomerism); LRMS (+ESI) m/z'. 314 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₆H₁₃N₅₀ [M+Na]⁺: 314.1018, found: 314.1013.

309 - l-((Adamantan-l-yl)methyl)-2-cyano-3-(l-methyl-lH-benzo[d]imidazol-6- yl)guanidine

[0154] 309 was prepared from 360 (101 mg, 0.347 mmol, 1.00 eq) and 64 (65 mg, 0.39 mmol, 1.1 eq) according to the general procedure D, providing the title compound as a white solid which was collected by vacuum filtration (86 mg, 68%). mp 258-259 °C; Rf= 0.33 (CHCl₃/hexane/MeOH, 9:9:2 v/v); IR (diamond cell): 3218, 2898, 2844, 2167, 1596, 1562, 1444, 1351 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 9.01 (1H, br s, -NH), 8.17 (1H, s, H8), 7.61 (1H, d, J= 8.5 Hz, H6), 7.44 (1H, d, J= 1.7 Hz, H7), 7.07 (1H, dd, J= 8.6, 1.9 Hz, H5), 6.79 (1H, brt, J= 6.0 Hz, -NH), 3.80 (3H, s, H9), 2.96 (2H, d, J= 6.3 Hz, H4), 1.95 (3H, br s, H2), 1.74-1.57 (6H, m, Hl), 1.47 (6H, s, H3) ppm; ¹³C NMR (101 MHz, DMSO- dd): 8 159.0, 145.0, 141.1, 134.7, 132.3, 119.5, 119.1, 117.4, 106.3, 52.3, 39.7, 36.5, 34.5, 30.6, 27.6 ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2112.

[0155] 361 was prepared from 355 (87 mg, 0.59 mmol, 1.0 eq), sodium hydride (60% w/w dispersion in mineral oil; 78 mg, 2.0 mmol, 3.3 eq), and diphenyl /V-cyanocarbonimidate (282 mg, 1.18 mmol, 2.00 eq) according to the general procedure C. The crude brown-grey residue was purified by flash chromatography (5% v/v MeOH sat. w/NH₃ in CH₂CI₂), eluting before a blue by-product on the column, isolating the title compound as a grey -white solid (166 mg, 97%). R_ƒ= 0.09 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂); ¹H NMR (400 MHz, DMSO-d₆): δ 10.86 (1H, br s, -NH), 8.22 (1H, s, H7), 7.65 (1H, d, J= 8.0 Hz, H6), 7.43 (2H, br s, H2), 7.37-7.14 (4H, m, Hl + H3 + H4), 7.24 (1H, t, J= 7.8 Hz, H5), 4.01 (3H, s, H8) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 162.0 (br), 151.2, 146.0 (br), 145.3 (br), 130.2, 129.8 (br), 129.4, 126.5, 122.8 (br), 121.6, 121.4 (br), 120.6 (br), 119.4 (br), 115.2, 113.6 (br), 32.7 ppm.

310 - l-((Adamantan-l-yl)methyl)-2-cyano-3-(l-methyl-lH-benzo [d]imidazol-7- yl)guanidine

[0156] 310 was prepared from 361 (47 mg, 0.16 mmol, 1.0 eq) and 64 (57 mg, 0.34 mmol, 2. 1 eq) according to the general procedure D, collecting the title compound as a white solid (14 mg, 22%) with the remainder eluting through the filter. The neutral compound is virtually insoluble in all organic solvents, including DMSO, diglyme, and N- methylpyrrolidine when heated to their respective boiling points. The retentate was dissolved in concentrated aqueous hydrochloric acid (32% w/v, 1 mL), and the acid was evaporated under a stream of nitrogen gas to afford the title compound as the hydrochloride salt. Biological analysis of this derivative could not be undertaken due to its poor physicochemical properties. ¹H NMR (400 MHz, DMSO-d₆): δ 9.02 (1H, br s, -NH), 8.15 (1H, s, H8), 7.62 (1H, d, J= 8.1 Hz, H7), 7.21 (1H, t, J= 7.8 Hz, H6), 7.03 (1H, d, J= 7.6 Hz, H5), 6.54 (1H, br s, -NH), 3.89 (3H, s, H9), 2.91 (2H, d, J= 6.1 Hz, H4), 1.92 (3H, s, H2), 1.70-1.53 (6H, m, Hl), 1.40 (6H, br s, H3) ppm; LRMS (+ESI) m/z: 385 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₂₁H₂₆N₆ [M+Na]⁺: 385.2117, found: 385.2115; ¹³C NMR spectra could not be obtained for this compound due to its poor solubility in various deuterated solvents even after extended acquisition times.

363 - l-Methyl-7-nitro-lH-indole

[0157] A yellow solution of 7-nitro- 1 //-indole 362 (501 mg, 3.09 mmol, 1.00 eq) in tetrahydrofuran (10 mL) at 0 °C was treated portion-wise with sodium hydride (caution-. effervescence; 60% w/w dispersion in mineral oil; 181 mg, 4.5 mmol, 1.5 eq), immediately forming a blood-red suspension. This was stirred at 0 °C for 5 min, and then treated dropwise with methyl iodide (0.30 mL, 4.8 mmol, 1.6 eq). The cooling bath was removed, and the reaction mixture stirred at room temperature for 20 h, after which the reaction had gone to completion. Volatiles were removed under a stream of nitrogen gas, and the crude residue was purified by flash chromatography (hexane/ethyl acetate, 9: 1 v/v), and then repurified by flash chromatography (10% v/v CHCl₃ in hexane) to yield the title compound as a yellow solid (276 mg, 51% yield), mp 47—49 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.85 (1H, dd, 7= 7.8, 1.0 Hz, Hl), 7.81 (1H, dd, 7= 7.9, 0.9 Hz, H3), 7.12 (1H, t, 7= 7.8 Hz, H2), 7.12 (1H, d, 7= 2.8 Hz, H5), 6.63 (1H, d, 7 = 3.2 Hz, H4), 3.85 (3H, s, H6) ppm; ¹³C NMR (101 MHz, CDCh): 8 136.7, 133.64, 133.63, 127.7, 127.1, 119.8, 118.6, 102.7, 37.5 ppm. The spectroscopic data matched those reported in the literature.¹⁶

SI 8 - 7 -Amino- 1 -methyl- IH-indole

[0158] S18 was prepared from 363 (206 mg, 1.17 mmol, 1.00 eq) according to the general procedure N to afford the title compound (170 mg, quant.) as a brown oil which was used immediately in the subsequent coupling step without further characterisation.

[0159] 364 was prepared from S18 (170 mg, 1.16 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil; 154 mg, 3.9 mmol, 3.3 eq), and diphenyl N- cyanocarbonimidate (609 mg, 2.56 mmol, 2.20 eq) according to the general procedure C. The crude brown residue was purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to provide the title compound (274 mg, 81%) as an off-white solid, mp 172-175 °C; R_ƒ= 0.42 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; ninhydrin stain); IR (diamond cell): 2924, 2854, 2193, 1639, 1419, 1309, 1195, 726 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.86 (1H, m, -NH), 7.55 (1H, dd, J= 7.9, 0.7 Hz, H6), 7.51-7.36 (2H, m, H2), 7.33 (1H, d, J= 2.3 Hz, H8), 7.32-7.07 (4H, m, H1+H3+H4), 7.05 (1H, t, J= 7.7 Hz, H5), 6.48 (1H, d, J= 3.0 Hz, H7), 4.01 (3H, m, H9) ppm; ¹³C NMR (101 MHz, DMSO-d₆): 5 162.3 (br), 151.2 (br), 131.8 (br), 131.6 (br), 131.0, 130.8, 129.7 (br), 129.5, 126.4, 121.9, 121.4 (br), 120.7 (br), 120.1 (br), 119.1 (br), 118.4, 114.1, 108.9, 100.7, 35.2, 34.7 ppm; LRMS (+ESI) m/z-. 313 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C17H14N4O [M+Na]⁺: 313.1066, found: 313.1061.

[0160] 365 was prepared from 364 (126 mg, 0.434 mmol, 1.00 eq) and 64 (110 mg, 0.666 mmol, 1.53 eq) according to the general procedure D to afford the title compound. The compound did not precipitate out of solution in contrast to all of the other heteroaromatic analogues, and was purified by flash chromatography (0.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to yield the title compound (74 mg, 47%) as a white solid, mp 180-181 °C; Rf= 0.37 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; ninhydrin stain); IR (diamond cell): 3412, 3211, 2900, 2845, 2173, 1596, 1567, 1304, 716 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 9.01 (1H, br s, -NH), 7.53 (1H, d, J= 7.5 Hz, H7), 7.28 (1H, d, J= 3.1 Hz, H9), 7.03 (1H, m, H6), 6.90 (1H, d, J= 7.3 Hz, H5), 6.46 (1H, d, J= 3.1 Hz, H8), 6.23 (1H, br s, -NH), 3.89 (3H, s, H10), 2.91 (2H, d, J= 6.1 Hz, H4), 1.91 (3H, br s, H2), 1.70-1.51 (6H, m, H3), 1.37 (6H, br s, Hl) ppm; ¹³C NMR (101 MHz, DMSO-d₆): 160.0, 132.5, 131.6, 131.0, 122.4, 120.9, 120.5, 119.3, 117.5, 100.6, 52.2, 39.7, 36.5, 34.9, 34.2, 27.6 ppm; LRMS (+ESI) m/z-. 384 ([M+Na]⁺, 100%); HRMS (+ESI) m/z'. Calc, for C22H27N5 [M+Na]⁺: 384.2164, found: 384.2160.

366 - N¹ -methyl-6-nitrobenzene-l ,2-diamine*

[0161] A solution of 352 (500 mg, 2.54 mmol, 1.00 eq) and ammonium formate (848 mg, 13.5 mmol, 5.31 eq) in absolute ethanol (5 mL) was evacuated and purged three times with nitrogen, and then palladium on carbon (10% w/w, 30 mg, 25 pmol, 5 mol% Pd) was added in one portion. After stirring at room temperature for 4 h, solvent was evaporated under a stream of nitrogen gas. The black residue was purified by flash chromatography (0.25% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to isolate the title compound 366 as a dark red crystalline solid (237 mg, 56%) which was used immediately in the subsequent cyclisation step without further characterisation. Rf 0.53 (hexane/ethyl acetate, 2: 1 v/v).

[0162] To a red suspension of 366 (200 mg, 1.20 mmol, 1.00 eq) in water (2 mL) at 0 °C was added concentrated hydrochloric acid (32% w/w, 0.26 mL, 2.7 mmol, 2.2 eq), forming an orange suspension. A solution of sodium nitrite (103 mg, 1.49 mmol, 1.25 eq) in water (1 mL) was then added to this reaction mixture dropwise via a syringe, forming an orange foamy mixture which was stirred at room temperature for 1 h. The reaction mixture was diluted with aqueous sodium hydroxide (1 M, 20 mL), and this was extracted with diethyl ether (4 x 15 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO₄ and reduced in vacuo to yield a pale brown solid. This was purified by flash chromatography (CH₂CI₂/hexane, 9: 1 v/v), eluting as a pale orange band on silica. This was reduced in vacuo to yield a tan brown solid (180 mg, 85%). ¹H NMR (400 MHz, CDCl₃): δ 8.38 (1H, dd, J= 8.2, 1.0 Hz, Hl or H3), 8.33 (1H, dd, J= 7.8, 0.9 Hz, Hl or H3), 7.48 (1H, dd, J= 8.2, 7.8 Hz, H2) 4.58 (3H, s, H4) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 149.5, 134.9, 127.6, 126.1, 125.4, 123.2, 39.9 ppm. The spectroscopic data matches that reported in the literature.¹⁷

[0163] The title compound was prepared from 367 (144 mg, 0.808 mmol, 1.00 eq) according to the general procedure N, purifying the crude product by flash chromatography (1% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to isolate the title compound 368 (102 mg, 85%) as a white solid which was used immediately in the subsequent coupling step below. Rf= 0.25 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂; ninhydrin stain).

[0164] The title compound was prepared from 368 (100 mg, 0.675 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil; 81 mg, 2.0 mmol, 3.0 eq), and diphenyl N- cyanocarbonimidate (330 mg, 1.39 mmol, 2.05 eq) according to the general procedure C. The crude brown-grey residue was crudely purified by flash chromatography (5% v/v MeOH sat. w/ NH₃ in CH₂CI₂ until phenol eluted, and then increased to 10% v/v MeOH sat. w/ NH₃ in CH₂CI₂). Despite the high polarity of the eluent, the compound eluted slowly over many fractions. The product was isolated as a colourless viscous oil, which under high- vacuum dried to a white solid (188 mg, 95%). mp 157-159 °C; R_ƒ= 0.20 (2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂); IR (diamond cell): 3561, 3129, 2972, 2197, 1631, 1613, 1587, 1408, 1196 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 10.96 (1H, br s, -NH), 8.02 (1H, d, J= 8.3 Hz, H6), 7.67 (1H, d, J= 7.3 Hz, H4), 7.54-7.39 (3H, m, H2+H5), 7.38-7.24 (3H, m, H1+H3), 4.44 (3H, s, H7) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 151.2 (br), 147.0, 129.7 (br), 129.6, 126.9 (br), 126.5, 124.3, 121.5 (br), 118.8 (br), 36.0 ppm (3 aromatic carbons unresolved due to extensive broadening of some resonances); LRMS (+ESI) m/z-. 315 ([M+Na]⁺, 100%); HRMS (+ESI) m/z-. Calc, for C₁₅H₁₂N₆O [M+Na]⁺: 315.0971, found: 315.0966.

370 - 1-((A damantan-l-yl)methyl)-2-cyano-3-(l-methyl-lH-benzo [d] [1 , 2, 3 ]triazol-7- yl)guanidine

[0165] 370 was prepared from 369 (59 mg, 0.20 mmol, 1.0 eq) and 1- adamantylmethylamine (55 mg, 0.33 mmol, 1.7 eq) according to the general procedure D. The precipitate was collected to afford the title compound 370 (14 mg, 13%) as a white solid, which as for the benzimidazole analogue 310 was insoluble in organic solvents.

[0166] For the next section, the following two compounds were prepared using previously reported procedures: 2-amino-6-nitrophenol (35)¹ and 4-nitrobenzoxadiazole (44).² The following compounds were prepared using adaptions of previously reported procedures: benzodioxol-4-carboxylic acid (48), ³ 7-nitrobenzoxazole (34), ⁴ and tert-butyl benzodioxol- 4-ylcarbamate (50).⁵

39 - Phenyl N-(benzo[d]oxazol-4-yl)-N’-cyanocarbamimidate

[0167] 39 was prepared from 4-benzoxazolamine 36 (239 mg, 1.78 mmol), diphenyl cyanocarbonimidate (864 mg, 3.63 mmol, 2.0 eq) and sodium hydride (247 mg, 6.23 mmol) according to general procedure C. Purification by flash chromatography (1.0 - 1.5% v/v MeOH sat. NH₃ in CH₂CI₂) afforded the title compound as a white, crystalline solid (350 mg, 71%). mp 171-174 °C; R_ƒ= 0.20 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂); IR (neat, diamond cell): 2193, 1633, 1596, 1432, 1407, 1199, 1084, 687 cm^-1; ¹H NMR (400 MHz, DMSO-d₆): δ 11.23 (s, 1H, NH), 8.84 (s, 1H, H7), 7.74 (dd, J = 8.1, 1.0 Hz, 1H, H5), 7.49 (t, J = 8.0 Hz, 1H), 7.45-7.39 (m, 3H, Hl, H3), 7.33-7.23 (m, 3H, H2, H6); ¹³C NMR (101 MHz, DMSO-d₆): δ 160.9, 154.4, 151.5, 150.2, 135.3, 129.8, 127.8, 126.4, 125.8, 121.3, 121.3, 113.5, 110.1 ppm; LRMS (+ESI) m/z: 301 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C₁₅H₁₀N₄O₂ [M+Na]⁺: 301.0696, found: 301.0697.

11 - l-((Adamantan-l-yl)methyl)-3-(benzo[d]oxazol-4-yl)-2-cyanoguanidine

[0168] 11 was prepared from 39 (200 mg, 0.68 mmol) and 1-adamantylmethylamine (148 mg, 0.90 mmol) according to general procedure D, providing the title compound as a white solid, collected by vacuum fdtration (104 mg, 41%). mp 193-196 ° C; Rf= 0.11 (1% v/v MeOH sat. NH₃ in CH₂C₁₂); IR (neat, diamond cell): 2891, 2845, 2183, 1552, 1430, 1313, 741 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 9.26 (s, 1H, NH), 8.76 (s, 1H, H8), 7.56 (dd, J = 8.1, 1.0 Hz, 1H, H7), 7.41 (t, J = 8.0 Hz, 1H, H6), 7.33 (dd, J = 7.9, 1.0 Hz, 1H, H5), 7.13 (s, 1H, NH), 3.02 (d, J = 6.1 Hz, 2H, H4), 2.01-1.92 (m, 3H, H2), 1.71-1.57 (m, 6H, Hl), 1.49 (d, J = 2.8 Hz, 6H, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 158.1, 153.4, 150.2, 133.5, 129.7, 125.8, 119.0, 116.8, 107.6, 52.8, 36.5, 34.1, 27.7 ppm; LRMS (+ESI) m/z: 372 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C20H23N5O [M+Na]⁺: 372.1795, found: 372.1794. Anal. Calc, for C₂₀H₂₃N₅: C, 68.74; H, 6.63; N, 20.05; Found: C, 68.97; H, 6.70; N, 19.97.

40 - Phenyl N-(benzo [d]oxazol-5-yl)-N ’-cyanocarbamimidate

[0169] 40 was prepared from 5-benzoxazolamine 37 (200 mg, 1.49 mmol), diphenyl cyanocarbonimidate (746 mg, 3.13 mmol, 2.6 eq) and sodium hydride (209 mg, 5.22 mmol) according to general procedure C. Purification by flash chromatography (2.5% v/v MeOH sat. NH₃ in CH₂C1₂) afforded an off white solid, which was recrystallised from MeOH and hexane to yield the title compound as a white, crystalline solid (221 mg, 53%). mp 182-183 °C; R_ƒ= 0.22 (2.5% v/v MeOH sat. NH₃ in CH₂Cl₂); IR (neat, diamond cell): 3149, 2189, 1644, 1607, 1464, 1084, 688 cm⁴; ¹H NMR (500 MHz, DMSO-d₆): δ 10.94 (s, 1H, NH), 8.79 (s, 1H, H7), 7.93 (d, J = 2.1 Hz, 1H, H5), 7.81 (d, J = 8.7 Hz, 1H, H6), 7.53 (dd, J = 8.7, 2.1 Hz, 1H, H4), 7.48-7.41 (m, 2H, H2), 7.34-7.26 (m, 3H, Hl, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 155.4, 151.5, 147.5, 139.9, 132.9, 129.8, 126.2, 122.7, 120.9 (br), 116.2 (br), 111.1 ppm; LRMS (+ESI) m/z: 301 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C15H10N4O2 [M+Na]⁺: 301.0696, found: 301.0695.

12 - l-((Adamantan-l-yl)methyl)-3-(benzo[d]oxazol-5-yl)-2-cyanoguanidine

[0170] 12 was prepared from 40 (190 mg, 0.68 mmol) and 1-adamantyhnethylamine (142 mg, 0.85 mmol) according to general procedure D, providing the title compound as a white solid, collected by vacuum filtration (120 mg, 50%). mp 290-293 °C; Rf= 0.28 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂); IR (neat, diamond cell): 3350, 3211, 3100, 2896, 2943, 2173, 1568, 1072, 648 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 9.20 (s, 1H, NH), 8.75 (s, 1H, H8), 7.76 (d, J = 8.7 Hz, 1H, H6), 7.64 (d, J = 2.1 Hz, 1H, H7), 7.29 (dd, J = 8.7, 2.1 Hz, 1H, H5), 7.09 (t, J = 6.4 Hz, 1H, NH), 2.98 (d, J = 6.1 Hz, 2H, 4H), 1 .98-1.93 (m, 3H, H2), 1.76-1.58 (m, 7H, Hl), 1.48 (d, J = 2.8 Hz, 6H, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 159.4, 155.5, 147.3, 140.5, 135.1, 122.9, 117.7, 116.0, 111.6, 52.9, 39.5, 36.9, 34.9, 28.1 ppm; LRMS (+ESI) m/z-. 372 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C20H23N5O [M+Na]⁺: 372.1795, found: 372.1794; Anal. Calc, for C20H23N5: C, 68.74; H, 6.63; N, 20.05; Found: C, 68.67; H, 6.62; N, 20.05.

41 - Phenyl N-(benzo [d]oxazol-6-yl)-N ’-cyanocarbamimidate

[0171] 41 was prepared from 6-benzoxazolamine 38 (300 mg, 2.24 mmol), diphenyl cyanocarbonimidate (1.12 g, 4.71 mmol, 2.1 eq) and sodium hydride (313 mg, 7.83 mmol) according to general procedure C. Purification by flash chromatography (2% v/v MeOH sat. NH₃ in CH₂CI₂) afforded the title compound as a white, crystalline solid (307 mg, 49%). mp 178-179 °C; Rf= 0.06 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂); IR (neat, diamond cell): 3087, 2176, 1607, 1074, 616 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 11.05 (s, 1H, NH), 8.76 (s, 1H, H7), 7.96 (d, J = 2.0 Hz, 1H, H6), 7.82 (d, J = 8.6 Hz, 1H, H5), 7.50 (dd, J = 8.6, 2.0 Hz, 1H, H4), 7.48-7.38 (m, 2H, 16, H2), 7.38-7.20 (m, 3H, Hl, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 155.0, 151.6, 149.2, 137.7, 133.8, 129.8, 126.3, 121.3, 119.9, 113.2, 107.3 ppm; LRMS (+ESI) m/z: 301 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C15H10N4O2 [M+Na]⁺: 301.0696, found: 301.0699.

14 - l-((Adamantan-l -yl)methyl)-3-(benzo[d]oxazol-6-yl)-2-cyanoguanidine

[0172] 14 was prepared from 41 (220 mg, 0.79 mmol) and 1-adamantylmethylamine (163 mg, 0.98 mmol) according to general procedure D. Purification by flash chromatography (1% v/v MeOH sat. NH₃ in CH₂CI₂) provided the title compound as a white solid (107 mg, 36%). mp 291-294 °C; R_ƒ= 0.13 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂); IR (neat, diamond cell): 2893, 2172, 1596, 1570, 1071, 593 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 9.15 (s, 1H, NH), 8.70 (s, 1H, H8), 7.76 (d, J = 8.5 Hz, 1H, H6), 7.65 (d, J = 2.0 Hz, 1H, H7), 7.25 (dd, J = 8.5, 2.0 Hz, 1H, H5), 7.09 (t, J = 6.3 Hz, 1H, NH), 2.98 (d, J = 6.3 Hz, 2H, H4), 1.97-1.93 (m, 3H, H2), 1.73-1.57 (m, 6H, H3), 1.47 (d, J = 2.8 Hz, 6H, Hl) ppm; ¹³C NMR

(126 MHz, DMSO-d₆): δ 158.7, 154.3, 149.5, 136.6, 135.8, 121.3, 120.9, 120.0, 117.1,

106.4, 52.6, 36.5, 34.4, 27.7 ppm; LRMS (+ESI) m/z: 372 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C20H23N5O [M+Na]⁺: 372.1795, found: 372.1798.

29 - 2,6-Dinitrophenol

[0173] A solution of 2-chloro-l,3-dinitrobenzene 30 (325 mg, 1.60 mmol) in THF (20 mL) was treated with aq. NaOH (4 M, 2.4 mL, 6 eq) and aq. tetrabutylammonium hydroxide (40% v/v, 1.0 mL, 0.5 eq), then heated at reflux for 5 h. The reaction mixture was then concentrated in vacuo, redissolved in aq. HC1 (8 mL, 3 M), and extracted with CH₂CI₂ (3 x 10 mL). The organic layers were combined and washed with aq. HC1 (20 mL, 3 M), brine (20 mL), dried over MgSO₄ and concentrated in vacuo to yield the title compound 29 as a deep red solid (295 mg, quant.), which was used immediately in the subsequent reaction without further characterisation. R_ƒ= 0.35 (EtOAc/Hex, 2: 1).

35 - 2-Amino-6-nitrophenol

[0174] A mixture of 2,6-dinitrophenol 29 (295 mg, 1.60 mmol), activated carbon powder (300 mg) and iron(III) chloride hexahydrate (10 mg, 37 μM 2 mol%) in ethanol (5 mL) was heated to reflux and treated dropwise with hydrazine hydrate (85% v/v, 0. 18 mL, 2 eq) over 2 h. After 6 h at reflux the reaction mixture was concentrated in vacuo and purified by flash chromatography (EtOAc/Hex, 30:70, l% NEt3), eluting rapidly, to yield the title compound 35 as red crystals (212 mg, 86%), which was used immediately in the subsequent reaction following ¹H NMR analysis. Rf= 0.51 (EtOAc/Hex, 2:3, significant dragging on plate), 0.63 (10% v/v MeOH sat. NH₃ in CH₂CI₂, orange spot visible without UV or staining, quickly discolours to brown); ¹H NMR (300 MHz, DMSO-d₆): δ 7.14 (dd, J = 8.4, 1.6 Hz, 1H), 6.93 (dd, J = 7.8, 1.6 Hz, 1H), 6.76 (t, J = 8.1 Hz, 1H) ppm.

34 -7 -Nitrobenzo [d] oxazole

[0175] A mixture of 2-amino-6-nitrophenol 35 (373 mg, 2.42 mmol) in trimethyl orthoformate (1 mL, 9.42 mmol, 3.9 eq) was treated with p-toluenesulfonic acid (83 mg, 0.48 mmol, 20 mol%) and heated at 100 °C for 1 h. The remaining trimethyl orthoformate was removed in vacuo providing an orange solid which was dissolved in CH₂CI₂ (20 mL), washed with sat. aq. NaHCO₃ (2 x 20 mL), brine (20 mL), dried over MgSC>4 and concentrated in vacuo. The remaining crude product was purified by flash chromatography (EtOAc/Hex, 2:3) to yield the title compound 34 as an orange powder (300 mg, 76%). Rf= 0.48 (EtOAc/Hex, 2:3, stains purple with Goofy's allowing differentiation with starting material); ¹H NMR (500 MHz, DMSO-d₆): δ 19.04 (s, 1H), 8.27 (m, 2H), 7.64 (t, J = 8.1 Hz, 1H) ppm; ¹³C NMR (126 MHz, DMSO-X): 8 156.0, 143.0, 142.3, 133.2, 127.6, 125.0, 121.7 ppm; HRMS (+ESI) m/z: Calc, for C7H4N2O3 [M+H]⁺: 165.0295, found: 165.0295. 27 - Benzo [d]oxazol-7 -amine

[0176] 27 was prepared 34 (368 mg, 2.24 mmol) according to general procedure N to yield the title compound as a red solid (301 mg, quant.), which was used immediately in the subsequent coupling reaction without further characterisation. Rf= 0.51 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, single spot stains brown with Goofy's).

42 - Phenyl N-(benzo[d]oxazol-7-yl)-N’-cyanocarbamimidate

[0177] 42 was prepared from 7-benzoxazolamine 27 (301 mg, 2.24 mmol), diphenyl cyanocarbonimidate (1.87 g, 7.85 mmol, 3.5 eq) and sodium hydride (314 mg, 7.85 mmol) according to general procedure C. Purification by flash chromatography (Hex/CH₂CI₂/ MeOH sat. NH₃, 9:9:2) afforded the title compound as a white, crystalline solid (277 mg, 44%). mp 154-158 °C; R_ƒ= 0.18 (Hex/CH₂CI₂/MeOH sat. NH₃, 9:9:2); IR (neat, diamond cell): 2921, 2852, 2199, 1650, 1624 1415, 690 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 11.30 (s, 1H, NH), 8.85 (s, 1H, H7), 7.77 (dd, J = 7.5, 1.5 Hz, 1H, H5), 7.52-7.38 (m, 4H, H2, H4, H6), 7.35-7.19 (m, 5H, Hl, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 154.3, 151.2, 143.7, 141.0, 129.8, 129.4, 126.5, 124.9, 122.6, 121.2, 120.5, 119.0, 115.2 ppm; LRMS (+ESI) m/z-. 301 ([M+Na]⁺, 100%); HRMS (+ESI) m/z'. Calc, for C15H10N4O2 [M+Na]⁺: 301.0696, found: 301.0694.

13 - l-((Adamantan-l -yl)methyl)-3-(benzo[d]oxazol- 7-yl)-2-cyanoguanidine

[0178] 13 was prepared from 42 (100 mg, 0.36 mmol) and 1-adamantylmethylamine (89 mg, 0.54 mmol) according to general procedure D. Purification by flash chromatography (1% v/v MeOH sat. NH₃ in CH₂CI₂) provided the title compound as a white solid (53 mg, 42%). mp 243-245 °C; R_ƒ= 0.25 (1% v/v MeOH sat. NH₃ in CH₂CI₂); IR (neat, diamond cell): 3282, 3110, 2908, 2177, 1583, 1085, 635 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 9.42 (s, 1H, NH), 8.76 (s, 1H, H8), 7.64 (dd, J = 8.0, 1.0 Hz, 1H, H5), 7.37 (t, J = 7.9 Hz, 1H, H6), 7.26 (d, J = 7.8 Hz, 1H, H7), 7.15 (d, J = 6.5 Hz, 1H, NH), 2.96 (d, J = 5.8 Hz, 2H, H4), 2.03-1.91 (m, 3H, H2), 1.70-1.57 (m, 6H, H3), 1.46 (d, J = 2.8 Hz, 6H, Hl) ppm; ¹³C

NMR (126 MHz, DMSO-d₆): δ 158.4, 153.8, 143.7, 141.1, 124.9, 122.5, 122.0, 117.4,

117.1, 52.7, 36.5, 34.3, 27.7 ppm; LRMS (+ESI) m/z: 372 ([M+Na]⁺, 100%); HRMS (+ESI) m/z: Calc, for C20H23N5O [M+Na]⁺: 372.1795, found: 372.1791; Anal. Calc, for C20H23N5: C, 68.74; H, 6.63; N, 20.05; Found: C, 68.55; H, 6.62; N, 19.99.

44 - 4-Nitrobenzo[c] [1,2, 5] oxadiazole

[0179] A solution of benzoxadiazole 43 (1.00 g, 8.33 mmol) in cone, sulfuric acid (4.05 mL, 18 M, 8.8 eq) cooled to 0 °C was treated dropwise with cone, nitric acid (1.58 mL, 15.8 M, 3.0 eq) while maintaining the temperature below 20 °C. The mixture was stirred at rt for 20 min, then cooled to 0 °C and water (20 mL) was added dropwise to form a yellow precipitate. The precipitate was collected by filtration, washing with water (3 x 20 mL), and dried in a vacuum desiccator (24 h) to yield the title compound 44 as a yellow solid (1.11 g, 81%). ¹H NMR (500 MHz, DMSO-d₆): δ 8.69 (d, J = 7.2 Hz, 1H), 8.59 (d, J = 9.0 Hz, 1H), 7.85 (dd, J = 9.0, 7.3 Hz, 1H) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 150.4, 142.7, 136.4 (br), 131.8, 131.5, 124.5 ppm; HRMS (+ESI) m/z: Calc, for C₆H₃N₃O₃ [M+H]+: 166.0247, found: 166.0247.

45 - Benzo [c] [1 ,2,5]oxadiazol-4-amine

[0180] 45 was prepared from 44 (500 mg, 3.03 mmol) according to general procedure N. TLC analysis of the crude product indicated multiple UV active compounds, so flash chromatography (EtOAc/Hex, 1:4, 1% NEti) was performed to yield the title compound as yellow and orange crystals (311 mg, 76%) which did not degrade upon exposure to air, permitting characterisation. Rf= 0.72 ((EtOAc/Hex, 1:4, l% NEt3), stains brown with ninhydrin); ¹H NMR (500 MHz, DMSO-d₆): δ 7.28 (dd, J = 8.8, 7.3 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.49 (s, 1H, NH), 6.32 (d, J = 7.2 Hz, 1H) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 149.9, 144.7, 137.0, 135.2, 104.3, 99.7 ppm; LRMS (-ESI) m/z 276 ([2M-H]’, 100%).

46 - Phenyl N-(benzo[c] [l,2,5]oxadiazol-4-yl)-N’-cyanocarbamimidate

[0181] This procedure used lithium bis(trimethylsilyl)amide (LiHMDS) instead of sodium hydride as a base in an analogous procedure as outlined by general procedure C. 46 was prepared from benzoxadiazol-4-amine 45 (288 mg, 2.13 mmol), diphenyl cyanocarbonimidate (1.07 g, 4.48 mmol, 2.1 eq) and LiHMDS in THF (2.34 mL, I M, 1.1 eq) according to general procedure C. Purification by flash chromatography (2.5% v/v MeOH sat. NH₃ in CH₂CI₂) afforded the title compound as a white, crystalline solid (354 mg, 60%). mp 192-194 °C; Rf= 0.08 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, stains white with Goofy's); IR (neat, diamond cell): 3150, 3078, 3004, 2190, 1635, 1621, 1423, 658 cm’ 1H NMR (500 MHz, DMSO-d₆): δ 11.64 (s, 1H, NH), 7.99 (d, J = 9.0 Hz, 1H, H6), 7.67 (dd, J = 9.0, 7.0 Hz, 1H, H5), 7.60 (d, J = 7.0 Hz, 1H, H4), 7.50-7.40 (m, 2H, H2), 7.34- 7.24 (m, 3H, Hl, H3) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 160.3, 151.2, 149.8, 146.4, 133.0, 129.9, 126.7, 126.6, 124.4, 121.1, 114.6, 113.0 ppm; LRMS (+ESI) m/z: 302 ([M+Na]⁺, 100%); HRMS (+ESI) m/z. Calc, for C14H9N5O2 [M+Na]⁺: 302.0649, found: 302.0646.

15 - l-((Adamantan-l-yl)methyl)-3-(benzo[c] [l,2,5]oxadiazol-4-yl)-2-cyanoguanidine

[0182] 15 was prepared from 46 (175 mg, 0.63 mmol) and 1-adamantylmethylamine (139 mg, 0.85 mmol) according to general procedure D, providing the title compound as an off white solid, collected by vacuum filtration (192 mg, 88%). This was recrystallised from MeOH and EtOAc to yield the title compound as a white solid (66 mg, 30%). mp 241-245 °C; Rf= 0.5 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, stains white with Goofy's); IR (neat, diamond cell): 3365, 3163, 2902, 2846, 2177, 1608, 1545, 739, 571 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 9.64 (s, 1H, NH), 7.79 (d, J = 9.0 Hz, 1H, H7), 7.57 (dd, J = 9.0, 7.1 Hz, 1H, H6), 7.44 (s, 1H, NH), 7.35 (d, J = 7.0 Hz, 1H, H5), 3.01 (d, J = 6.2 Hz, 2H, H4), 2.01-

1.87 (m, 6H, H3), 1.73-1.57 (m, 6H, Hl) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 157.8, 150.0, 146.1, 133.4, 126.6, 122.5 (br), 116.7, 111.8, 53.0, 36.4, 34.3, 27.7 ppm; LRMS (+ESI) m/z: 313 ([M+Na]⁺, 100%); HRMS (+ESI) m/z'. Calc, for C₁₉H₂₂N₆O [M+Na]⁺: 373.1747, found: 373.1744.

48 - Benzo [d] [l,3]dioxole-4-carboxylic acid

[0183] A solution of benzodioxole-4-carbaldehyde 47 (1.00 g, 6.66 mmol), potassium carbonate (3.68 g, 26.6 mmol, 4.0 eq) and aq. hydrogen peroxide (15.0 , 30 m%L w/w) in methanol (15 mL) was stirred for 16 h at rt. The reaction mixture was partially concentrated in vacuo to remove methanol, and washed with diethyl ether (3 x 20 mL). The remaining aqueous mixture was acidified to pH 2 with aq. HC1 (14 mL, 4 M) and extracted with ethyl acetate (3 x 15 mL). The organic extracts were combined and washed with aq. HC1 (2 x 20 mL, 1 M), brine (20 mL), dried over MgSO-i. and concentrated in vacuo to yield the title compound as an off white solid (872 mg, 79%). This was recrystallised from EtOAc to yield the title compound 48 as fine white crystals (600 mg, 54%). mp 220-223 °C; Rf= 0.17 (5% v/v MeOH in CH₂CI₂, stains yellow with bromocresol green); ¹H NMR (500 MHz, DMSO- d₆): δ 12.97 (br s, 1H, OH), 7.28 (dd, J = 8.1, 1.2 Hz, 1H), 7.09 (dd, J = 7.7, 1.2 Hz, 1H),

6.87 (t, J = 7.9 Hz, 1H), 6.11 (s, 2H) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 165.2, 148.5, 148.1, 122.6, 121.2, 113.4, 112.0, 101.7 ppm; LRMS (-ESI) m/z: 165 ([M-H]-, 100%). 50 - Tert-butyl benzo [d] [1,3] dioxol-4-ylcarbamate

[0184] A solution of benzodioxole-4-carboxylic acid 48 (600 mg, 3.61 mmol) and triethylamine (731 mg, 7.22 mmol, 2.0 eq) in CH₂CI₂ (10 mL) at rt was treated with DPPA (0.70 mL, 3.25 mmol, 0.9 eq), and stirred for 2 h. The reaction mixture was concentrated in vacuo and purified via flash chromatography (1% v/v MeOH in CH₂CI₂) to afford the acyl azide as a resinous solid (662 mg, quant.). A solution of the acyl azide in tert-butanol (8.0 mL, ~24 eq) was heated at reflux for 10 h, then the excess tert-butanol was removed in vacuo to yield the title compound 50 as a clear, crystalline solid (577 mg, 70%). mp 92-94 °C; Rf= 0.85 (1% v/v MeOH in CH₂CI₂, spot turns dark brown with ninhydrin and excessive heating); ¹H NMR (500 MHz, DMSO-d₆): δ 8.79 (s, 1H, NH), 6.92 (d, J = 8.3 Hz, 1H), 6.75 (t, J = 8.0 Hz, 1H), 6.67 (dd, J = 7.8, 1.2 Hz, 1H), 5.98 (s, 2H), 1.44 (s, 9H) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 152.9, 147.7, 139.2, 121.2, 117.1, 104.5, 100.7, 79.0, 28.1 ppm; LRMS (+ESI) m/z: 260 ([M+Na]⁺, 100%).

51*HCl - Benzo [d] [1 ,3]dioxol-4-amine hydrochloride

[0185] A solution of tert-butyl carbamate 50 (280 mg, 1.18 mmol) in methanol cooled to 0 °C was treated dropwise with HCI in dioxane (2 mL, 4 M, 6 eq) to form a white precipitate. After stirring at rt for 30 mins, the precipitate was collected via filtration, washing with diethyl ether (2 x 15 mL) to afford the title compound 51 'HCI as a white solid (204 mg, quant.). Rf= 0.70 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, stains pink with Goofy's); ¹H NMR (500 MHz, DMSO-d₆): δ 6.97-6.82 (m, 3H), 6.11 (s, 2H) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 148.4, 140.0, 122.3, 116.4, 116.1, 107.2, 101.9 ppm; LRMS (+ESI) m/z- 138 ([M(free-base)+H]⁺, 100%). 56 - Phenyl N-(benzo[d][l,3]dioxol-4-yl)-N’-cyanocarbamimidate

[0186] 56 was prepared from 4-benzodioxolamine 51 (169 mg, 1.23 mmol), diphenyl cyanocarbonimidate (617 mg, 2.59 mmol, 2.1 eq) and sodium hydride (173 mg, 4.31 mmol) according to general procedure C. Purification by flash chromatography (2.5% v/v MeOH sat. NH₃ in CH₂CI₂) afforded the title compound as a white, crystalline solid (246 mg, 71%). mp 175-179 °C; R_ƒ= 0.40 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, stains yellow with Goofy's); IR (neat, diamond cell): 3149, 2890, 2191, 1604, 1419, 1166, 689 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 10.86 (s, 1H, NH), 7.44 (t, J = 7.8 Hz, 2H, H4, H6), 7.30 (t, J = 7.4 Hz, 1H, H5), 7.21 (m, J = 7.9 Hz, 2H, H2), 6.92-6.83 (m, 3H, Hl, H3), 6.09 (s, 2H, H7) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 151.3, 148.1, 141.8, 129.8, 129.4, 128.8, 126.4, 121.7, 118.3, 115.2, 107.5, 101.6 ppm; LRMS (+ESI) m/z 304 ([M+Na]⁺, 100%); HRMS (+ESI) m/z. Calc, for C15H11N3O3 [M+Na]⁺: 304.0693, found: 304.0690.

17 - l-((Adamantan-l-yl)methyl)-3-(benzo [d] [1 , 3 ]dioxol-4-yl)-2-cyanoguanidine

[0187] 17 was prepared from 56 (175 mg, 0.62 mmol) and 1-adamantylmethylamine (134 mg, 0.81 mmol) according to general procedure D, providing the title compound as a white solid, collected by vacuum fdtration (78 mg, 36%). mp 199-200 °C; P/= 0.47 (2.5% v/v MeOH sat. NH₃ in CH₂CI₂, stains white with Goofy's); IR (neat, diamond cell): 3428, 3403, 2900, 2847, 2173, 1599, 1569, 1148, 494 cm^-1; ¹H NMR (500 MHz, DMSO-d₆): δ 8.86 (s, 1H, NH), 6.88-6.77 (m, 3H, H5, H6, H7), 6.71 (d, J = 7.3, 2.1 Hz, 1H, NH), 6.03 (s, 2H, H8), 2.92 (d, J = 6.3 Hz, 2H, H4), 2.08 (s, 6H, Hl), 1.71-1.53 (m, 6H, H2), 1.45 (m, 3H, H3) ppm;¹³C NMR (126 MHz, DMSO-d₆): δ 206.0, 157.5, 147.7, 140.8, 121.1, 119.2, 119.1, 116.7, 105.8, 100.6, 52.0, 39.1, 36.0, 33.8, 30.2, 27.2 ppm; LRMS (+ESI) m/z: 375 ([M+Na]⁺, 100%); HRMS (+ESI) m/z-. Calc, for C20H24N4O2 [M+H]⁺: 353.1972, found: 353.1969. 55 - Phenyl N-(benzo[d][l,3]dioxol-5-yl)-N’-cyanocarbamimidate

[0188] 55 was prepared from 5-benzodioxolamine 54 (301 mg, 2.19 mmol), diphenyl cyanocarbonimidate (1.10 g, 4.61 mmol, 2.1 eq) and sodium hydride (307 mg, 7.68 mmol) according to general procedure C. Purification by flash chromatography (1% v/v MeOH sat. NH₃ in CH₂CI₂) yielded a white solid, which was recrystallised from EtOAc to yield the title compound as a white, crystalline solid (220 mg, 36%). mp 181-185 °C; Rf= 0.10 (1% v/v MeOH sat. NH₃ in CH₂CI₂, stains yellow with Goofy's); IR (neat, diamond cell): 3157, 2191, 1620, 1490, 1199, 1032, 787, 688 cm^-1; ¹HNMR (500 MHz, DMSO-d₆): δ 10.67 (s, 1H, NH), 7.43 (t, J = 7.7 Hz, 2H, H2), 7.31-7.23 (m, 3H, Hl, H3), 7.08 (t, J = 1.6 Hz, 1H, H6), 6.95-6.84 (m, 2H, H4, H5), 6.04 (s, 2H, H7) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 151.7, 147.3, 145.5, 130.2, 129.7, 126.1, 121.7, 120.9, 117.8, 107.9, 106.1, 101.5 ppm; LRMS (+ESI) m/z-. 304 ([M+Na]+, 100%); HRMS (+ESI) m/z: Calc, for C15H11N3O3 [M+Na]+: 304.0693, found: 304.0692.

18 - l-((Adamantan-l-yl)methyl)-3-(benzo[d] [l,3]dtoxol-5-yl)-2-cyanoguanidine

[0189] 18 was prepared from 55 (191 mg, 0.68 mmol) and 1-adamanfyhnethylamine (140 mg, 0.85 mmol) according to general procedure D, providing the title compound as a white solid, collected by vacuum filtration (214 mg, 90%). mp 254-256 °C; R_ƒ= 0.21 (2.5% v/v MeOH sat. NH₃ in CH₂C1₂); IR (neat, diamond cell): 3397, 3169, 2897, 2172, 1599, 1571, 1192, 1034, 588 cm^-1; ¹ H NMR (500 MHz, DMSO-d₆): δ 8.76 (s, 1H, NH), 6.89 (d, J = 8.2 Hz, 1H, H7), 6.81 (d, J = 2.1 Hz, 1H, H6), 6.74 (t, J = 6.3 Hz, NH), 6.67 (dd, J = 8.3, 2.1 Hz, 1H, H5), 6.02 (s, 2H, H8), 2.93 (d, J = 6.3 Hz, 2H, H4), 1.97-1.91 (m, 3H, H2), 1.71-1.55 (m, 6H, H3), 1.44 (d, J = 2.8 Hz, 6H, Hl) ppm; ¹³C NMR (126 MHz, DMSO-d₆): δ 158.9, 147.4, 144.8, 131.3, 117.7, 117.3, 108.1, 106.2, 101.3, 52.3, 39.2, 36.5, 34.4, 27.6 ppm;

LRMS (-ESI) m/z-. 351 ([M-H]’, 100%); HRMS (+ESI) m/z: Calc, for C20H24N4O2 [M+Na]⁺: 375.1791, found: 375.1790; HPLC: 95.4%, 215 (nm), 22.0 min. JSG B24 - phenyl (Z)-N-(benzo[d]thiazol-7-yl)-N’-cyanocarbamimidate

[0190] JSG_B24 was prepared from laa (283 mg, 1.88 mmol, 1.00 eq), sodium hydride (60% w/w dispersion in mineral oil, 264 mg, 6.6 mmol, 3.5 eq), and diphenyl N- cyanocarbonimidate (943 mg, 3.96 mmol, 2.1 eq) in anhydrous tetrahydrofuran (6 ) mL according to general procedure C, and then purified by flash chromatography (1% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to yield the title compound (300 mg, 54%) a white solid, mp 168 °C (decomp.) ¹H NMR (400 MHz, DMSO-d₆): δ 11.33 (s, 1H), 9.45 (s, 1H), 8.08 (dd, J = 7.6, 1.6 Hz, 1H), 7.67-7.56 (m, 2H), 7.49-7.40 (m, 2H), 7.34-7.24 (m, 3H) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 156.30, 154.28, 151.22, 131.27, 130.62, 129.80, 126.71, 126.44, 123.46, 122.07, 121.05 ppm; LRMS (+ESI) m/z: 317.06 ([M+Na]⁺, 100%).

JSG B25 - (E)-l-(((3r,5r, 7r)-adamantan-l-yl)methyl)-3-(benzo[d]thiazol-7-yl)-2- cyanoguanidine

[0191] JSG_B25 was prepared from JSG_B24 (160 mg, 0.544 mmol) and 1- adamantylmethylamine (98.8 mg, 0.598 mmol, 1.10 eq) according to general procedure D to provide the title compound (86 mg, 43%) as a white solid. ¹ H NMR (400 MHz, DMSO-d₆): δ 9.39 (d, J= 4.5 Hz, 2H), 7.98 (dd, J= 8.1, 0.9 Hz, 1H), 7.56 (t, J= 7.9 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 2.97 (d, J= 6.4 Hz, 2H), 1.95 (s, 3H), 1.77-1.54 (m, 6H), 1.48 (s, 6H) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 158.17, 156.01, 154.40, 132.36, 131.08, 126.69, 122.66, 120.87, 116.82, 61.98, 52.56, 39.62, 36.45, 34.46, 27.62 ppm; LRMS (+ESI) m/z: 388.11 ([M+Na]+, 100%). JSG B37 - (3s, 5s, 7s)-3,5, 7-trifluoroadamantane-l-carboxamide™

[0192] (3s,5s,7s)-3,5,7-trifluoroadamantane-l-carboxylic acid (2.33 g, 9.94 mmol) and 1,1'- carbonyldiimidazole (4.84 g, 29.83 mmol) were dissolved in anhydrous THF (~0.3 mmol of acid/mL) and stirred under a nitrogen atmosphere for 1 h. The solution was cooled in an ice bath, and aqueous ammonia (28% w/v, 0.2 mL per mmol of acid) was added. The reaction mixture was allowed to stir for a further 1.5 h, and THF was removed in vacuo. The crude product was dissolved in dichloromethane, washed with aqueous sodium hydroxide (1 M, 3 x 100 mL), aqueous hydrochloric acid (1 M, 2 x 150 mL), and water (2 x 150 mL). The organic fraction was dried over MgSO₄, and dichloromethane was removed by rotary evaporation to yield JSG_B37 (1.02 g, 44%) as a white solid, mp 178.5-179.5 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 7.27 (1H, br s, -NH_a), 7.10 (1H, br s, -NH_b), 2.24-1.97 (6H, m, Hl), 1.89 (6H, br s, H2) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 174.6-174.2 (m), 92.8 (dd, J = 188.8, 15.0 Hz), 46.04.7 (m), 42.8 (q, J = 10.9 Hz), 41.6-40.8 (m) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆): δ -140.2 (s) ppm; LRMS (-ESI) m/z: 232 ([M-H]-, 100%). All spectroscopic data for this compound matched those previously reported in the literature.

JSG B38 - ((3s, 5s, 7s)-3,5, 7 -trifluoroadamantan-l-yl)me thanamine hydrochloride™

[0193] JSG_B37 (1.42 g, 6.08 mmol) in anhydrous THF (0.25 mmol/mL) was treated with lithium aluminium hydride (924 mg, 24.34 mmol) at 0 °C with stirring, warmed to room temperature over 30 min, and then heated at reflux for 20 h. The reaction mixture was allowed to cool to room temperature, and then chilled water (0. 17 mL/mmol) was added dropwise, followed by aqueous sodium hydroxide (4 M, 0.17 mL/mmol), and additional water (0.5 mL/mmol). THF was added to mobilise the viscous slurry, and after stirring for 30 min, MgSO₄ was added directly to the reaction mixture. The product mixture was filtered through a Celite® plug, washing with dichloromethane, and the filtrate was reduced in vacuo to yield the amine. The amine was dissolved in diethyl ether (1 mL/mmol), and then washed with aqueous sodium hydroxide (1 M, 1 mL/mmol), water (1 mL/mmol), brine (1 mL/mmol), dried over MgSO₄ and filtered. The filtrate was cooled in an ice bath, and then HC1 in dioxane (4 M, 0.4 mL/mmol) was added dropwise with stirring to form a white precipitate. The precipitate was collected by vacuum filtration and washed with diethyl ether to afford JSG_B38 (907 mg, 58%) as a white solid, mp 270 °C (sublimation); ¹H NMR (400 MHz, DMSO-d₆): δ 8.29 (3H, br s, -NH₃ +), 2.81 (2H, d, J = 4.8 Hz, H3), 2.24-2.12 (3H, m), 2.06-1.91 (3H, m), 1.76 (6H, br s) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 92.4 (dt, J = 188.5, 15.7 Hz), 46.5 (q, J = 2.2 Hz), 45.4-44.8 (m), 41.5-41.0 (m), 35.6 (q, J = 12.0 Hz) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆): δ -140.8 ppm; LRMS (+ESI) m/z: 220 ([M+H]+, 100%). All spectroscopic data for this compound matched those previously reported in the literature.¹⁸

JSG B29 - (E)-l-(benzo[d]thiazol-7-yl)-2-cyano-3-(((3s,5s, 7s)-3,5, 7-trifluoroadamantan-l- yl)methyl)guanidine

[0194] JSG_B29 was prepared from JSG_B24 (98 mg, 0.333 mmol) and JSG_B38 (120 mg, 0.549 mmol, 1.65 eq) according to general procedure D, and then purified by flash chromatography (1-2.5% v/v MeOH sat. w/ NH₃ in CH₂CI₂) to provide the title compound (49 mg, 35%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) 8 9.56 (s, 1H), 9.42 (s, 1H), 8.02 (dd, J= 8.2, 0.9 Hz, 1H), 7.59 (t, J= 7.9 Hz, 1H), 7.36 (d, J= 7.6 Hz, 1H), 7.22 (s, 1H), 3.21 (d, J= 6.3 Hz, 2H), 2.19 - 1.98 (m, 7H), 1.61 (s, 6H) ppm; ¹³C NMR (101 MHz, DMSO-d₆): δ 158.31, 155.96, 154.51, 131.46, 126.92, 123.34, 121.37, 116.69, 49.16, 45.60- 42.24 (m) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆) 8 -140.35 ppm; LRMS (+ESI) m/z: 418.07 ([M-H]-, 100%); HPLC 95.1% (λ_max1 = 254 nm), RT: 21.3 min. JSG D06 - (E)-2-cyano-l-(l-methyl-lH-indazol-7-yl)-3-(((3s,5s, 7s)-3,5, 7-

[0195] JSG D06 was prepared from 336 (115 mg, 0.395 mmol) and JSG B38 (93.6 mg, 0.427 mmol, 1.08 eq) according to general procedure D, and then purified by flash chromatography (0.75-1.75% v/v MeOH in CH₂CI₂) to provide the title compound (48 mg, 29%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.23 (s, 1H), 8.09 (s, 1H), 7.76 (dd, .7= 8.0, 1.0 Hz, 1H), 7.25 (d, J= 7.2 Hz, 1H), 7.15 (t, J= 7.6 Hz, 1H), 6.90 (s, 1H), 5.75 (s, OH), 4.09 (s, 3H), 3.19 (d, J= 6.0 Hz, 2H), 2.24-1.84 (m, 6H), 1.56 (s, 6H) ppm; ¹³C NMR (101 MHz, DMSO-d₆) 8 160.06, 136.52, 132.66, 127.14, 126.35, 121.04, 117.06, 93.74, 93.59, 91.71, 49.05, 45.41, 42.35-37.60 (m) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆) 8 - 140.37 ppm; LRMS (+ESI) m/z: 415.19 ([M-H]-, 100%); HPLC 99.8% (λ_max1 = 254 nm), RT: 22.3 min; 99.7% (λ_max1 = 230 nm), RT: 22.4 min.

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(18) Wilkinson, S. M.; Barron, M. L.; O'Brien-Brown, J.; Janssen, B.; Stokes, L.; Werry, E. L.; Chishty, M.; Skarrat, K. K.; Ong, J. A.; Hibbs, D. E.; Vugts, D. J.; Fuller, S.; Windhorst, A. D.; Kassiou, M. Pharmacological Evaluation of Novel Bioisosteres of an Adamantanyl Benzamide P2X 7 Receptor Antagonist. ACS Chem. Neurosci. 2017, 8 (11), 2374-2380. htps : //doi . org/ 10.1021 /acschemneuro .7b00272. Industrial Applicability

[0196] As the P2X7R has been implicated in a wide range of disease states for which no good treatment currently exists (e.g., stroke, ALS, MS, Alzheimer's disease, Huntington's disease, atherosclerosis, diabetic retinopathy, dilated cardiomyopathy, ischemic injury and left ventricular hypertrophy post myocardial infarction), development of a small molecule capable of modulating the signaling pathway is of significant commercial interest. In fact, the diverse involvement of the P2X7R in disease ensures any compound developed from this invention has no shortage of options for therapeutic application.

[0197] The Inventors are confident that the inventive series of 5,6-fused heterocyclic analogues have never been explored in any previous P2X7R SAR study. This series has to date revealed valuable pharmacophoric criteria for potent P2X7R inhibition within this scaffold: the optimum position and identity of distal hydrogen bond acceptors. The activity achieved by these adamantyl heterocycles is likely to extend beyond the current cyanoguanidine linker backbone.

[0198] To the best of the Inventors' knowledge, there are no clinical trials underway investigating inhibitors of the P2X7R for any cardiovascular disease. There is one trial currently underway for Major Depressive Disorder (Inflammation) by Janssen (Phase 2 clinical trial with API JNJ-54175446).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:-

1. A compound of Formula (I) :

wherein Ri is a bicyclic fused heterocyclic moiety; and wherein R2 is H or F.

2. A compound according to claim 1, wherein the Ri moiety is a 5,6-fused heterocyclic moiety.

3. A compound according to claim 1, wherein the Ri moiety is selected from the group consisting of: benzothienyl, benzoxadiazolyl, cinnolinyl, dibenzofuranyl, furopyridinyl, furyl, imidazolyl, indazolyl, indolyl, isoxazolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyridinium N-oxide, pyrrolyl, quinolinyl, tetrazolyl, thiadiazolyl, thiazolyl, thienopyridinyl, thienyl, triazolyl, triazinyl and benzothiazolyl.

4. A compound according to claim 1, wherein the Ri moiety is selected from the group consisting of: indazolyl, benzimidazolyl, benzotriazolyl, indolyl, benzoxazolyl, benzoxadiazolyl and benzothiazolyl.

5. A compound according to claim 1, wherein the Ri moiety is selected from the group consisting of: 4-NH-l-Me-indazolyl; 4-NH-l-Me-benzimidazolyl; 4-NH-2- Me-indazolyl; 7-NH-2-Me-indazolyl; 7-NH-l-Me-indazolyl; 7-NH-l-Me- benzimidazolyl; 7-NH-l -Me -benzotriazolyl; 7-NH-l-Me-indolyl; 6-NH-l-Me- indazolyl; 6-NH-l-Me-benzimidazolyl; 6-NH-2-Me-indazolyl; 5-NH-2-Me- indazolyl; 5 -NH-1 -Me -indazolyl; 5-NH-l-Me-benzimidazolyl; 4-NH- benzoxazolyl; 5-NH-benzoxazolyl; 6-NH-benzoxazolyl; 7-NH-benzoxazolyl; 4- NH-benzoxadiazolyl; 4-NH-benzodioxolyl; 5-NH-benzodioxolyl; and 7-NH- benzothiazolyl. A compound according to claim 1, wherein the compound is selected from the group consisting of:

7. A compound according to claim 1, wherein the Ri moiety is selected from the group consisting of: 7-NH-benzoxazolyl; 4-NH-l-Me-indazolyl; 6-NH-l-Me- indazolyl; 7-NH-l-Me-indazolyl; 5-NH-2-Me-indazolyl; 6-NH-l-Me- benzimidazolyl; and 7-NH-benzothiazolyl.

8. A compound according to claim 1, wherein R2 is H.

9. A compound according to claim 1, wherein R2 is F. 10. A compound according to claim 1, wherein the compound is selected from the group consisting of:

A compound according to claim 7, wherein Ri is selected from the group consisting of: 7-NH-benzoxazolyl; 7-NH-l-Me-indazolyl; and 4-NH-2-Me-indazolyl. A compound according to claim 1, wherein the compound is selected from the group consisting of:

A compound according to claim 1, wherein Ri is 7-NH-benzoxazolyl and the compound is

A compound according to claim 1, wherein Ri is 7-NH-l-Me-indazolyl and the compound is

A compound according to claim 1, wherein Ri is 4-NH-2-Me-indazolyl and the compound is

A compound, being l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7-yl)-2- cyanoguanidine

A compound, being l-(adamantan-l-yhnethyl)-2-cyano-3-(l -methyl- lH-indazol-7- yl)guanidine

A compound, being l-((adamantan-l-yl)methyl)-2-cyano-3-(2-methyl-2H-indazol-

4-yl)guanidine

A compound, being (E)-l-(benzo[d]thiazol-7-yl)-2-cyano-3-(((3s,5s,7s)-3,5,7- trifluoroadamantan- 1 -yl)methyl)guanidine

A compound according to any one of the preceding claims, wherein the compound is a P2X7 receptor antagonist. A method for the treatment of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction, the method comprising administering to a patient in need to such treatment and effective amount of a compound as defined according to claim 1, or a pharmaceutically acceptable salt thereof. Use of a compound as defined according to claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction. A compound as defined according to claim 1, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease or condition selected from the group consisting of: stroke; amyotrophic lateral sclerosis; multiple sclerosis; Alzheimer's disease; Huntington's disease, atherosclerosis, diabetic retinopathy; dilated cardiomyopathy; ischemic injury and left ventricular hypertrophy post myocardial infarction. A method for the synthesis of a compound according to claim 1, the method comprising the steps of: a) forming adamant- 1-ylmethylamine hydrochloride wherein R2 is H or ((3s,5s,7s)-3,5,7-trifluoroadamantan-l-yl)methanamine hydrochloride wherein R2 is F; b) forming a cyanocarbamimidate by reduction of an aryl amine and subsequent reaction with diphenyl A-cyanocarbonimidate; and c) coupling the adamant- 1-ylmethylamine hydrochloride or ((3s, 5s, 7s)- 3,5,7-trifluoroadamantan-l-yl)methanamine hydrochloride to the cyanocarbamimidate . A method for the synthesis of l-((adamantan-l-yl)methyl)-3-(benzo[d]oxazol-7- yl)-2 -cyanoguanidine

the method comprising the steps of: a) reacting 2-chloro-l,3-dinitrobenzene under basic conditions to form 2,6-dinitrophenol; b) aminating the 2,6-dinitrophenol to form 2-amino-6-nitrophenol; c) treating the 2-amino-6-nitrophenol with trimethyl orthoformate to give 7 -nitrobenzo [d] oxazole; d) reducing the 7-nitrobenzo[d]oxazole to benzo [d]oxazol-7-amine; e) reacting the benzo [d]oxazol-7-amine with diphenyl cyanocarbonimidate to give phenyl N-(benzo[d]oxazol-7-yl)-N'- cyanocarbamimidate; and f) coupling the phenyl N-(benzo[d]oxazol-7-yl)-N'- cyanocarbamimidate with 1-adamantyhnethylamine to give l-((adamantan-l- yl)methyl)-3 -(benzo [d] oxazol-7 -yl) -2-cy anoguanidine . A method for the synthesis of l-(adamantan-l-ylmethyl)-2-cyano-3-(1-methyl-lH- indazol-7 -yl)guanidine

the method comprising the steps of: a) methylating 7-nitroindazole to give 1-methyl-7-nitro-lH-indazole; b) reducing the 1-methyl-7-nitro-lH-indazole to provide 1-methyl-lH- indazol-7-amine; c) treating the 1-methyl-lH-indazol-7-amine with diphenyl N- cyanocarbonimidate to provide phenyl N'-cyano-N-(1-methyl-lH-indazol-7- yl)carbamimidate; and d) coupling the phenyl N'-cyano-N-(1-methyl-lH-indazol-7- yl)carbamimidate with 1-adamantyhnethylamine to give l-(adamantan-l- ylmethyl)-2-cyano-3 -( 1 -methyl- lH-indazol-7 -yl)guanidine . A method for the synthesis of l-((adamantan-l-yl)methyl)-2-cyano-3 -(2 -methyl - 2H-indazol-4-yl)guanidine

the method comprising the steps of: a) preparing 2-methyl-4-nitro-2H-indazole by methylating 4- nitroindazole; b) reducing the 2-methyl-4-nitro-2H-indazole to provide 2-methyl-2H- indazol -4 -amine ; c) reacting the 2-methyl-2H-indazol-4-amine with diphenyl N- cyanocarbonimidate to provide phenyl N'-cyano-N-(2-methyl-2H-indazol-4- yl)carbamimidate; and d) coupling the phenyl N'-cyano-N-(2-methyl-2H-indazol-4- yl)carbamimidate with 1-adamantyhnethylamine to provide l-((Adamantan-l- yl)methyl)-2-cyano-3 -(2 -methyl -2H-indazol-4-yl)guanidine .