6-Substituted derivatives of hexamethylene amiloride as inhibitors of uPA and uses thereof

Field of the Invention

The present invention broadly relates to 6-substituted derivatives of hexamethylene amiloride, the preparation thereof, and their use in the treatment of diseases such as cancer.

Background of the Invention

Cancer continues to claim many lives annually throughout the world. Whilst there have been many significant breakthroughs made in the treatment and prevention of a wide variety of cancers, there remains a need for new and improved therapeutics and treatment strategies to more effectively target the disease, increase survival rates in aggressive neoplastic disease and reduce the incidence of harmful side effects.

The plasminogen activation system (PAS) comprises the serine protease urokinase plasminogen activator (uPA), its cognate cell surface receptor (uPAR) and two endogenous serpin inhibitors; plasminogen activator inhibitor 1 (PAI-1 ) and PAI-2. Activation of zymogen pro-uPA to uPA is facilitated upon binding to uPAR, which serves to cleave cell surface-associated plasminogen generating the broad-spectrum serine protease plasmin. Plasmin activates a downstream cascade of extracellular proteases (e.g. matrix metalloproteinases) and latent growth factors and together these provide controlled pericellular proteolysis and directional remodeling of the local extracellular environment. The PAS plays a central role in many cell migration and invasion processes and, not surprisingly, its upregulation is heavily implicated in tumour metastasis and invasive disease. To highlight this, upregulated uPA is one of the strongest prognostic biomarkers of shortened disease-free survival and overall survival in breast cancer and one of the most accurate predictors of metastasis in lymph-node- negative breast tumours.

In view of the above, it is apparent that inhibition of uPA may provide an effective anticancer therapy. Against this background, the present inventors have discovered that selected 6-substituted hexamethylene amiloride derivatives are potent inhibitors of uPA, thereby offering a new therapeutic option for treating cancer. Furthermore, these compounds may also be useful in the treatment of any disease or condition associated with uPA. Summary of the Invention

In a first aspect the present invention provides a compound of the general formula (I):

or a pharmaceutically acceptable salt, hydrate, derivative, solvate, tautomer or prodrug thereof, wherein:

R is -T-Ft _! ;

T is absent or is selected from the group consisting of: -NH-, -S-, -0-, -CH ₂ -, -S(=0)-, - S(=0) ₂ -, -NH-S(=0) ₂ -, -S(=0) ₂ NH-, -C(=0)-, -C(=0)-NH, -NH-C(=0)-;

Ri is selected from the group consisting of: an aryl group, a heteroaryl group, a carbocyclyl group and a heterocycyl group, each of which may be optionally substituted, and with the proviso that 3-amino-5-(azepan-1 -yl)-A/-carbamimidoyl-6-phenylpyrazine-2- carboxamide is excluded.

In a second aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) according to the first aspect together with a pharmaceutically acceptable carrier, diluent or excipient.

In a third aspect the present invention provides a method for the treatment of a disease or condition associated with uPA in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.

The disease or condition may be associated with upregulation or increased expression of uPA.

The disease or condition may be cancer.

The cancer may be locally advanced.

The cancer may be metastatic.

The cancer may be, for example, breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.

The disease or condition may be an inflammatory disease or condition.

The inflammatory disease or condition may be, for example, chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.

The disease or condition may be amyotrophic lateral sclerosis.

The disease or condition may be a wound.

The wound may be a chronic wound. In one embodiment the wound is a dermal ulcer.

In a fourth aspect the present invention provides a method for preventing or slowing cancer spread in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.

The cancer may be associated with uPA.

The cancer may be associated with upregulation or increased expression of uPA.

The cancer may be localised.

The cancer may be locally advanced.

The cancer may be metastatic.

In a fifth aspect the present invention provides a method for inducing apoptosis in a cancer cell, the method comprising contacting the cancer cell with an effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.

In a sixth aspect the present invention provides a method for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence, the method comprising administration to the subject of an effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.

The subject deemed to be at risk of cancer recurrence may be a subject who is in cancer remission.

In a seventh aspect the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for the treatment of a disease or condition associated with uPA.

The disease or condition may be associated with upregulation or increased expression of uPA.

The disease or condition may be cancer.

The cancer may be locally advanced.

The cancer may be metastatic. The cancer may be, for example, breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.

The disease or condition may be an inflammatory disease or condition.

The inflammatory disease or condition may be, for example, chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.

The disease or condition may be amyotrophic lateral sclerosis.

The disease or condition may be a wound.

The wound may be a chronic wound. In one embodiment the wound is a dermal ulcer.

In an eighth aspect the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for preventing or slowing cancer spread.

The cancer may be associated with uPA.

The cancer may be associated with upregulation or increased expression of uPA.

The cancer may be localised.

The cancer may be locally advanced.

The cancer may be metastatic.

In a ninth aspect the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for inducing apoptosis in a cancer cell.

In a tenth aspect the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence.

In an eleventh aspect the present invention provides a method for inhibiting uPA in a subject, the method comprising administration to the subject of a compound of formula (I) according to the first aspect, or a composition of the second aspect.

In a twelfth aspect the present invention provides use of a compound of formula (I) according to the first aspect for inhibiting uPA.

In a thirteenth aspect the present invention provides a method for preparing a compound of the formula (I) as defined in the first aspect comprising the steps of: (i) reacting a compound of the following formula (IV):

wherein X and Y are halogens and may be the same or different, with hexamethyleneimine to give a compound of the following formula (III):

wherein Y is a halogen;

(ii) reacting a compound of the formula (III) with a boronic acid of the following formula,

HO .g .OH

R , or a pinacol ester or salt thereof,

wherein R is as defined in the first aspect, to give a compound of formula (II):

wherein R is as defined in the first aspect;

(iii) reacting a compound of the formula (II) with guanidine to provide a compound of formula (I).

In a fourteenth aspect the present invention provides a compound of the general formula (II):

(ii) or a pharmaceutically acceptable salt, hydrate, derivative, solvate or prodrug thereof, wherein R is as defined in the first aspect and R ₂ is C C ₂₀ alkyl, with the proviso that methyl 3-amino-5-(azepan-1 -yl)-6-phenylpyrazine-2-carboxylate is excluded

In one embodiment, R ₂ is C C ₁₀ alkyl.

In another embodiment, R ₂ is C C ₆ alkyl.

In a further embodiment, R ₂ is C C ₃ alkyl.

In another embodiment R ₂ is methyl.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of this specification, the term "alkyl" is taken to mean straight chain or branched chain monovalent saturated hydrocarbon groups having the recited number of carbon atoms, such as methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert- butyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 -dimethylpropyl, pentyl, isopentyl, hexyl and the like.

In the context of this specification, the terms "halo" and "halogen" are synonomous and refer to fluoro, chloro, bromo and iodo.

In the context of this specification, the term "haloCrCe alkyl" is taken to mean straight chain or branched chain monovalent saturated hydrocarbon groups having between 1 and 6 carbon atoms wherein one or more hydrogens are replaced by a halogen.

In the context of this specification, the term "C ₂ -C ₆ alkenyl" is taken to mean straight chain or branched chain monovalent hydrocarbon radicals having between 2 and 6 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenyl, 2- methyl-2-propenyl, butenyl and the like. The group may contain a plurality of double bonds and the geometry about each double bond is independently cis or trans, E or Z.

In the context of this specification, the term "C ₂ -C ₆ alkynyl" is taken to mean straight chain or branched chain monovalent hydrocarbon radicals having between 2 and 6 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, propargyl and the like. The group may contain a plurality of triple bonds.

In the context of this specification, the term "aryl group" is taken to mean monovalent aromatic hydrocarbon groups having between 6 and 20 ring carbon atoms, or between 6 and 14 ring carbon atoms, or between 6 and 10 ring carbon atoms. The aryl group may have a single ring or multiple rings. "Aryl" also includes bicyclic radicals comprising an aromatic ring fused to a saturated or partially unsaturated carbocyclic ring. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, phenanthrenyl, azulenyl, anthracenyl, 1 ,2-dihydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl and the like.

In the context of this specification, the term "heteroaryl group" is taken to mean monovalent aromatic groups having between 5 and 20 ring atoms, or between 5 and 14 ring atoms, or between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S, the remaining ring atoms being carbon. The heteroaryl group may have a single ring or multiple rings. "Heteroaryl" also includes bicyclic radicals comprising a heteroaromatic ring fused to a saturated, partially unsaturated ring or aromatic carbocyclic ring. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazolyl, triazolyl, pyrimidinyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, benzothienyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl and the like.

In the context of this specification, the term "carbocyclyl group" is taken to mean monovalent, non-aromatic, saturated or partially unsaturated carbocyclic rings having between 3 and 12 carbon atoms, or between 5 and 10 carbon atoms. The carbocyclyl ring may be monocyclic or bicyclic. Bicyclic carbocycles may be arranged, for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6], or as bridged systems, such as bicyclo[2.2.1 ]heptane. Examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent-1 -enyl, 1 -cyclopent-3-enyl, 1 -cyclohex- 2-enyl, cyclohexadienyl, cyclooctyl, cyclodecyl and the like. In the context of this specification, the term "heterocyclyl group" is taken to mean monovalent, non-aromatic, saturated or partially unsaturated rings having between 3 and 12 ring atoms, or between 5 and 10 ring atoms, in which one or more of the ring atoms is O, N or S, the remaining ring atoms being carbon. The heterocyclyl ring may be monocyclic or bicyclic. Examples of heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, tetrahydropyrimidinyl, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3- dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl and the like.

In the context of this specification, the term "tautomer" refers to structural isomers of different energies which are interconvertible via a low energy barrier.

In the context of this specification, the term "optionally substituted" is taken to mean that the group to which it refers may or may not be further substituted with one or more non- hydrogen groups. Examples of optional substituents include: Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, SH, SC C ₆ alkyl, SC ₂ -C ₆ alkenyl, SC ₂ -C ₆ alkynyl, halo, =0, OH, halod- C ₆ alkyl, OC C ₆ alkyl, OC ₂ -C ₆ alkenyl, OC ₂ -C ₆ alkynyl, methylenedioxy, ethylenedioxy, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, (CH ₂ ) ₃ -morpholinyl, NH ₂ , -NHCrC ₆ alkyl, NH(CH ₂ )OH, NH(CH ₂ ) ₂ OH, N(C C ₆ alkyl) ₂ , -NHS0 ₂ C C ₆ alkyl, -S0 ₂ NHC C ₆ alkyl, C(0)NH ₂ , NHC(0)Ci-C ₆ alkyl and C(0)NHCi-C ₆ alkyl, including any and all combinations thereof.

In the context of this specification, the term "prodrug" means a compound which is able to be converted in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of the formula (I).

In the context of this specification, the term "cancer" refers to a physiological condition characterised by unregulated cell growth.

In the context of this specification, the term "effective amount" includes a non-toxic but sufficient amount of an active compound to provide the stated effect. When used in reference to cancer recurrence "effective amount" means an amount of a compound of formula (I) that is required to reduce the incidence of, or risk of an individual experiencing cancer recurrence. Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact "effective amount". However, for any given case an appropriate "effective amount" may be determined by one of ordinary skill in the art.

In the context of this specification, the term "therapeutically effective amount" includes a non-toxic but sufficient amount of an active compound to provide the desired therapeutic effect. Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact "therapeutically effective amount". However, for any given case an appropriate "therapeutically effective amount" may be determined by one of ordinary skill in the art.

In the context of this specification, the terms "treating" and "treatment" refer to any and all uses, which remedy the stated disease or symptoms thereof, hinder, retard or otherwise reverse the progression of the disease or other undesirable symptoms in any way whatsoever. Thus, the terms "treating" and "treatment" are to be considered in their broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery.

In the context of this specification the term "locally advanced" is synonomous with the term "locally invasive" and means a cancer that has spread from where it originated to nearby tissues or lymph nodes, but which has not metastasized.

In the context of this specification the term "recurrence" as it relates to cancer is understood to mean the return of cancerous cells and/or a cancerous tumour after cancerous cells and/or a cancerous tumour have been successfully treated previously.

In the context of this specification the term "associated with" when used in the context of a disease or condition "associated with" uPA means that the disease or condition, or a symptom thereof, may result from, result in, be characterized by, or otherwise related to uPA. Thus, the association between the disease or condition and uPA activity may be direct or indirect and may be temporally separated.

In the context of this specification, the term "subject" includes human and also non- human animals. As such, in addition to being useful in the treatment of diseases such as cancer in humans, the compounds of the present invention also find use in the treatment of diseases in non-human animals, for example mammals such as companion animals and farm animals. Non-limiting examples of companion animals and farm animals include dogs, cats, horses, cows, sheep and pigs. Preferably, the subject is a human.

In the context of this specification the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism by any appropriate means.

Brief Description of the Drawings

Figure 1 : Inhibition of low molecular weight (LMW) human uPA proteolytic activity by compounds 19 and 26 as determined by fluorometric enzyme inhibition assay. LMW active human uPA (0.75 nM) was added to drug dilutions or vehicle control containing Urokinase III Fluorescent substrate (250 μΜ) in assay buffer (20 mM HEPES (pH 7.6), 100 mM NaCI, 0.5 mM EDTA, 0.01 % v/v Tween-20) in a final volume of 200 μΙ and 1 % v/v DMSO. Reads were conducted every minute at 37°C for 45 min, ex. 355 nn, em. 460 nm. Data points represent mean ± SEM (n = 3). Blank subtracted data from the linear portion of the reaction progress curve was entered into Graphpad PRISM v.6.0 and data fitted with the log inhibitor vs normalized response - variable slope algorithm to calculate IC ₅₀ values. All data was normalized to the uninhibited vehicle control (100% activity) included in each assay.

Figure 2: Dose-dependent effects of compounds of the formula (I) on cell viability and LDH release in U-937 Human histiocytic lymphoma cells. Panels A) and B) show results from a CellTitre 96® Aqueous One Solution Cell Proliferation Assay experiment where colour intensity (490 nm) is indicative of cell metabolism. Cells were harvested 48 h post-passage, plated at a density of 5000 cells/well and incubated at 37 °C, 5% C0 ₂ for 18h prior to drug treatment. Vehicle (DMSO) was present at concentration of 1 %. Cells were cultured for 48 h post-drug treatment, followed by 2 h incubation with MTS reagent and reading at 490 nm. Panels C) and D) show results from assays using the Roche Cytotoxicity Detection Kit ^(PLUS) . Abs ₄₉₀ corresponds to degree of cytotoxic cell death in response to drug treatment. Cells were harvested and subsequently treated for 15 min with 5 μΙ lysis Buffer (Lysis control) followed by 10 min incubation with reagent mixture and subsequent reading at 490 nm. Bars represent mean (n = 4, ± SEM). All assays were conducted using a Molecular Devices SpectraMax Plus 384-well plate reader and data analyzed using GraphPad PRISM v6.0.

Figure 3: Dose-dependent caspase 3/7 activation in HEK-293 cells treated with compound 19 or hexamethylene amiloride (HMA). Cells were plated at a density of 2.0x10 ⁴ cells/well in a black 96-well cell culture plate and incubated for 18 h at 37° C, 5% C0 ₂ . Cells were treated with compounds or DMSO (1 % v/v) for 5 h, shaken for 1 h at 500 rpm via a motorized plate shaker and incubated for 2 h at RT prior to addition of a caspase 3/7 fluorogenic substrate in lysis buffer. After incubation at RT for 3h the plate was read using a BMG Labtech POLARstar OMEGA Fluorescence Plate reader, ex. 485 nm, em 520 nm. Bars = mean ± SEM (n = 3).

Figure 4: Evaluation of the selectivity of compound 19 for a variety of trypsin-like serine proteases via a chromogenic enzyme activity assay. Enzymes were present at a concentration of 10 nM thrombin, trypsin, plasmin and t-PA, uPA. The substrate, S- 2288 was present at a concentration of 250 μΜ. All components were present in assay buffer (10 mM HEPES, 150 mM NaCI, 0.01 % Tween-20, pH 7.4) containing a final volume of 1 % DMSO. Change in absorbance overtime at 405 nm was measured at 37 °C using a Molecular Devices SpectraMax Plus 384-weil plate reader. IC ₅₀ values were determined by plotting the percentage of residual activity (V0) versus log drug concentration and fitted to a sigmoidal dose response curve using GraphPad Prism v.6.0.

Figure 5: ENaC inhibition by compounds of the formula (I) in vitro. Experiments were conducted on HEK-293 cells expressing the human α, β and γ ENaC subunits. All cells were stimulated with the ENaC agonist S3969. Values were normalized to uninhibited and benzamil treated (ENaC antagonist) controls. Bars = mean (n = 4) ± SD.

Figure 6: Effects of Amiloride, HMA, 19 and 24 on diuresis and urinary K7Na ⁺ in male acetazolamide-treated Sprague Dawley rats. Excepting the vehicle control cohort, all animals received 25 mg/kg acetazolamide IP followed by 1 .5 mg/kg amiloride, HMA or 6-HMA analogues treatment IV, all delivered at 1 ml/kg. Following administration animals were housed in metabolic cages and urine collected for urinalysis over 6 h.

Figure 7: Inhibition of MDA-MB-231 cell migration by HMA and compounds 19 and 24. Degree of migration was determined through measuring the % confluency of the masked scratch wound over time. Data points represent mean ± SD (n = 3). Migration into the scratch wound was significantly inhibited by HMA, 19 or 24 at 5 μΜ relative to vehicle control treated cells (0.2% v/v DMSO), p = 0.0206, one-way unpaired parametric ANOVA.

Figure 8: Representative images showing migration of SKOV-3 human ovarian carcinoma cells into collagen I matrix contracted by human-skin derived telomerase- immortalized fibroblasts. Cells were allowed to invade for 21 days prior to formalin fixing. Cytokeratin stained plugs were imaged using brightfield microscopy with 10 images collected across three plugs for each treatment group. Compound or vehicle containing media was refreshed every 72 h. Images for each treatment group were obtained from different replicate plugs that received identical drug or vehicle treatments. All drugs were present at 2 μΜ. Figure 9: Inhibition of SKOV-3 ovarian adenocarcinoma cell invasion by compounds of formula (I). Collagen I plugs contracted by human skin derived telomerase-immortalized fibroblasts were seeded with SKOV-3 cells at a density of 1 .875x10 ⁵ /plug and allowed to invade for 21 days prior to fixing. Stained plug sections were imaged and images quantified for invasion index (invasion index % = (# epithelial cells invaded into collagen plug/ # epithelial cells forming contiguous monolayer along top of the plug) x100). Data points represent invasive indices from individual images, central line = mean, bars = ± SEM.

Figure 10: Dose-tolerability of HMA, 19 and 24 in AlbPLGI humanized plasminogen mice. Compounds were administered at a dose of 10 mg/kg/day as single 100 μΙ_ i.p. injections in 20% v/v DMSO/saline over a course of 10 days. Cohorts consisted of four age matched female mice. Mice were monitored twice daily for signs of moribundity. Data points = mean ± SEM (n = 4). No acute adverse responses were observed over the course of the study.

Figure 11 : Effects of daily intraperitoneal injection (7.5 mg/kg) of Amiloride, 19 and 24 on experimental lung metastases in a fibrosarcoma mouse xenograft model over 21 days. Bars = mean ± SEM (n = 6), except sham control (n = 1 ).

Figure 12: X-ray co-crystal structures of compounds 19 and 24 bound to the active site of human uPA. A) 1 .6 A structure of 19 and B) 3.0 A structure of 24 respectively bound to the serine protease domain of uPA. Electron density maps (2mF ₀ -DF _c, blue wirebasket, contoured at 1.5 σ) showing fit of ligand. Ligand, Ser146 and the amino acid side chains within 6 A of the inhibitors are presented as cylinders. H-bonds are represented as dashed lines. Electrostatic potential surfaces of the protein are shown with blue = negative, red = positive. Bridging water molecules are represented as red ball. All non-bridging solvent molecules are omitted for clarity.

Detailed Description of the Invention

In one aspect the present invention provides a compound of the general formula (I)

(I)

or a pharmaceutically acceptable salt, hydrate, derivative, solvate, tautomer or prodrug thereof, wherein: R is -T-Ft _! ;

T is absent or is selected from the group consisting of: -NH-, -S-, -0-, -CH ₂ -, -S(=0)-, - S(=0) ₂ -, -NH-S(=0) ₂ -, -S(=0) ₂ NH-, -C(=0)-, -C(=0)-NH, -NH-C(=0)-;

Ri is selected from the group consisting of: an aryl group, a heteroaryl group, a carbocyclyl group and a heterocycyl group, each of which may be optionally substituted, and with the proviso that 3-amino-5-(azepan-1 -yl)-A/-carbamimidoyl-6-phenylpyrazine-2- carboxamide is excluded.

In one embodiment T is absent.

In another embodiment the aryl group may comprise between 6 and 14 ring carbon atoms, the heteroaryl group comprises between 5 and 14 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon , the carbocyclyl group comprises between 3 and 12 ring carbon atoms and the heterocycyl group comprises between 3 and 12 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon, and wherein each of the groups may be optionally substituted.

In still a further embodiment the aryl group may comprise between 6 and 10 ring carbon atoms, the heteroaryl group comprises between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon , the carbocyclyl group comprises between 5 and 10 ring carbon atoms and the heterocycyl group comprises between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon, and wherein each of the groups may be optionally substituted.

In yet another embodiment the aryl group may be phenyl or naphthyl, each of which may be optionally substituted.

In a further embodiment the aryl group may be optionally substituted with one or more substituents independently selected from: d-C ₆ alkyl, SH, SCi-C ₆ alkyl, halo, OH, haloCi-C ₆ alkyl, OCi-C ₆ alkyl, methylenedioxy, ethylenedioxy, CH ₂ -morpholinyl, (CH ₂ ) ₂ - morpholinyl, (CH ₂ ) ₃ -morpholinyl, NH ₂ , NHC C ₆ alkyl, NH(CH ₂ )OH, NH(CH ₂ ) ₂ OH, N(C C ₆ alkyl) ₂ , -NHS0 ₂ C C ₆ alkyl, -S0 ₂ NHC C ₆ alkyl, C(0)NH ₂ , NHC(0)C C ₆ alkyl and C(0)NHC C ₆ alkyl.

In another embodiment the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SC C ₆ alkyl, haloC C ₆ alkyl, OC C ₆ alkyl, halo, methylenedioxy, C C ₆ alkyl, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, (CH ₂ ) ₃ - morpholinyl, NHC C ₆ alkyl, NH ₂ , NH(CH ₂ )OH and NH(CH ₂ ) ₂ OH. In still a further embodiment the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, halomethyl, OMe, halo, methylenedioxy, (CH ₂ ) ₂ -morpholinyl, methyl, NHC C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In yet another embodiment the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, CF ₃ , OMe, halo, methylenedioxy and methyl.

In still a further embodiment the aryl group may be optionally substituted with 1 or 2 substituents independently selected from: SMe, CF ₃ , OMe, methylenedioxy and methyl.

In a further embodiment the carbocyclyl group may be cyclopentyl or cyclohexyl, each of which may be optionally substituted.

In another embodiment the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, Sd-C ₆ alkyl, haloCi-C ₆ alkyl, OC C ₆ alkyl, halo, methylenedioxy, Ci-C ₆ alkyl, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, (CH ₂ ) ₃ - morpholinyl, NHC C ₆ alkyl, NH ₂ , NH(CH ₂ )OH and NH(CH ₂ ) ₂ OH.

In yet another embodiment the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, halomethyl, OMe, halo, methylenedioxy, (CH ₂ ) ₂ -morpholinyl, methyl, NHCi-C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In still a further embodiment the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, CF ₃ , OMe, halo, methylenedioxy and methyl.

In another embodiment the carbocyclyl group may be optionally substituted with 1 or 2 substituents independently selected from: carbonyl, SMe, CF ₃ , OMe, methylenedioxy and methyl.

In a further embodiment the heteroaryl group may comprise between 5 and 10 ring atoms in which between 1 and 4 of the ring atoms is O, N or S and the remaining ring atoms are carbon.

In yet another embodiment the heteroaryl group may comprise between 5 and 10 ring atoms in which 1 or 2 of the ring atoms is O, N or S and the remaining ring atoms are carbon.

The heteroaryl group may be selected from the group consisting of:

each of which may be optionally substituted.

In the above embodiment, in the case of a bicyclic substituent it is to be understood that the substituent may be attached to the rest of the molecule via either ring.

In another embodiment the heteroaryl group may be selected from the group consisting of:

each of which may be optionally substituted.

In the above embodiment, in the case of a bicyclic substituent it is to be understood that the substituent may be attached to the rest of the molecule via either ring.

In still a further embodiment the heteroaryl group may be selected from the group consisting of:

each of which may be optionally substituted.

In the above embodiment, in the case of a bicyclic substituent it is to be understood that the substituent may be attached to the rest of the molecule via either ring.

In yet another embodiment the heteroaryl group may be selected from the group consisting of:

each of which may be optionally substituted.

In the above embodiment, in the case of a bicyclic substituent it is to be understood that the substituent may be attached to the rest of the molecule via either ring.

In still a further embodiment the heteroaryl group may be selected from the group consisting of:

each of which may be optionally substituted.

In a further embodiment the heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: Sd-C ₆ alkyl, haloCi-C ₆ alkyl, OCi-C ₆ alkyl, halo, methylenedioxy, C C ₆ alkyl, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, (CH ₂ ) ₃ - morpholinyl, NHC C ₆ alkyl, NH ₂ , NH(CH ₂ )OH and NH(CH ₂ ) ₂ OH.

In yet another embodiment the heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, halomethyl, OMe, halo, methylenedioxy, (CH ₂ ) ₂ -morpholinyl, methyl, NHC ₁ -C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In still a further embodiment the heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: halomethyl, OMe, halo, (CH ₂ ) ₂ - morpholinyl, methyl, NHC ₁ -C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In a further embodiment the heteroaryl group may be optionally substituted with 1 or 2 substituents independently selected from: halomethyl, OMe, halo, (CH ₂ ) ₂ -morpholinyl, methyl, NHC ₁ -C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In another embodiment the heterocycyl group may comprise 6 ring atoms in which one or more of the ring atoms is N and the remaining ring atoms are carbon, and wherein the heterocycyl group is optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, halomethyl, OMe, halo, methylenedioxy, (CH ₂ ) ₂ - morpholinyl, methyl, NHCi-C ₃ alkyl, NH ₂ and NH(CH ₂ ) ₂ OH.

In still a further embodiment the heterocycyl group may comprise 6 ring atoms in which 1 or 2 of the ring atoms is N and the remaining ring atoms are carbon, and wherein the heterocycyl group is optionally substituted with 1 or 2 substituents independently selected from : carbonyl, OMe, halo and methyl.

In another embodiment the heterocycyl group is:

In one embodiment Ri is an aryl group, a heteroaryl group or a heterocyclyl group, each of which may be optionally substituted. In this embodiment the aryl group, heteroaryl group and heterocyclyl group may be as defined in any of the foregoing embodiments and combinations thereof.

In another embodiment R is a heteroaryl group or a heterocyclyl group, each of which may be optionally substituted. In this embodiment the heteroaryl group and the heterocyclyl group may be as defined in any of the foregoing embodiments and combinations thereof.

In another embodiment R is a heteroaryl group, a heterocyclyl group or an aryl group, each of which may be optionally substituted. In this embodiment the heteroaryl group, the heterocyclyl group and the aryl group may be as defined in any of the foregoing embodiments and combinations thereof.

In still a further embodiment Ri is a heteroaryl group. In this embodiment the heteroaryl group may be as defined in any of the foregoing embodiments and combinations thereof.

In another embodiment Ri may be selected from :

each of which may be optionally substituted with one or more substitutents selected from the group consisting of: SC C ₆ alkyl, haloC C ₆ alkyl, OC C ₆ alkyl, halo, methylenedioxy, C C ₆ alkyl, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, (CH ₂ ) ₃ -morpholinyl, NHCi-C ₆ alkyl, NH ₂ , NH(CH ₂ )OH and NH(CH ₂ ) ₂ OH.

In a further embodiment Ri may be selected from:

each of which may be optionally substituted with one or more substitutents selected from the group consisting of: SMe, halomethyl, OMe, halo, methylenedioxy, C C ₆ alkyl, CH ₂ -morpholinyl, (CH ₂ ) ₂ -morpholinyl, NHCi-C ₆ alkyl, NH ₂ , NH(CH ₂ )OH and NH(CH ₂ ) ₂ OH.

Exemplary compounds of the formula (I) include:

21

The present invention further extends to compounds of formula (II), which are intermediates in the synthesis of the compounds of formula (I). Accordingly, in another aspect the present invention provides compounds of the formula (II):

or a pharmaceutically acceptable salt, hydrate, derivative, solvate or prodrug thereof, wherein R is as defined in the first aspect and R ₂ is Ci-C ₂₀ alkyl, with the proviso that methyl 3-amino-5-(azepan-1 -yl)-6-phenylpyrazine-2-carboxylate is excluded. In some embodiments R ₂ is C C ₁₀ alkyl. In other embodiments R ₂ is C C ₆ alkyl. In further embodiments R ₂ is C C ₃ alkyl. In other embodiments R ₂ is methyl or ethyl. In a further embodiment R ₂ is methyl.

Exemplary compounds of the formula (II) include: 

Compounds of the formula (I) and (II) are also taken to include hydrates and solvates. Solvates are complexes formed by association of molecules of a solvent with a compound of the formula (I) or (II). Examples of solvents that are capable of forming solvates include, but are not limited to water, isopropanol, ethanol, methanol, DMSO, ethyl acetate and ethanolamine.

In the case of compounds of the formula (I) and (II) that are solids, it will be understood by those skilled in the art that such compounds may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention.

The compounds of formula (I) and (II) may be in the form of pharmaceutically acceptable salts. Such salts are well known to those skilled in the art. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1 - 19. Pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of compounds of the formula (I) or (II), or separately by reacting the free base compound with a suitable organic acid. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, fumaric, maleic, pyruvic, alkyl sulfonic, arylsulfonic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, pantothenic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine and morpholine. Alternatively, organic salts made from Λ/,/V-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine), procaine, ammonium salts, quaternary salts such as tetramethylammonium salt, amino acid addition salts such as salts with glycine and arginine.

The compounds of formula (I) may also exist in different tautomeric forms, and all such forms are within the scope of the invention.

The compounds of formula (I) also extend to include all derivatives with physiologically cleavable leaving groups that can be cleaved in vivo to provide the compounds of the formula (I).

The compounds of formula (I) also extend to include prodrugs thereof. An exemplary prodrug of a compound formula (I) is the following compound:

(compound 30)

This compound includes an amino-1 ,2,4-oxadiazolyl group at the 2-position of the pyrazine ring which can be converted in vivo to a guanidino group, thereby liberating compound 19.

Compounds of the formula (I) may be synthesised from a commercially available starting material (methyl 3-amino-5,6-dichloropyrazine-2-carboxylate), as described in Scheme 1 below. In the first step of the synthesis, methyl 3-amino-5,6-dichloropyrazine-2- carboxylate (IVa) is reacted with hexamethyleneimine to provide a common 5-(azepan- 1 -yl) methyl ester intermediate (Ilia). This common intermediate is then subjected to Suzuki-Miyaura cross-coupling reactions with a suitably functionalised boronic acid or a pinacol ester or salt thereof, to afford 6-substituted methyl ester precursors (lla), which are then reacted with guanidine using either Methods A or B to provide compounds of the formula (I). Arylboronic acid,

Method A:

Guanidine HCf, / ^' -PrOH, Na

NH

i ^" NH,

Scheme 1 : Preparation of compounds of the formula (I).

The present inventors have discovered that compounds of the formula (I) are potent inhibitors of uPA. By virtue of this inhibitory activity, compounds of the formula (I) are useful in the treatment of diseases and conditions associated with uPA. Typically, the diseases or conditions are associated with upregulation or increased expression of uPA.

In some embodiments the disease or condition is cancer. The cancer may be, for example, breast cancer, lung cancer (NSCLC and SCLC), prostate cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, ovarian cancer, renal cancer, glioblastoma, cervical cancer, testicular cancer, lip cancer, tongue cancer, urinary tract cancer, laryngeal cancer, esophageal cancer, bone cancer, uterine cancer, brain cancer (including, for example, glioma and glioblastoma), skin cancer, thyroid cancer, bladder cancer, colorectal cancer, gastric cancer, liver cancer, head and neck cancer, cancer of the central nervous system, melanoma, lymphoma, follicular carcinoma, seminoma, neuroblastoma or mesothelioma. In particular embodiments, the cancer is breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.

The compounds of formula (I) may be used in conjunction with, or alternatively in the absence of, other chemotherapeutic agents.

u PA is a determinant of cell invasiveness and metastatic potential and is overexpressed in a variety of aggressive malignancies. The compounds of formula (I) inhibit uPA through the reversible competitive inhibition of the uPA active site. The 6-substitution increases the affinity of binding with uPA through the formation of additional favourable non-specific interactions between the inhibitor and the enzyme relative to HMA (as determined by X-ray crystallography herein). The compounds of formula (I) show a high therapeutic window between their uPA inhibitory potencies (IC ₅₀ 10's-100's nM) and their cytotoxic potencies (IC ₅₀ 10's-100 μΜ) and therefore find particular use in the treatment of metastatic cancers. In this regard, the compounds of formula (I) inhibited the migration and invasion of highly metastatic breast cancer cells in two dimensional in vitro and three dimensional ex vivo assays, demonstrating their ability as antimetastatic agents.

Compounds of the formula (I) may therefore also find use in preventing or slowing cancer spread, and in particular, slowing the spread of metastatic cancer. Compounds of the formula (I) may also find use in reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence.

In other embodiments the disease or condition may be an inflammatory disease or condition, such as for example chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.

In further embodiments the disease or condition may be amyotrophic lateral sclerosis.

In other embodiments the disease or condition may be a wound, and more particularly a chronic wound. In some embodiments the wound is a dermal ulcer.

Another embodiment of the invention involves a method for inhibiting uPA in a subject comprising administration to the subject of a compound of formula (I).

Advantageously, the compounds of formula (I) also show a high degree of selectivity (30-1000's fold) for uPA over related trypsin-like serine protease (TLSP) off-targets and are therefore not expected to suffer from side-effects relating to inhibition of these off- targets in vivo.

Pharmacokinetic analysis in rats demonstrated that certain compounds of formula (I) show improved metabolic stability and plasma half-lives relative to HMA. The resulting pharmacokinetic profiles are comparable to amiloride. Additionally, the compounds of formula (I) show no activity against epithelial sodium channels (ENaC), the diuretic and K ⁺ -sparing target of amiloride, and as such are unlikely to possess diuretic or antikaliuretic effects in vivo, an important characteristic as these properties are undesirable in an anticancer therapeutic.

Those skilled in the art will recognise that compounds and pharmaceutical compositions of the present invention may be administered via any route that delivers an effective amount of the compounds to the tissue or site to be treated. In general, the compounds and compositions may be administered by the parenteral (for example intravenous, intraspinal, subcutaneous or intramuscular), oral, inhalation, or topical route. Administration may be systemic, regional or local.

The particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the disease or condition to be treated, the severity and extent of the disease or condition, the required dosage of the particular compound to be delivered and the potential side-effects of the compound.

In general, suitable compositions may be prepared according to methods that are known to those of ordinary skill in the art and may include pharmaceutically acceptable carriers, diluents and/or excipients. The carriers, diluents and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; Cremaphor; cyclodextrins; lower alcohols, for example ethanol or /-propanol; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1 ,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

Pharmaceutical compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets and elixirs for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include cyclodextrins (for example Captisol®) Cremaphor, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1 ,2-propylene glycol. To aid injection and delivery, the compounds may also be added to PEG and non-PEGylated liposomes or micelles with specific targeting tags attached to PEG moieties, such as the RGD peptide or glutathione, for aiding passage across the blood brain barrier.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include cyclodextrins, Cremaphor, peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition, these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate that delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time- delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof. Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids, such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.

Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

A further suitable emulsifying agent for use in oral or parenteral formulations, which may also function as a solubilizer, is Kolliphor® HS 15.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, PA, hereby incorporated by reference herein.

Topical formulations may comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by autoclaving or maintaining at 90 °C to 100 °C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01 %) and chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisiteriser such as glycerol, or oil such as olive oil.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol, such as propylene glycol or macrogols.

The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant, such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inoraganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

In some embodiments the compositions are administered in the form of suppositories suitable for rectal administration of the compounds. These compositions are prepared by mixing the compound with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the zeolite or zeolite-like material. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

The compositions may also be administered or delivered to target cells in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), the phospholipid 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); Avanti Polar Lipids), the PEG lipid 3-N-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-1 ,2- dimyrestyloxy-propylamine (PEG-cDMA), and the cationic lipid 1 ,2-di-o-octadecenyl-3- (N,N-dimethyl)aminopropane (DODMA) or 1 ,2-dilinoleyloxy-3-(N,N- dimethyl)aminopropane (DLinDMA) in the molar ratios 55:20:10:15 or 48:20:2:30, respectively, PEG-cDMA, DODMA and DLinDMA. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stablisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this, specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference.

The compositions may also be administered in the form of microparticles or nanoparticles. Biodegradable microparticles formed from polyactide (PLA), polylactide- co-glycolide (PLGA), and epsilon-caprolactone (έ-caprlactone) have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J. Pharm. Pharmaceut. Sci. 3(2) 234-258). Microparticles have been formulated for the delivery of a range of drug candidates including vaccines, antibiotics, and DNA. Moreover, these formulations have been developed for various delivery routes including parenteral subcutaneous injection, intravenous injection and inhalation.

The compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and an organic solvent or organic solvents mixture. Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate. SAIB is a well known food additive. It is a very hydrophobic, fully esterified sucrose derivative, at a nominal ratio of six isobutyrate to two acetate groups. As a mixed ester, SAIB does not crystallise but exists as a clear viscous liquid. Mixing SAIB with a pharmaceutically acceptable organic solvent, such as ethanol or benzyl alcohol decreases the viscosity of the mixture sufficiently to allow for injection. An active pharmaceutical ingredient may be added to the SAIB delivery vehicle to form SAIB solution or suspension formulations. When the formulation is injected subcutaneously, the solvent differs from the matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to set up as an in situ forming depot.

For the purposes of the present invention compounds and compositions may be administered to subjects either therapeutically or preventively. In a therapeutic application compositions are administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the disease or condition and its complications. The composition should provide a quantity of the compound or agent sufficient to effectively treat the subject.

The therapeutically effective amount for any particular subject will depend upon a variety of factors including: the disease or condition being treated and the severity thereof; the activity of the compound administered; the composition in which the compound is present; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of sequestration of the compound; the duration of the treatment; drugs used in combination or coincidental with the compound, together with other related factors well known in medicine. One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a compound that would be required to treat or prevent a particular disease or condition.

Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750 mg per kg body weight per 24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250 mg per kg body weight per 24 hours; about 1 .0 mg to about 250 mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1 .0 mg to about 200 mg per kg body weight per 24 hours; about 1 .0 mg to about 100 mg per kg body weight per 24 hours; about 1 .0 mg to about 50 mg per kg body weight per 24 hours; about 1 .0 mg to about 25 mg per kg body weight per 24 hours; about 5.0 mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about 20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500 mg/m ² . Generally, an effective dosage is expected to be in the range of about 25 to about 500 mg/m ² , preferably about 25 to about 350 mg/m ² , more preferably about 25 to about 300 mg/m ² , still more preferably about 25 to about 250 mg/m ² , even more preferably about 50 to about 250 mg/m ² , and still even more preferably about 75 to about 150 mg/m ² .

Typically, in therapeutic applications, the treatment would be for the duration of the disease or condition.

Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

In the treatment of cancer, the compounds of formula (I) may be used alone or alternatively in combination with photodynamic therapy and/or radiotherapy and/or surgery and/or other therapeutic agents, for example chemotherapeutic agents and immunostimulatory agents, as part of a combination therapy. The compounds of formula (I) may sensitise cancer cells to other chemotherapeutic agents and/or radiotherapy.

The terms "combination therapy" and "adjunct therapy" are intended to embrace administration of multiple therapeutic agents in a sequential manner in a regimen that will provide beneficial effects and is intended to embrace administration of these agents in either a single formulation or in separate formulations.

Combination therapy may involve the active agents being administered together, sequentially, or spaced apart as appropriate in each case. Combinations of active agents, including compounds of the invention, may be synergistic.

The co-administration of compounds of the formula (I) with other therapeutic agent(s) may be effected by a compound of the formula (I) being in the same unit dose form as the other therapeutic agent(s), or the compound of the formula (I) and the other therapeutic agent(s) may be present in individual and discrete unit dosage forms that are administered sequentially, at the same, or at a similar time. Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial agent to be current when the second or later agent is administered, especially where a cumulative or synergistic effect is desired. When administered separately, it may be preferred for the compound of formula (I) and the other agent to be administered by the same route of administration, although it is not necessary for this to be so.

In accordance with various embodiments of the present invention one or more compounds of formula (I) may be included in combination therapy with surgery and/or radiotherapy and/or one or more chemotherapeutic agents.

There are large numbers of chemotherapeutic agents that are currently in use, in clinical evaluation and in pre-clinical development, which could be selected for treatment of cancers in combination with compounds of the formula (I). Such agents fall into several major categories, namely, antibiotic-type agents, alkylating agents, anti-metabolite agents, hormonal agents, immunological agents, interferon-type agents and a category of miscellaneous agents. Suitable agents which may be used in combination therapies include those listed, for example, in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 15th Ed., 2014 the entire contents of which are incorporated herein by reference.

When used in the treatment of cancer, compounds of the formula (I) may be administered with vinblastine. The present invention is further described below by reference to the following non- limiting examples.

Examples

Example 1 - Method for the synthesis of compounds of formulae (Ha) and (I)

Preparation of methyl 3-amino-5-(azepan- 1-yl)-6-chloropyrazine-2-carboxylate (Ilia) To a suspension of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (IVa) (5.55 g, 25.0 mmol) in 2-propanol (50 mL) was added hexamethyleneimine (2.73 g, 3.1 0 mL, 27.5 mmol) and the mixture was stirred at room temperature. Diisopropyethylamine (3.55 g, 4.79 mL, 27.5 mmol) was then added and the reaction mixture was heated at reflux. After 2 h the reaction was allowed to cool to room temperature, which caused the product to crystallize. The solid product was collected by vacuum filtration and washed with cold 2-propanol followed by diethyl ether. After drying, the desired product (Ilia) was obtained as pink crystals (5.91 g, 83%).

General method for the preparation of Suzuki-Miyaura cross-coupled products (Ma)

Methyl 3-amino-5-(azepan-1 -yl)-6-chloropyrazine-2-carboxylate (Ilia) was combined with K ₂ C0 ₃ (1 0 mol eq) _, 1 .5 mole equivalents of the appropriately substituted (hetero)aryl boronic acid and Pd(PPh ₃ ) ₄ (5 mole %) in a round-bottom flask under argon. Depending on the reaction scale, either 15 mL or 30 mL of 4:1 dry toluene:MeOH was then added and the mixture heated at reflux (30 min - 48 h), while monitoring by TLC. The reaction mixture was filtered through celite, washed with EtOAc (20 mL) and the filtrate evaporated to dryness. The crude residue was purified by silica gel flash column chromatography using EtOAc/pet. spirit to afford the product (Ma).

General method for guanidinylation to give compounds of formula (I)

Method A

Na metal (2 mole eq) was added in portions to dry 'PrOH (10 mL) and stirred at 70 °C until complete dissolution was achieved. Guanidine.HCI (2 mole eq) was added to this solution and stirring continued for a further 30 min. The solution was then filtered and the filtrate added to the the 6-substituted methyl ester precursor (Ma). The mixture was stirred at 70°C for 1 -3 h (monitoring by TLC) and then allowed to cool to room temperature. The solvent was removed under reduced pressure and the residue taken up in 2 mL of 50:50 solvent A:B and purified by preparative reverse phase HPLC (Waters Sunfire Ci ₈ OBD 50 mm x 1 9 mm, 5 μηι, Solvent A H ₂ O:0.1 % TFA, Solvent B Acetonitrile:0.1 % TFA; gradient elution 70:30 A: B→100% B). Method B

Dry CH ₃ OH (50 mL) was slowly added with stirring to small pieces of sodium (2.3 g, 0.1 mol) in an ice bath under nitrogen. After the complete dissolution of sodium in CH ₃ OH, guanidine hydrochloride (9.55 g, 0.1 mol) was added, and the resulting mixture was stirred at room temperature for 1 .5 h. Filtration of the white precipitate gave a 2 M solution of free guanidine in methanol, which was collected in a sealed dry flask under nitrogen and stored in a refrigerator. To a suspension of the 6-substituted methyl ester precursor (Ma) (1 .0 mmol) in DMF (5 mL) was added a 2 M methanol solution of guanidine (5 mL, 10 mmol) and the resulting mixture was stirred at room temperature overnight. Brine (20 mL) was added and the mixture extracted with ethyl acetate (3 x 30 mL). The organic layer was washed with 10% NaCI (2 x 30 mL), dried over anhydrous MgS0 ₄ and concentrated under reduced pressure. Preparative reverse phase HPLC was performed as above, when necessary.

Example 2- Characterisation data for compounds of formula (Ha) and (I)

Compound 1a - methyl 3-amino-5-(azepan- 1-yl)-6-(4-(methylthio)phenyl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (gradient elution: 2.5:97.5 EtOAc:Pet. Spirit → 5:95 EtOAc:Pet. Spirit) to give 1 a as a yellow solid (104 mg, 77% yield): MP: 132- 134° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.42 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 6.20 (bs, 2H), 3.88 (s, 3H), 3.36 (t, J = 5.5 Hz, 4H,), 2.48 (s, 3H), 1 .62 (s, 4H), 1 .43 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.42, 154.77, 153.43, 137.77, 137.47, 131 .19, 128.29, 126.77, 1 12.45, 51 .90, 51 .01 , 27.96, 27.08, 16.05; ES TOF MS m/z (M + H) ⁺ 373, Anal, for Calc. Mass 373.1698. Found 373.1688. Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA→100% ACN/0.1 % TFA over 30 min, t _r = 36.1 min).

Compound 2a - methyl 3-amino-5-(azepan-1-yl)-6-(2,4-dioxo-1, 2,3,4- tetrahydropyrimidin-5-yl)pyrazine-2-carboxylate

¹ H NMR (500 MHz, DMSO-cQ: δ 1.40 (s, 4H), 1.59 (m, 4H), 3.42 (br s, 4H), 3.70 (s, 3H), 6.95 (br s, 2H, NH ₂ ), 7.51 (s, 1 H), 1 1 .20 (br s, 2H, 2 X NH); ¹³ C NMR (126 MHz, DMSO- de): δ 26.8, 28.2, 50.0, 51 .6, 1 1 1 .2, 1 15.1 , 123.6, 140.8, 151 .8, 154.8, 155.4, 164.1 , 167.3; MS (ESI): m/z 344 (M+H) ⁺ , 366 (M+Na) ⁺ ; Yellow solid (Yield 53%).

Compound 3a - methyl 3-amino-5-(azepan-1-yl)-6-(1H-indol-2-yl)pyrazine-2-carboxyl ate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 3a as a orange solid (87.5 mg, 34% yield); MP 152-154° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 9.14 (s, 1 H), 7.57 (d, 1 H), 7.37 (d, 1 H, J = 7.7 Hz), 7.16 (t, 1 H, J = 7.0 Hz), 7.08 (t, 1 H, J = 7.0 Hz), 6.52 (s, 1 H), 3.93 (s, 3H), 3.58 (t, J = 5.75 Hz, 4H), 1 .70 (s, 4H), 1.50 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.18, 155.09, 153.15, 135.96, 135.68, 128.95, 124.51, 122.36, 120.67, 119.92, 112.71, 111.06, 101.95, 52.05, 51.65, 28.12, 27.53; ES TOF MS m/z (M + H) ⁺ 388, Anal, for C ₂ oH ₂₄ N ₅ 0 ₂ : Calc. Mass 388.1730. Found 388.1731; Anal HPLC (100 H ₂ O/0.1% TFA:→· 100% ACN/0.1% TFA over 30 min, t _r = 29.8 min).

Compound 5a - methyl 3-amino-5-(azepan-1-yl)-6-(4-(trifluoromethyl)phenyl)pyrazin e-2- carboxylate

Purified by silica gel flash chromatography (5:95 EtOAc:Pet. Spirit 10:90 EtOAc:Pet. Spirit) to give 5a as a white solid (238 mg, 64%); ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.45 (s, 4H), 1.65 (s, 4H), 3.34 (s, 4H), 3.89 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 7.63 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.3, 28.1, 51.3, 52.1, 113.2, 125.1, 125.6, 128.2 (ArCH), 129.3, 129.9, 144.6, 153.7, 154.9, 167.4; MS (ESI): m/z 395 (M+H) ⁺ , 417 (M+Na) ⁺ ; mp 158-160 °C.

Compound 6a - methyl 3-amino-5-(azepan-1-yl)-6-(isoquinolin-4-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (5:95 EtOAc:Pet. Spirit 10:90 EtOAc:Pet. Spirit) to give 6a as a brown solid (155.7 mg, 58% Yield); MP 140-142° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 9.23 (s, 1H), 8.58 (s, 1H), 8.00 (d, J= 8.0 Hz, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.68 (t, J= 7.5 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 3.86 (s, 3H), 3.21 (t, J= 5.5 Hz, 4H), 1.47 (s, 4H), 1.38 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.29, 155.77, 154.02, 152.06, 143.01, 134.26, 131.78, 130.82, 128.42, 127.97, 127.37, 126.66, 124.50, 112.90, 52.02, 50.38, 27.87, 26.83; ES TOF MS m/z (M + Na) ⁺ 400, Anal, for C ₂ iH ₂₄ N ₅ 0 ₂ Na ⁺ : Calc. Mass 400.1749. Found 400.1747; Anal HPLC (100 H ₂ O/0.1% TFA: 100% ACN/0.1% TFA over 30 min, t _r = 22.3 min).

Compound 7a - methyl 3-amino-5-(azepan-1-yl)-6-(benzo[b]thiophen-2-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography using 5-20% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.48 (s, 4H), 1.69 (s, 4H), 3.51 (s, 4H), 3.91 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 7.22 (s, 1H), 7.21 (m, 2H), 7.71 (d, J= 6.9 Hz, 1H), 7.80 (d, J = 7.0 Hz, 1H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.4, 28.2, 51.3, 52.3, 113.2, 122.0, 122.3, 123.6, 124.4, 124.5, 125.4, 140.1, 140.2, 143.3, 153.6, 155.2, 167.3; MS (ESI): m/z 383 (M+H) ⁺ , 405 (M+Na) ⁺ ; Yellow solid (Yield 78%) mp 146-148 °C.

Compound 8a - methyl 3-amino-5-(azepan-1-yl)-6-(3,5-dimethoxyphenyl)pyrazine-2- carboxylate Purified by silica gel flash chromatography (2.5:97.5 EtOAc:Pet. Spirit 10:90 EtOAc:Pet. Spirit) to give 8a as a light yellow solid (166.1 mg, 62% yield); MP: 96-98 ° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 6.64 (s, 2H), 6.40 (s, 1H), 3.88 (s, 3H), 3.81 (s, 6H), 3.38 (t, J= 6.0 Hz, 4H), 1.64 (s, 4H), 1.44 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.50, 160.98, 154.73, 153.67, 142.75, 131.59, 111.97, 106.32, 99.95, 55.61, 51.98, 50.89, 28.09, 27.14. ES TOF MS m/z (M + Na) ⁺ 409, Anal, for C ₂₀ H ₂₆ N ₄ NaO ₄ : Calc. Mass 409.1852. Found 409.1867 Anal HPLC (70:30 H ₂ O/0.1% TFA:ACN/0.1% TFA→· 100% ACN/0.1% TFA over 30 min, t _r = 30.0 min).

Compound 9a - methyl-3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(thiophen-2 - yl)pyrazine-2-carboxamide

Purified via silica gel flash chromatography (gradient elution, 5:95 to 10:90 EtOAc:Hexane) to yield 9a as a light yellow solid (99.2 mg, 85% Yield): MP: 118-120 °C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.27 (d, J= 5 Hz, 1 H), 7.03 (d, J= 3 Hz, 1 H), 6.98 (m, 1 H), 3.89 (s, 3H), 3.43 (t, J= 5.7 Hz, 4H), 1.67 (s, 4H), 1.47 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.28, 155.24, 153.52, 142.83, 126.89, 125.96, 125.78, 125.54, 112.70, 52.13, 51.11, 28.15, 27.26; ES TOF MS m/z (M + Na) ⁺ 355, Anal, for C ₁₆ H ₂₀ N ₄ NaO ₂ : Calc. Mass 355.1205. Found 355.1193; Anal HPLC (100 H ₂ O/0.1% TFA: →· 100% ACN/0.1 % TFA over 30 min, t _r = 28.8 min).

Compound 10a - methyl 3-amino-5-(azepan-1-yl)-6-(2-fluoropyridin-3-yl)pyrazine-2- carboxylate

Purified via silica gel flash chromatography (gradient elution, 5:95 to 15:85 EtOAc:Hexane) to yield 10a as an light brown solid (92.3 mg, 76% yield): MP: 156- 158°C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.17 (d, J= 4.3 Hz, 1H), 8.06 (m, 1H), 7.29 (t, J = 5.5 Hz, 1H), 3.89 (s, 3H), 3.36 (s, J= 5.7 Hz, 4H), 1.63 (s, 4H), 1.46 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.21, 161.32, 155.23, 154.10, 146.80, 141.07, 124.54, 123.84, 122.15, 113.34, 52.27, 50.34, 28.13, 27.29; ES TOF MS m/z (M + Na) ⁺ 368, Anal, for C ₁₇ H ₂₀ FN ₅ NaO ₂ : Calc. Mass 368.1499. Found 368.1489; Anal HPLC (100 H ₂ O/0.1% TFA: 100% ACN/0.1% TFA over 30 min, t _r = 26.3 min).

Compound 11a - methyl 3-amino-5-(azepan-1-yl)-6-(2,4-dimethoxyphenyl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 11a as a light yellow solid (142.8 mg, 53% yield): MP: 106-108 °C; ¹ H NMR (500 MHz, CDCI ₃ ): ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.33 (d, J= 8.3 Hz, 1H), 6.54 (dd, J= 8.3 Hz, J= 2.1 Hz 1H), 6.42 (d, J= 2.1 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.70 (s, 3H), 3.44 (s, 2H), 3.26 (s, 2H), 1.58 (s, 2H), 1.53 (s, 2H), 1.46 (s, 2H), 1.39 (s, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) ¹³ C NMR (126 MHz, CDCI ₃ ) δ 165.95, 160.93, 158.21, 155.42, 153.99, 131.10, 129.22, 123.71, 111.77, 105.07, 98.67, 55.74, 55.66, 51.99, 50.62, 27.81, 26.93. ES TOF MS m/z (M + Na) ⁺ 409, Anal, for C ₂₀ H ₂₇ N ₄ NaO ₄ : Calc. Mass 409.1852. Found 409.1866; Anal HPLC (100 H ₂ O/0.1% TFA:→· 100% ACN/0.1% TFA over 30 min, t _r = 28.6 min).

Compound 12a - methyl 3-amino-5-(azepan-1-yl)-6-(naphthalen-2-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (5:95 EtOAc:Pet. Spirit 10:90 EtOAc:Pet. Spirit) to give 12a as a brown solid (249.2 mg, 92% Yield); MP 160-162° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.95 (s, 1H), 7.81 (m, 3H), 7.64 (dd, J= 8.5, 1.5 Hz, 1H), 7.44 (m, 2H), 6.23 (bs, 2H), 3.89 (s, 3H), 3.36 (t, J= 5.75, 4H), 1.59 (s, 4H), 1.41 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.45, 154.93, 153.49, 138.16, 133.49, 132.69, 131.58, 128.14, 128.06, 127.65, 126.51, 126.17, 126.13, 125.93, 112.46, 51.97, 51.01, 27.91, 27.03; ES TOF MS m/z (M + H) ⁺ 377, Anal, for C ₂₂ H ₂₅ N ₄ 0 ₂ Na ⁺ : Calc. Mass 399.1794. Found 399.1790 Anal HPLC (70:30 H ₂ O/0.1% TFA:ACN/0.1% TFA→· 100% ACN/0.1% TFA over 30 min, t _r = 31.8 min).

Compound 13a - methyl 3-amino-5-(azepan-1-yl)-6-(thiophen-3-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (gradient elution, 5:95 to 10:90 EtOAc:Hexane) to yield 13a as an off white solid (100.4 mg, 85% yield). MP: 118-120°C: ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.38 (s, 1H), 7.29 (m, 1H), 7.25 (d, J= 5 Hz, 1H), 3.88 (s, 3H), 3.38 (t, J= 5.8 Hz, 4H), 1.63 (s, 4H), 1.44 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.35, 155.30, 153.53, 141.34, 128.06, 127.86, 125.39, 122.83, 112.16, 51.97, 50.90, 28.01, 27.05; ES TOF MS m/z (M + H) ⁺ 355, Anal, for d ₆ H ₂₁ N ₄ Na0 ₂ S: Calc. Mass 355.1205. Found 355.1197; Anal HPLC (100 H ₂ O/0.1% TFA:→· 100% ACN/0.1% TFA over 30 min, t _r = 28.3 min).

Compound 14a - methyl 3-amino-5-(azepan-1-yl)-6-(benzo[d][1,3]dioxol-5-yl)pyrazine -2- carboxylate

Purified by silica gel flash chromatography (2.5 EtOAc:97.5 Pet spirit - 10:90 EtOAc:Pet. spirit) to give 14a as a yellow solid (65.9 mg, 51% yield); MP: 156-158° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.02 (s, 1H), 6.93 (d, J= 8.0 Hz, 1H), 6.80 (d, J= 8.0 Hz, 1H), 5.96 (s, 2H), 3.88 (s, 4H), 3.38 (s, 4H), 1.63 (s, 4H), 1.44 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.55, 154.89, 153.46, 147.86, 147.05, 134.93, 131.51, 121.69, 112.20, 108.73, 108.34, 101.12, 51.94, 50.99, 28.08, 27.10; ES TOF MS m/z (M + H) ⁺ 371 , Anal, for C ₁₉ H ₂₃ N ₄ 0 ₄ : Calc. Mass 371 .1719. Found 371 .1734; Anal HPLC (100 H ₂ O/0.1 % TFA: -^100% ACN/0.1 % TFA over 30 min, t _r = 31 .8 min).

Compound 15a - methyl 3-amino-5-(azepan-1-yl)-6-(2,6-dimethoxypyridin-3-yl)pyrazin e- 2-carboxylate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 15a as a yellow solid (1 14.9 mg, 42% yield); MP: 134-136° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.66 (d, J = 8.0 Hz, 1 H), 6.38 (d, J = 8.0 Hz, 1 H), 3.92 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.45 (bs, 2H), 3.31 (bs, 2H), 1 .57 (s, 4H), 1 .44 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.52, 162.79, 159.68, 155.29, 153.92, 141.52, 127.61 , 1 16.24, 1 12.21 , 101 .33, 53.72, 53.48, 51 .90, 50.14, 28.12, 27.07; ESI MS m/z (M) ⁺ 388. ES TOF MS m/z (M + H) ⁺ 388, Anal, for C ₁₉ H ₂₅ N ₅ 0 ₄ : Calc. Mass 388.1985. Found 388.1983; Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA -^100% ACN/0.1 % TFA over 30 min, t _r = 31 .6 min).

Compound 16a - methyl 3-amino-5-(azepan- 1 -yl)-6-(1 -(2-morpholinoethyl)-1 H-pyrazol-4- yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography (EtOAc to 6% MeOH:EtOAc) to give 16a as a yellow solid (1 19.3 mg, 80% yield): MP: 132-134° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.70 (s, 1 H), 7.61 (s, 1 H), 6.24 (bs, 2H), 4.25 (s, 2H), 3.89 (s, 3H), 3.69 (s, 4H), 3.46 (s, 4H), 2.84 (s, 2H), 2.50 (s, 4H), 1 .65 (s, 4H), 1 .47 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.20, 155.50, 153.33, 138.62, 128.67, 125.13, 122.10 1 12.41 , 66.94, 58.19, 53.68, 51 .91 , 51 .05, 49.55, 27.94 27.02 ES TOF MS m/z (M + H) ⁺ 430, Anal, for C ₂₂ H ₃ 6N ₇ 0 ₃ : Calc. Mass 430.2567. Found 430.2553; Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA 100% ACN/0.1 % TFA over 30 min, t _r = 21 .6 min).

Compound 17a - methyl 3-amino-5-(azepan-1 -yl)-6-(1 -methyl-1 H-pyrazol-4-yl)pyrazine- 2-carboxylate

Purified by silica gel flash chromatography (5:95 EtOAc:Pet spirit - 10:90 EtOAc:Pet. spirit) to give 17a 89.2 mg of a yellow solid (77% yield); MP: 134-136°; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.64 (s, 1 H), 7.57 (s, 1 H), 6.17 (bs, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 3.47 (t, J = 6 Hz, 4H), 1 .66 (s, 4H), 1 .47 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.34, 155.54, 153.40, 138.73, 129.1 1 , 125.25, 122.56, 1 12.42, 52.08, 51 .16, 39.00, 28.00, 27.17; ES TOF MS m/z (M + H) ⁺ 331 , Anal, for C ₁₆ H ₂₃ N ₆ 0 ₂ : Calc. Mass 331 .1882. Found 331 .1894; Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA→· 100% ACN/0.1 % TFA over 30 min, t _r = 25.2 min). Compound 18a - methyl 3-amino-5-(azepan-1-yl)-6-(3,6-dimethoxypyridazin-4- yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 18a as a yellow solid (94.2 mg, 68% Yield): MP: 152-154° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.17 (s, 1H), 4.08 (s, 3H), 4.00 (s, 3H), 3.88 (s, 3H), 3.43 (s, 2H), 3.29 (s, 2H), 1.63 (s, 4H), 1.44 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.97, 162.75, 159.61, 154.70, 154.14, 135.05, 122.88, 119.39, 113.08, 54.82, 54.62, 52.07, 49.74, 27.98, 27.20; ES TOF MS m/z (M + Na) ⁺ 411 , Anal, for C ₁₈ H ₂₄ N ₆ Na0 ₄ : Calc. Mass 411.1757. Found 411.1762; Anal HPLC (70:30 H ₂ O/0.1% TFA:ACN/0.1% TFA→· 100% ACN/0.1 % TFA over 30 min, t _r = 21.2 min).

Compound 19a - methyl 3-amino-5-(azepan-1-yl)-6-(benzofuran-2-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography using 5-25% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.46 (s, 4H), 1.66 (s, 4H), 3.45 (t, J= 5.8 Hz, 4H), 3.90 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 6.93 (s, 1H), 7.21 (t, J = 7.3 Hz, 1H), 7.25 (t, J= 7.2 Hz, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.56 (d, J= 7.5 Hz, 1H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.3, 28.2, 50.2, 52.2, 105.1, 111.5, 112.9, 121.2, 121.4, 123.1, 124.4, 129.2, 154.2, 154.6, 154.8, 164.9, 167.2; MS (ESI): m/z 367 (M+H) ⁺ , 389 (M+Na) ⁺ ; Yellow solid (Yield 57%) mp 121-123 °C.

Compound 20a - methyl 3-amino-5-(azepan-1-yl)-6-(furan-3-yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography (5:95 EtOAc:Pet spirit - 10:90 EtOAc:Pet. spirit) to give 20a of a yellow solid (114.4 mg , 93% yield); MP: 122-124° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.64 (s, 1H), 7.41 (s, 1H), 6.61 (s, 1H), 6.20 (bs, 2H), 3.89 (s, 3H), 3.48 (t, J= 5 Hz, 4H), 1.66 (s, 4H), 1.47 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.30, 155.56, 153.60, 142.62, 140.63, 125.82, 124.77, 112.55, 111.15, 51.97, 50.99, 28.02, 27.10; ES TOF MS m/z (M + H) ⁺ 317, Anal, for Ci ₆ H ₂₁ N ₄ 0 ₃ : Calc. Mass 316.1614. Found 316.1604; Anal HPLC (70:30 H ₂ O/0.1% TFA:ACN/0.1% TFA→· 100% ACN/0.1% TFA over 30 min, t _r = 30.9 min).

Compound 21a - methyl 3-amino-5-(azepan-1-yl)-6-(furan-2-yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography using 10-25% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.47 (s, 4H), 1.66 (s, 4H), 3.37 (t, J= 5.5 Hz, 4H), 3.89 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 6.45 (s, 1H), 6.54 (d, J= 2.3 Hz, 1H), 7.41 (s, 1H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 26.9, 28.0, 50.0, 52.0, 108.6, 111.6, 112.1, 122.0, 141.4, 152.2, 154.0, 154.8, 167.1 ; MS (ESI): m/z 317 (M+H) ⁺ , 339 (M+Na) ⁺ ; Yellow solid (Yield 79%) mp 1 16-1 18 °C.

Compound 22a - methyl 3-amino-5-(azepan- 1-yl)-6-(pyrimidin-5-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography using 20-60% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1 .48 (s, 4H), 1 .68 (s, 4H), 3.52 (t, J = 6.0 Hz, 4H), 3.91 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 8.89 (s, 2H), 9.13 (s, 1 H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.4, 28.0, 51 .5, 52.3, 1 14.5, 124.5, 134.9, 153.9, 155.3, 155.7, 157.1 , 167.1 ; MS (ESI): m/z 329 (M+H) ⁺ , 351 (M+Na) ⁺ ; Yellow solid (Yield 91 %) mp 176-178 °C.

Compound 23a - methyl 3-amino-5-(azepan- 1 -yl)-6-(4-fluorobenzofuran-2-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 23a as a yellow solid (79.7 mg, 58% Yield); MP: 108-1 10° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.27 (d, 1 H), 7.20 (m, 1 H), 6.99 (s, 1 H), 6.93 (t, 1 H, J = 8.5 Hz), 3.91 (s, 3H), 3.46 (s, 4H), 1 .69 (s, 4H), 1 .49 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.0, 156.9, 156.2, 154.7, 154.0, 138.3, 124.6, 120.5, 1 18.0, 1 15.9, 1 10.4, 108.6, 107.6, 52.2, 50.0, 28.0, 27.1 ; ES TOF MS m/z (M + Na) ⁺ 407, Anal, for C ₂₀ H ₂₁ FN ₆ NaO ₄ : Calc. Mass 407.1495 Found 407.1504 Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA 100% ACN/0.1 % TFA over 30 min, t _r = 21 .2 min).

Compound 24a - methyl 3-amino-5-(azepan- 1-yl)-6-(2-methoxypyrimidin-5-yl)pyrazine- 2-carboxylate

Purified by silica gel flash chromatography using 10-40% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1 .48 (s, 4H), 1 .68 (s, 4H), 3.38 (t, J = 5.5 Hz, 4H), 3.90 (s, 3H), 4.05 (s, 3H), 6.13 (br s, 2H, NH ₂ ), 8.66 (s, 2H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.3, 28.0, 51 .4, 52.2, 55.2, 1 13.9, 125.2, 128.8, 153.8, 155.3, 158.3, 164.7, 166.4; MS (ESI): m/z 359 (M+H) ⁺ , 381 (M+Na) ⁺ ; Yellow solid (Yield 80%) mp 168-170 °C.

Compound 25a - methyl 3-amino-5-(azepan-1-yl)-6-(2-(methylamino)pyrimidin-5- yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography using 30-60% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.47 (s, 4H), 1.67 (s, 4H), 3.04 (d, J = 5.1 Hz, 3H), 3.42 (t, J = 5.9 Hz, 4H), 3.90 (s, 3H), 5.28 (q, J = 4.9 Hz, 1 H, NH), 6.22 (br s, 2H, NH ₂ ), 8.45 (s, 2H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.3, 28.0, 28.8, 51.3, 52.3, 1 13.3, 124.2, 126.9, 153.7 (ArC), 155.3, 157.2, 162.0, 167.4; MS (ESI): m/z 358 (M+H) ⁺ , 380 (M+Na) ⁺ ; Yellow solid (Yield 87%). Compound 26a - methyl 3-amino-5-(azepan- 1-yl)-6-(2,4-dimethoxypyrimidin-5- yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography (10:90 EtOAc:Pet. Spirit 20:80 EtOAc:Pet. Spirit) to give 26a as a light yellow solid (124.3 mg, 91 % yield): MP: 150-152° C; ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.36 (s, 1 H), 4.03 (s, 3H), 3.94 (s, 3H), 3.87 (s, 3H), 3.39 (s, 4H), 1 .60 (s, 4H), 1 .46 (s, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 168.30, 167.18, 164.76, 158.09, 155.34, 154.05, 123.75, 1 17.06, 1 12.64, 54.96, 54.10, 51 .95, 50.06, 27.98, 26.95; ES TOF MS m/z (M + H) ⁺ 389, Anal, for C ₁₈ H ₂₅ N ₆ 0 ₄ : Calc. Mass 389.1937. Found 389.1929; Anal HPLC (70:30 H ₂ O/0.1 % TFA:ACN/0.1 % TFA→· 100% ACN/0.1 % TFA over 30 min, t _r = 27.1 min).

Compound 27a - methyl 3-amino-5-(azepan- 1-yl)-6-(2-(isopropylamino)pyrimidin-5- yl)pyrazine-2-carboxylate

Purified by silica gel flash chromatography using 10-25% EtOAc/pet spirit; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.25 (d, J = 7.0 Hz, 6H), 1 .47 (s, 4H), 1 .67 (s, 4H), 3.43 (t, J = 5.9 Hz, 4H), 3.89 (s, 3H), 4.16 (septet, J = 6.8 Hz, 1 H), 5.10 (d, J = 7.8 Hz, 1 H, NH), 6.25 (br s, 2H, NH ₂ ), 8.43 (s, 2H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 22.9, 27.1 , 27.8, 43.0, 51.1 , 52.0, 1 13.1 , 123.8, 126.8, 153.5, 155.1 , 157.0, 161 .7, 167.2; MS (ESI): m/z 386 (M+H) ⁺ , 408 (M+Na) ⁺ ; Yellow solid (Yield 83%).

Compound 28a - methyl 3-amino-5-(azepan-1-yl)-6-(2-(2-hydroxyethyl)amino)pyrimidin - 5-yl)pyrazine-2-carboxylate

Product was crystallized from /-PrOH, washed with diethylether; ¹ H NMR (500 MHz, CDCI ₃ ): δ 1.48 (s, 4H), 1.68 (s, 4H), 3.42 (t, J = 5.6 Hz, 4H), 3.62 (q, J = 4.9 Hz, 2H) 3.85 (t, J = 4.4 Hz, 2H), 3.90 (s, 3H), 5.64 (t, J = 5.5 Hz, 1 H, NH), 6.23 (br s, 2H, NH ₂ ), 8.44 (s, 2H); ¹³ C NMR (126 MHz, CDCI ₃ ): δ 27.1 , 27.8, 44.9, 51.1 , 52.1 , 63.6, 113.3, 124.6, 126.2, 153.5, 155.1 , 156.9, 161 .7, 167.1 ; MS (ESI): m/z 388 (M+H) ⁺ , 410 (M+Na) ⁺ ; Yellow solid (Yield 84%).

Compound 29a - methyl 3-amino-6-(2-aminopyrimidin-5-yl)-5-(azepan-1-yl)pyrazine-2- carboxylate

Purified by silica gel flash chromatography using 70-100% EtOAc/pet spirit; ¹ H NMR (500 MHz, DMSO-d ₆ ): δ 1.41 (s, 4H), 1.59 (s, 4H), 3.41 (t, J = 5.9 Hz, 4H), 3.74 (s, 3H), 6.76 (br s, 2H, NH ₂ ), 7.04 (br s, 2H, NH ₂ ), 8.27 (s, 2H); ¹³ C NMR (126 MHz, DMSO-d ₆ ): δ 27.0, 27.9, 51.0, 51.9, 112.3, 123.9, 126.5, 154.2, 155.1 , 157.4, 163.0, 167.3; MS (ESI): m/z 344 (M+H) ⁺ , 366 (M+Na) ⁺ ; Yellow solid (Yield 83%). Compound 1 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(4-(methylthio)phe n pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.31 (s, 1H), 9.44 (s, 2H), 8.47 (s, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 3.40 (t, J= 5.5 Hz, 4H) 1.60 (s, 4H), 1.46 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.04, 156.63, 155.71, 153.56, 138.99, 136.40, 131.19, 128.14, 126.61, 110.96, 51.47, 27.89, 27.11, 15.45. IR 3332.99, 2924.09, 2854.65, 2360.87, 1651.07, 1573.91, 1512.19, 1427.32, 1350.17, 1242.16, 1195.87. HRMS (M + H) ⁺ Anal for C ₁₉ H ₂₆ N ₇ OS, calc mass: 400.1920, found mass: 400.1935. MP: 152-154 °C. Anal. HPLC {t _r = 28.4 min). Isolated 5.5 mg as a yellow oil. Yield 5% (2 steps, TFA salt).

Compound 2 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(2,4-dioxo-1, 2,3,4- tetrahydropyrimidin-5-yl)pyrazine-2-carboxamide

Yellow solid (Yield 65%); ¹ H NMR (500 MHz DMSO-d ₆ ): δ 1.40 (s, 4H, H4\ H5'), 1.59 (m, 4H, H3\ H6'), 3.44 (br s, 4H, H2\ H7'), 7.28 (s, 1 H, ArH); MS (ESI): m/z 388 (M+H) ⁺ ; HRMS (ESI+) calcd for Ci ₆ H ₂₂ N ₉ 0 ₃ 388.1846, found 388.1838; Anal. HPLC (t _r 17.9 min).

Compound 3 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(1H-indol-2-yl)pyr azine-2- carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 11.09 (s, 1H), 10.82 (s, 1H), 8.82 (s, 2H), 7.92 (s, 2H), 7.55 (d, 1H, J= 7.7 Hz), 7.41 (d, 1H, J= 7.7 Hz), 7.12 (t, 1H, J= 7 Hz), 7.03 (t, 1H), 6.44 (s, 1H), 3.64 (t, J= 5.0 Hz, 4H), 1.73 (s, 4H), 1.50(s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.66, 156.22, 155.69, 152.98, 136.62, 134.89, 128.32, 125.72, 122.71, 120.49, 119.81, 111.74, 110.83, 102.31, 52.13, 27.88, 27.55. HRMS (M + H) ⁺ Anal for C ₂ oH ₂₈ N ₇ 0 ₃ , calc. mass: 393.2151, found mass: 393.2159. Anal HPLC (t _r =24.4 min). MP 152-154°C. Isolated 29.0 mg as an orange solid. Yield: 31% (2 steps, TFA salt).

Compound 5 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(4-(trifluoromethy l)phenyl) pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD): δ 7.68 (d, J= 8.0 Hz, 2H), 7.65 (d, J= 8.0 Hz, 2H), 3.38 (t, J= 5.0 Hz, 4H), 1.65 (s, 4H), 1.46 (s, 4H). ¹³ C NMR (CD ₃ OD): δ 167.9, 160.2, 155.8, 155.0, 145.6, 130.1, 130.0 (d, J = 32 Hz), 129.2, 125.8 (d, J = 2.7 Hz), 124.3 (d, J = 170), 112.9, 51.6, 28.6, 27.6. FT!R 3317.561 ', 1681.93, 1597.06, 1535.34, 1496.76, 1319.31 , 848.68. HRMS (M+ H) ⁺ Anal for Ci ₉ H ₂₃ F ₃ N ₇ 0, calc. mass: 422.1916, found mass: 422.1911. Anal. HPLC (t _r 27.9 min). Isolated 385 mg as a yellow solid. Yield 92% (free base). Compound 6- 3-amino-5-(azepan- 1 -yl)-N-carbamimidoyl-6-(isoquinolin-4-yl)pyrazine-2- carboxamide

¹ H NMR (500 MHz, CD ₃ OD) δ 10.47 (s, exchange with solvent), 9.57 (bs, 1H), 9.19 (s, exchange with solvent), 9.09 (s, exchange with solvent) 8.69 (bs, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.01 (t, J= 7.5 Hz, 1H), 7.91 (t, J= 7.5 Hz, 1H), 7.81 (d, J= 8.2 Hz, 1H), 3.37 (s, 2H), 3.23 (s, 2H), 1.53 (s, 4H), 1.44 (s, 4H). ¹³ C NMR (126 MHz, CD ₃ OD) δ 167.51, 157.74, 156.87, 156.36, 149.67, 137.60, 136.92, 136.87, 136.82, 131.61, 131.42, 129.044, 125.72, 124.73, 112.97, 51.58, 28.94, 27.75. HRMS (M + H) ⁺ Anal for C ₂ oH ₂₄ N ₇ OS, calc. mass: 410.1763, found mass: 410.1758. Anal. HPLC ( _r 22.7 min). MP 58-160°C. Isolated 31.2 mg as an orange solid. Yield 15% (TFA salt).

Compound 7 - 3-amino-5-(azepan-1-yl)-6-(benzo[b]thiophen-2-yl)-N-carbamim idoyl pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.48 (s, 1H), 8.92 (s, 2H), 8.76 (s, 2H), 7.60 (d, J= 7.5 Hz, 1H), 7.43 (d, J= 7.5 Hz, 1H), 7.31 (t, J= 7.5 Hz, 1H), 7.26 (t, J= 7.5 Hz, 1H), 7.20 (s, 1H), 3.50 (t, J= 5.75 Hz, 4H), 1.69 (s, 4H), 1.49 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 168.2. 164.9, 160.7, 156.5, 155.4, 144.1, 141.3, 141.2, 125.8, 125.5, 124.7, 123.5, 123.0, 113.5, 52.0, 29.2, 28.1. HRMS (M + H) ⁺ Anal for C ₂₀ H ₂₄ N ₇ OS, calc mass: 410.1763, found mass: 410.1758. Anal. HPLC (t _r 27.5 min). MP 220-224°C (dec) Isolated 320 mg as a yellow solid. Yield 78% (free base).

Compound 8 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(2,4-dimethoxyphen yl) pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.19 (s, 1H), 9.10 (bs, 2H), 7.79 (bs, 2H), 7.23 (d, J= 8.1 Hz), 6.58 (d, J= 8.1 Hz), 6.48 (s, 1H) 3.86 (s, 3H), 3.74 (s, 3H), 3.51 (s, 2H), 3.36 (s, 2H), 1.59 (s, 4H), 1.50 (s, 2H), 1.45 (s, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 165.95, 161.54, 157.83, 155.71, 155.40, 153.75, 130.62, 129.59, 122.11, 109.78, 104.99, 98.72, 55.67, 55.64, 50.62, 27.81 , 26.93. HRMS (M + H) ⁺ Anal for C ₂₀ H ₂₈ N ₇ O ₃ , calc mass: 414.2254, found mass 414.2269. Anal. HPLC (t _r 26.7 min). MP: 156-158 °C. Isolated 11.5 mg as a yellow oil. Yield 8% (2 steps, HCI salt).

Compound 9 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(thiophen-2-yl)pyr azine-2- carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.10 (s, 1H), 10.04 (bs, 2H), 8.08 (bs, 2H), 7.26 (d, J= 5 Hz, 1H), 7.03 (d, J= 3 Hz, 1H), 3.47 (t, J= 5.75 Hz, 4H), 1.68 (m, 4H), 1.49 (m, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.97, 156.30, 156.05, 153.39, 139.86, 128.44, 128.18, 125.24, 124.54, 111.07, 51.04, 27.82, 27.12. HRMS (M + H) ⁺ Anal for Ci ₆ H ₂₁ N ₇ 0: calc. mass: 360.1607, found mass: 360.1610. Anal. HPLC (f, 24.2 min). Isolated 1 .9 mg as a yellow oil. Yield 2% (2 steps, TFA salt).

Compound 10 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(2-fluoropyridin-3- yl)pyrazine-2-carboxamide.

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.42 (s, 1 H), 8.59 (bs, 4H), 8.17 (m, 2H), 7.29 (t, J = 5.4 Hz) 5.13 (bs, 2H), 3.38 (s, 4H), 1 .64 (s, 4H), 1 .47 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.45, 160.67, 158.76, 155.98, 155.90, 154.01 , 146.91 , 141 .41 , 124.19, 123.41 , 122.45, 1 1 1 .79, 50.40, 27.86, 27.16. LRMS m/z (M+ H) ⁺ 373. Anal HPLC (f _r 22.2 min). Isolated 17.7 mg as a yellow oil. Yield 13% (2 steps, TFA salt).

Compound 11 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(3,5-dimethoxyphenyl) pyrazine-2-carboxamide.

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.75 (s, 1 H), 8.81 (bs, 2H), 8.60 (bs, 2H), 6.73 (d, J = 2.2 Hz), 6.58 (t, J = 2.2 Hz, 1 H), 3.84 (s, 6H), 3.38 (s, 2H), 1 .64 (s, 4H), 1 .45 (s, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 167.04, 160.96, 156.35, 155.60, 153.77, 141.64, 132.92, 1 10.93, 106.46, 100.28, 55.92, 50.99, 27.95, 27.12. FTIR 3340.71 , 2924.09 1 ', 2854.65, 2360.87, 1681 .93, 1589.34, 1535.34, 1496.76, 1419.61 , 1242.16, 1 188.15, 1 149.57, 1095.57, 1064.71 , 1033.85, 709.80. HRMS (M + H) ⁺ Anal for C ₂₀ H ₂₈ N ₇ O ₃ : calc. mass 414.2254, found mass: 414.2244. Anal. HPLC (f, 25.8 min). MP: 138-140 °C. Isolated 43.9 mg as a yellow oil. Yield 26% (2 steps, HCI salt).

Compound 12 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(naphthalen-2-yl)p yrazine- 2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.31 (s, 1 H), 8.62 (bs, 2H), 8.29, (bs, 2H), 7.78 (m, 3H), 7.64 (dd, J = 8.5, 1 .5 Hz, 1 H,), 7.44 (m, 2H), 6.23 (bs, 2H), 3.38 t, J = 5.75, 4H), 1 .59 (s, 4H), 1 .42 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.49, 155.85, 155.71 , 153.67, 137.15, 133.15, 132.95, 132.22, 129.75, 128.67, 128.16, 127.93, 127.81 , 126.66, 126.50, 126.41 , 125.74, 123.48, 1 18.05, 1 10.94, 109.64, 51 .39, 27.81 , 27.07. HRMS (M + H) ⁺ Anal for C ₂ i H ₂₅ N ₈ 0: calc. mass: 404.2185, found mass: 404.2199. Anal. HPLC { 28.3 min). MP: 158-160 °C. Isolated 81 .4 mg as a yellow oil. Yield 26% (2 steps, HCI salt).

Compound 13 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(thiophen-3-yl)pyr azine-2- carboxamide.

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.31 (s, 1 H), 8.62 (bs, 2H), 8.29, (bs, 2H), 7.78 (m, 3H), 7.64 (dd, J = 8.5, 1 .5 Hz, 1 H,), 7.44 (m, 2H), 6.23 (bs, 2H), 3.38 t, J = 5.75, 4H), 1 .59 (s, 4H), 1 .42 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.66, 156.24, 156.02, 153.58, 140.12, 128.40, 127.79, 125.88, 123.57, 1 10.74, 51 .27, 27.92, 27.14. HRMS (M + H) ⁺ Anal for C ₁₆ H ₂₁ N ₇ 0, calc. mass: 360.1607, found mass: 360.1611. Anal HPLC (f _r = 25.2 min). 24.2 mg isolated as an orange oil. Yield 17% (2 steps, TFA salt).

Compound 14 - 3-amino-5-(azepan-1-yl)-6-(benzo[d][1,3]dioxol-5-yl)-N-carba mimidoyl pyrazine-2-carboxamide

¹ H NMR (500 MHz, DMSO) δ 10.39 (s, 1H), 8.71 (s, 2H), 8.27 (s, 2H), 7.31 (s, 1H), 6.96 (m, 2H), 6.08 (s, 2H), 3.40 (t, J= 5.75 Hz, 4H), 1.60 (s, 2H), 1.39 (s, 2H). ¹³ C NMR (126 MHz, DMSO) δ 166.23, 155.15, 154.75, 153.61, 147.26, 146.71, 133.39, 130.28, 121.43, 112.73, 108.47, 107.96, 101.15, 50.44, 27.21, 26.07. FTIR 3741.90, 3340.71, 2924.09, 2862.36, 2360.87, 2322.29, 1681.93, 1535.34, 1496.76, 1419.61, 1365.60, 1242.16, 1188.15, 1095.57, 671.23. HRMS (M + H) ⁺ Anal for C ₁₉ H ₂₄ N ₇ 0 ₃ , calc. mass: 398.1941, found mass: 398.1929. Anal. HPLC { 26.4 min). MP: 154-156 °C. Isolated 5.7 mg as a yellow oil. Yield 9% (2 steps, HCI salt).

Compound 15 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(2,6-dimethoxypyri din-3- yl)pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.28 (s, 1H), 8.77 (s, 2H), 8.14 (s, 2H), 7.61 (d, J = 8.0 Hz), 6.39 (d, J = 8.0 Hz, 1H), 3.94 (s, 3H), 3.87 (s, 3H), 3.52 (s, 2H), 3.35 (s, 2H), 1.60 (s, 4H), 1.51 (s, 2H), 1.44 (s, 2H). ¹³ C NMR (126 MHz, CDCI3) δ 166.00, 163.16, 159.19, 155.44, 153.54, 141.10, 127.98, 114.64, 110.19, 101.28, 53.82, 53.54, 50.39, 27.65, 26.81. HRMS (M + H) ⁺ Anal for calc. mass: 415.2206, found mass: 415.2191. Anal HPLC (f _r 26.1 min). MP: 152-154° C. Isolated 14.8 mg as a yellow oil. Yield 13% (2 steps, HCI salt).

Compound 16 - 3-amino-5-(azepan-1 -yl)-N-carbamimidoyl-6-(1 -(2-morpholinoethyl)-1 H- pyrazol-4-yl)pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.48 (s, 1H), 8.86 (s, 2H), 8.65 (s, 2H), 7.89 (s, 1H), 7.56 (s, 1H), 4.67 (s, 2H), 3.93 (s, 4H), 3.69 (s, 2H,), 3.47 (s, 4H), 3.42 (bs, 2H), 3.05 (bs, 2H,) 1.67 (s, 4H), 1.49 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.63, 156.35, 156.06, 153.63, 139.96, 130.58, 124.10, 122.90, 111.31, 63.78, 57.19, 53.29, 51.40, 46.38, 27.89, 27.17. HRMS (M + H) ⁺ Anal for C ₂ iH ₃₃ N ₁₀ O2, calc. mass: 457.2788, found mass: 457.2796. Anal. HPLC (t _r 19.0 min). MP: 157-159 °C. Isolated 22.3 mg as orange oil. Yield 20% (2 steps, HCI salt).

Compound 17 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(1-methyl-1H-pyraz ol-4- yl)pyrazine-2-carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.49 (s, 1H), 8.83 (s, 2H), 8.52 (s, 2H), 8.00 (s, 1H), 7.57 (s, 1H), 5.49 (bs, 2H), 3.93 (s, 3H), 3.50 (t, J = 5.75 Hz, 4H), 1.69 (s, 4H), 1.49 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.57, 156.35, 156.07, 153.40, 137.39, 130.73, 125.04, 121.70, 111.08, 51.54, 38.67, 27.85, 27.27. FTIR 3302.13, 2924.09, 2854.65, 2360.87, 1674.21, 1535.34, 1496.76, 1419.61, 1242.16, 1195.87, 1095.57, 725.23. HRMS (M + H) ⁺ Anal for calc. mass: 358.2104, found mass: 358.2118. Anal HPLC (f _r = 21.0 min). MP: 150-152 °C. Isolated 31.2 mg as a yellow oil. Yield 38% (2 steps, HCI salt).

Compound 18 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(3,6-dimethoxypyri dazin-4- yl)pyrazine-2-carboxamide

¹ H NMR (500 MHz, CD ₃ OD) δ 10.38 (s, exchange with solvent), 8.77 (s, exchange with solvent), 8.21 (s, exchange with solvent), 7.35 (s, 1 H), 4.06 (s, 3H), 3.99 (s, 3H), 3.35 (s, 4H), 1.66 (s, 4H), 1.55 (s, 2H), 1.45 (s, 2H). ¹³ C NMR (126 MHz, CD ₃ OD) δ 167.31, 164.10, 160.89, 156.90, 156.75, 156.10, 136.33, 123.95, 121.38, 112.19, 55.44, 55.26, 50.98, 28.89, 27.92. HRMS (M + H) ⁺ Anal for C ₁₈ H ₂₆ N ₉ 0 ₃ : calc. mass: 416.2159, found mass: 416.2150. Anal HPLC (f _r = 19.0 min). MP: 202-204 °C. Isolated 20.9 mg as a yellow solid. Yield 17% (2 steps, TFA salt).

Compound 19 - 3-amino-5-(azepan-1-yl)-6-(benzofuran-2-yl)-N-carbamimidoylp yrazine- 2-carboxamide

¹ H NMR (CD ₃ OD): δ 7.64 (d, J= 7.5 Hz, 1H), 7.50 (d, J= 8.0 Hz, 1H), 7.31 (t, J= 8.0 Hz, 1H), 7.26 (t, J= 7.5 Hz, 1H), 7.05 (s, 1H), 3.51 (t, J= 5.0 Hz, 4H), 1.71 (s, 4H), 1.54 (s, 4H); ¹³ C NMR (CD ₃ OD): δ 167.4, 164.3, 154.6, 154.5, 154.4, 154.1, 129.7, 124.9, 123.7, 121.6, 121.1, 112.4, 111.5, 105.6, 50.3, 28.6, 27.3. HRMS (M + H) ⁺ Anal for C20H24N7O2, calc. mass: 394.1991, found mass: 394.1986. Anal HPLC (f _r 27.5 min). MP: °C. Isolated 338 mg as a yellow solid. Yield 86% (free base).

Compound 20 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(furan-3-yl)pyrazi ne-2- carboxamide

¹ H NMR (500 MHz, CDCI ₃ ) δ 10.42 (s, 1H), 8.54 (s, 4H), 7.63 (s, 1H), 7.42 (s, 1H), 6.63 (s, 1H), 5.35 (bs, 2H), 3.52 (t, J= 5.75 Hz, 4H), 1.68 (s, 4H), 1.49 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.44, 156.29, 155.84, 153.61, 143.19, 141.10, 125.32, 124.87, 110.82, 110.72, 51.37, 27.86, 27.14. FTIR 3294.42, 2924.09, 2854.65, 2360.87, 1674.21, 1535.34, 1496.76, 1419.61, 1242.16, 1195.87, 1095.57, 725.23. HRMS (M + H) ⁺ Anal for C ₁₆ H ₂₂ N ₇ 0 ₂ , calc. mass: 344.1835, found mass: 344.1837. Anal HPLC (f _r 25.5 min). MP 146-148 °C. Isolated 40.8 mg as a yellow oil. Yield 35% (2 steps, HCI salt). Compound 21 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(furan-2-yl)pyrazine-2- carboxamide

¹ H NMR (CD ₃ OD): δ 7.50 (s, 1 H), 6.55 (d, J = 3.0 Hz, 1 H), 6.50 (s, 1 H), 3.37 (t, J = 6.0 Hz, 4H), 1 .64 (s, 4H), 1 .47 (s, 4H); ¹³ C NMR (CD ₃ OD): δ 168.1 , 160.7, 156.3, 156.0, 153.4, 142.8, 122.4, 1 12.6, 1 12.4, 109.9, 50.9, 29.2, 27.8. FTIR 3332.99, 1681 .93, 1597.06, 1535.34, 1365.60. HRMS (M + H) ⁺ Anal for Ci ₆ H ₂₂ N ₇ 0 ₂ , calc. mass: 344.1835, found mass: 344.1830. Anal HPLC (f _r 24.3 min). Isolated 310 mg as a yellow solid. Yield 91 % (free base).

Compound 22 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(pyrimidin-5-yl)pyrazine-2- carboxamide

¹ H NMR (CD ₃ OD): δ 8.98 (s, 1 H), 8.97 (s, 2H), 3.35 (t, J = 5.5 Hz, 4H), 1 .67 (s, 4H), 1 .47 (s, 4H); ¹³ C NMR (CD ₃ OD): δ 162.2, 156.5, 156.3, 155.7, 155.5, 154.0, 153.5, 135.2, 122.2, 51 .0, 28.0, 27.1 . HRMS (M + H) ⁺ Anal for C ₁₆ H ₂₂ N ₉ 0, calc. mass: 356.1947, found mass: 356.1942. Anal HPLC (f _r 20.1 min). MP: °C Isolated 305 mg as a yellow solid. Yield 87% (free base).

Compound 23 - 3-amino-5-(azepan- 1 -yl)-N-carbamimidoyl-6-(4-fluorobenzofuran-2- yl)pyrazine-2-carboxamide

¹ H NMR (500 MHz, CD ₃ OD) δ 10.47 (s, exchange with solvent), 8.98 (s, exchange with solvent), 8.33 (s, exchange with solvent), 7.36 (t, J = 8.7Hz, 1 H), 7.32 (q, J = 5.5Hz, 1 H), 7.22 (s, 1 H), 7.02 (t, J = 8.7Hz,1 H), 3.44 (t, J = 5.8 Hz, 4H) 1 .72 (s, 4H), 1 .51 (s, 4H). ¹³ C NMR (126 MHz, CD ₃ OD) δ 167.32, 158.20, 157.38, 156.95, 156.89, 156.23, 156.18, 155.73, 126.57, 121 .13, 1 12.13, 109.75, 108.65, 102.44, 51 .08, 29.02, 27.88. HRMS (M + H) ⁺ Anal for C ₂₀ H ₂₂ FN ₇ O ₂ , calc. mass: 412.1897, found mass: 412.1909. Anal HPLC (f _r 25.6 min). MP: 132-136°C Isolated 15.2 mg as a orange solid. Yield 26% (TFA salt).

Compound 24 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(2-methoxypyrimidin-5- yl)pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD): δ 8.77 (s, 2H), 4.08 (s, 3H), 3.46 (t, J = 5.5 Hz, 4H), 1 .72 (s, 4H), 1 .54 (s, 4H). ¹³ C NMR (CD ₃ OD): δ 166.7, 164.1 , 163.4, 158.1 , 154.8, 153.6, 128.6, 124.2, 1 12.8, 54.3, 27.7, 26.7. FTIR 3332.99, 2924.09.37 1 ', 2854.65, 2360.87, 1681 .93, 1589.34, 1535.34, 1496.76, 1473.62, 1404.18, 1280.73, 1242.16, 1 195.87,1 188.15, 1018.41 , 671 .23. HRMS (M + H) ⁺ Anal for Ci ₇ H ₂₄ N ₉ 0 ₂ , calc. mass: 386.2053, found mass: 386.2048. Anal HPLC (f _r 20.7 min). MP: 148-150 °C. Isolated 345 mg as a yellow solid. Yield 90% (free base). Compound 25 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(2-(methylamino)py rimidin- 5-yl)pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD): δ 1.51 (s, 4H), 1.69 (s, 4H), 2.97 (s, 3H), 3.48 (t, J = 5.6 Hz, 4H), 8.47 (s, 2H); ¹³ C NMR (CD ₃ OD): δ 28.0, 28.5, 29.0, 52.0, 1 13.5, 124.4, 127.3, 155.4, 156.7, 158.3, 158.5, 162.9, 168.3; MS (ESI): m/z 385 (M+H) ⁺ , 402 (M+Na) ⁺ ; HRMS (ESI+) calcd for Ci ₇ H ₂ 5N ₁₀ O 385.2213, found 385.2195; Anal. HPLC (t _r 19.0 min); Yellow solid. Yield 85% (free base).

Compound 26 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(2,4-dimethoxypyrimidin-5- yl)pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD) δ 10.76 (s, 1 H), 9.27 (s, 2H), 8.51 (s, 2H), 8.48 (s, 2H), 4.08 (s, 3H), 3.35 (t, J = 5.75 Hz, 4H), 1 .66 (s, 4H), 1 .46 (s, 4H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 166.62, 163.99, 157.84, 156.07, 153.87, 127.83, 124.71 , 1 12.51 , 55.59, 51 .59, 27.76, 27.13. FTIR 3332.99, 2931 .80, 2854.65, 2360.87, 1651 .07, 1597.06, 1535.34, 1496.76, 1473.62, 1404.18, 1280.73, 1242.16, 1 172.72, 1010.70, 671 .23. HRMS (M + H) ⁺ Anal for C ₁₈ H ₂₆ N ₉ 0 ₃ , calc. mass: 416.2159, found mass: 416.2158. Anal HPLC (f _r 21 .4 min). MP: 184-186 °C. Isolated 64.5 mg as a yellow solid. Yield 54% (2 steps, HCI salt).

Compound 27 - 3-amino-5-(azepan- 1-yl)-N-carbamimidoyl-6-(2-

(isopropylamino)pyrimidin-5-yl)pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD): δ 1.25 (d, J = 6.5 Hz, 6H), 1 .51 (s, 4H), 1 .69 (s, 4H), 3.47 (t, J = 5.6 Hz, 4H), 4.14 (septet, J = 6.8 Hz, 1 H), 8.46 (s, 2H); ¹³ C NMR (CD ₃ OD): δ 21.6, 26.8, 27.8, 42.8, 50.9, 1 12.3, 123.2, 126.2, 154.2, 155.5, 157.1 , 158.8, 160.6, 167.1 ; MS (ESI): m/z 413 (M+H) ⁺ , 435 (M+Na) ⁺ ; HRMS (ESI+) calcd for Ci ₉ H ₂₉ N ₁₀ O 413.2526, found 413.2525, Anal. HPLC (f _r 20.8 min); Yellow solid. Yield 89% (free base).

Compound 28 - 3-amino-5-(azepan-1-yl)-N-carbamimidoyl-6-(2-((2- hydroxyethyl)amino)pyrimidin-5-yl)pyrazine-2-carboxamide

¹ H NMR (CD ₃ OD): δ 1.50 (s, 4H, H4', H5'), 1.68 (s, 4H, H3', H6'), 3.45 (t, J = 5.8 Hz, 4H, H2', H7'), 3.54 (t, J = 5.6 Hz, 2H, CH ₂ NH) 3.72 (t, J = 5.6 Hz, 2H, CH ₂ OH), 8.48 (s, 2H, ArH); ¹³ C NMR (CD ₃ OD): δ 27.8, 28.9, 44.6, 51.9, 61.7, 1 17.5, 124.7, 126.5, 154.9, 156.3, 158.2, 160.8, 162.3, 167.1 ; MS (ESI): m/z 415 (M+H) ⁺ ; HRMS (ESI+) calcd for Ci ₈ H ₂₇ N ₁₀ O ₂ 415.2318, found 415.2324, Anal. HPLC ( 18.7 min); Yellow solid. Yield 86% (free base). Compound 29 - 3-amino-6-(2-aminopyrimidin-5-yl)-5-(azepan-1-yl)-N- carbamimidoylpyrazine-2-carboxamide

¹ H NMR (DMSO-cfe): δ 1.41 (s, 4H, H4', H5'), 1.60 (s, 4H, H3', H6'), 3.38 (t, J = 5.9 Hz, 4H, H2', H7'), 6.73 (br s, 2H, NH ₂ ), 7.25 (br s, 1 H, NH), 8.40 (s, 2H, ArH); ¹³ C NMR (DMSO-d ₆ ): δ 27.0 (2 X CH ₂ , C4', C5'), 28.0 (2 X CH ₂ , C3', C6'), 51.1 (2 X CH ₂ , C2', C7'), 1 12.9 (ArC), 123.6 (ArC), 126.0 (ArC), 153.7 (ArC), 154.9 (ArC), 157.5 (2 X ArCH), 159.1 (C=N), 162.9 (ArC), 167.5 (C=0); MS (ESI): m/z 371 (M+H) ⁺ ; HRMS (ESI+) calcd for C ₁₆ H ₂₃ N ₁₀ O3 371.2056, found 371 .2067; Anal. HPLC (t _r 18.6 min). Yellow solid (Yield 78%).

Compound 30 - 5-(3-amino-5-(azepan-1-yl)-6-(benzofuran-2-yl)pyrazin-2-yl)- 1,2,4- oxadiazol-3-amine

The title compound was prepared according to the following scheme:

Hydroxylamine (50%) in water (40 μΙ_ 0.6 mmol) was added to cyanamide (50%) in water (25 mg, 0.6 mmol) in methanol (3 mL) and the mixture was refluxed for 4.5 hours. The mixture was concentrated to remove methanol/water, followed by co-evaporation with methanol (2 x 5 mL) to afford hydroxyguanidine as an amber

011 which was used without further purification (MS (ESI) m/z 76 |M+H]). Sodium metal (14 g, 0.6 mmol) was added to a suspension of molecular sieves (type 4A,

12 g) in dry methanol (10 mL) stirred under nitrogen. After 15 min at room temperature a solution of hydroxyguanidine (0.6 mmol) in methanol (2 mL) was added and stirring was continued a further 1 h. A solution of methyl 3-amino-5- (azepan-1 -yl)-6-(benzofuran-2-yl)pyrazine-2-carboxylate (146 mg, 0.4 mmol) in MeOH (3 mL) was added to the mixture, which was then heated at reflux for 12 h. After removal of the molecular sieves by filtration, the filtrate was evaporated, and the residue was partitioned between dichloromethane and water. The organic layer was separated and the aqueous layer was extracted with dichloromethane (2 x 200 mL). The combined organic layers were dried and evaporated. The residue was purified by silica gel column chromatography using 20-40% EtOAc/Petrolium spirit to give 30 as a yellow solid (12.4 mg, 8% yield); MP: 122-124° C; ¹ H NMR (CDCI ₃ , 400): 7.59 (d, 1 H), 7.48 (d, 1 H), 7.28 (t, 1 H), 7.24 (t, 1 H), 7.00 (s, 1 H), 6.32 (br s, 2H), 4.38 (s, 2H), 3.49 (t, 4H), 1 .70 (s, 4H), 1 .50 (dr t, 4H); ¹³ C NMR (126 MHz, CDCI ₃ ) δ 172.15, 167.23, 154.70, 154.34, 154.31 , 151.51 , 128.99, 124.20, 123.00, 122.07, 121 .13, 1 1 1 .27, 109.93, 105.12, 77.34, 77.02, 76.70, 49.97, 27.99, 27.13; ES TOF MS m/z (M + H) ⁺ 392.

Example 3 - Determination of uPA affinity and inhibitory potency for selected compounds

uPA inhibitory potency was determined for compounds 1 to 29 using a 96-well plate in vitro enzymatic assay. A commercially available fluorogenic substrate (Cat # 672159 - Urokinase substrate III (Fluorogenic) (Merck Millipore, Merck KgaA, Darmstadt, Germany)) was diluted in HEPES assay buffer (20 mM HEPES, 100 mM NaCI, 0.5 mM EDTA, 0.01 % (v/v) Tween-20, pH 7.6) to give a final concentration of 250 μΜ used with active low molecular weight uPA (Cat # U4010 - Urokinase from Human Kidney Cells, Sigma-Aldrich, St. Louis, Missouri, USA) in all assays. Briefly, compounds were dissolved in DMSO to create 50 mM stock solutions which were diluted with buffer in series in a separate 96-well plate and transferred to a Greiner CELLSTAR black 96 well microtitre plate (Cat. # M9936, Greiner Bio-One GmbH, Kremsmunster, Austria), giving a final DMSO concentration of 1 % v/v. Two compounds were assayed per plate with triplicate measurements taken for each inhibitor concentration. Assay blanks (no enzyme) were included to account for the intrinsic fluorescence of some inhibitors. Assay plates were read at 355 nm (excitation), 460 nm (emission) using a BMG Labtech POLARStar Omega Fluorescence Plate Reader (BMG Labtech, Ortenberg, Germany), with cycle time 60 s, 45-60 cycles and 3s orbital shaking before each read. Absorbance values were recorded for time points (AF/min) taken from the linear region of plots of absorbance versus time and IC ₅₀ values were calculated from sigmoidal dose response curves of absorbance versus log nhibitor] using GraphPad PRISM v6.0 (Graphpad Software, La Jolla, California, USA). The results are shown in Table 1 and Figure 1 .

Inhibitory constants (K,) against uPA were determined for select compounds using a 96- well plate in vitro enzyme assay. Urokinase substrate III (Fluorogenic) (Cat #672159 - Merck Millipore, Merck KgaA, Darmstadt, Germany) was serially diluted in HEPES assay buffer (20 mM HEPES, 100 mM NaCI, 0.5 mM EDTA, 0.01 % (v/v) Tween-20, pH 7.6) to give final concentrations between 2.0 μΜ and 250 μΜ. 200x inhibitor stocks in DMSO were diluted 1 in 100 into HEPES assay buffer to yield a 2x working stock in 1 % DMSO. The diluted substrate was added to a Greiner CELLSTAR black 96 well microtitre plate (Cat. # M9936, Greiner Bio-One GmbH, Kremsmunster, Austria), followed by addition of 2x working stock drug dilutions or equivalent volumes of assay buffer for controls such that all wells contained 200 μΙ solution, 0.5% DMSO v/v. The assay plate was allowed cool on a bed of ice and LMW Human uPA from a 900 nM stock was diluted in cold assay buffer to give a concentration of 15 nM. 10 μΙ of 15 nM enzyme stock was added to appropriate wells (final enzyme concentration = 0.75 nM) and the plate immediately read at ex. 355 nm, em. 460 nm using a BMG Labtech POLARStar Omega Fluorescence Plate Reader (BMG Labtech, Ortenberg, Germany), cycle time 60 s, 45-60 cycles, 3s orbital shaking before each read). Blank subtracted data from the linear portion of the kinetic curve was used to determine initial velocity and data plotted using GraphPad PRISM v6.0 (Graphpad Software, La Jolla, California, USA). Data was fitted using the 'competitive inhibition' function to determine K, for each inhibitor. The results are shown below in Table 1 .

Table 1 : uPA inhibitory potencies of compounds 1 -29

Compound uPA IC ₅₀ (nM) [n] uPA K, (nM) [n]

HMA 2206 ± 298 [2] 2408 ± 49.5 [2]

1 26717 1 2130 [2] -

2 13971 ± 2751 [2] -

3 9214 ± 1689 [2] -

5 6914 ± 316 [1 ] -

6 5751 ± 312 [2] -

7 5375 ± 487 [2] -

8 3571 ± 86 [2] -

9 3297 ± 408 [1 ] -

10 2889 ± 405[1 ] -

11 2143 ± 504 [1 ] -

12 1869 ± 74 [2] -

13 1669 ± 277 [2] -

14 1518 ± 246 [1 ] -

15 1437 ± 23[2] 1066 ± 168 [3]

16 1070 ± 108 [2] -

17 825 ± 15 [2] 621 ± 31 [3]

18 512 ± 55 [2] 310 ± 18 [3]

19 297 ± 16 [2] 139 ± 8 [3]

20 270 ± 16 [2] 258 ± 32 [3]

21 254 ± 33 [2] 235 ± 17 [3]

22 175 ± 26 [2] 82 ± 9 [3]

23 143 ± 54 [2] 123 ± 14 [3]

24 86 ± 8 [2] 77 ± 4 [3]

25 70 ± 7 [2] -

26 69 ± 4 [4] 56 ± 5 [3]

27 68 ± 14 [2] -

28 66 ± 4 [2] -

29 34 ± 5 [21 28 ± 2 [21

HMA is hexamethylene amiloride.

Numbers in square brackets indicate number of identical repeat results used to calculate mean. Errors are presented as standard errors of the mean. The data presented in Table 1 and Figure 1 illustrates the gains in uPA inhibitory activity achieved by substitution at the 6-position of HMA. Increases approaching 3-orders of magnitiude relative to HMA were observed across IC ₅₀ and K, experiments (e.g. 29 IC ₅₀ 34 nM, K, 28 nM). X-ray crystallographic analysis (presented in Example 16) revealed that increases in affinity were contributed to by the formation of favourable interactions with the S1 β subsite of the uPA active site. The finding that a variety of structurally unrelated 6-aryl substituents were well tolerated and resulted in increased activity illustrates the targetability of S1 β for the design of amiloride-based small molecule inhibitors of uPA. Given the central role of uPA overexpression as a determinant of cell invasiveness across a variety of metastatic cancers, compounds of formula (I) showing increased potency against uPA would be expected to show improved efficacy as antimetastatic cancer treatments relative to less potent related compounds (e.g. amiloride and HMA).

Example 4 - Determination of effect of compounds 19 and 26 on neoplastic cell proliferation and viability

In order to the measure the cytotoxic effects of compounds of the formula (I) on human cells, MDA-MB-231 breast adenocarcinoma cell viability and LDH release was measured following treatment with 19 or 26 (Figure 2). Cell viability was determined using the CellTitre 96 ^® Aqueous One Solution Cell Proliferation Assay (Cat # G3581 , Promega Corporation, Fitchburg, Wisconsin, USA). Sub-confluent cells (« 70-80% confluent) were harvested and counted and dissociated into a single cell suspension as described above. Cells were seeded at a density of 5000 cells/well (final volume 90 μΙ) into a Greiner CELLSTAR ^® 96 well plate (Greiner Bio-One GmBH, Kremsmunster, Austria) via multichannel pipette and incubated for 18h prior to the addition of compounds. Compounds were serially diluted from 20-50 mM DMSO stocks in RPMI 1640 media containing 5% v/v heat inactivated FBS in a separate 96-well plate under sterile conditions to give 10x stocks (10 nM-1 mM) on the day of compound addition. 10 μΙ of the 10x compound/media stocks or matched vehicle/media solutions (for vehicle controls) were then transferred via multichannel pipette into the assay plate containing 90 μΙ cell suspension via multichannel pipette to give the treated assay plate at the final 1 X drug concentrations (1 nM-100 μΜ, n = 4 wells at each concentration). Relevant drug blanks or vehicle media blanks were included at each concentration to correct for the intrinsic colour of the compounds and of the Phenol Red containing media. 200 μΙ media was added to wells A1 -12 and H1 -12 to decrease evaporation and were not used in the assay itself. Compound-treated plates were incubated for 48h prior to development. After 48h treatment time, plates were removed from the incubator and 20 μΙ CellTitre 96 ^® Aqueous One Solution Cell Proliferation Assay (Cat # G3581 , Promega Corporation, Fitchburg, Wisconsin, USA) was added to each well. Plates were incubated for a further 2h prior to reading at 490 nm using a SpectraMax Plus 384-well plate reader (Molecular Devices LLC, Sunnyvale, California, USA). Blank subtracted data was analyzed and graphed using GraphPad PRISM v6.0 (GraphPad Software, San Diego, California, USA). Concentrations of compound 19 exceeding 10 μΜ significantly decreased cell viability, with total cell death observed at 100 μΜ (Figure 2A). Compound 26 did not significantly effect cell viability at concentrations up to 100 μΜ (Figure 2B).

Lactate Dehydrogenase (LDH) is a cytosolic enzyme whose release can be used to measure plasma membrane damage caused by drug treatment and thus can be used to determine the cytotoxic activity of a compound. In order to confirm results obtained from MTS assays, MDA-MB-231 cells were assayed for LDH release following treatment with compounds 19 or 26 (Figures 2C and D). Assay and compound dilution plates were prepared as described for the MTS assay described in Example 4. After 48h treatment time assay plates were removed from the incubator and 5 μΙ of Lysis solution (Bottle 3) of the Roche Cytotoxicity Detection Kit ^(PLUS) (LDH) (Roche Diagnostics Australia Pty. Ltd., NSW, Australia), was added to cell lysis controls. The plate was gently mixed by hand in a circular motion atop a bench (similar to the manner in which one might wax a car) for 30 s and incubated for a further 15 min. During this period 2.5 μΙ Catalyst solution (Bottle 1 ) was added to 1 12.5 μΙ Dye solution (Bottle 2) to form Reaction mixture for each well to be assayed. The plate was removed from the incubator and 100 μΙ Reaction mixture added to each well. The plate was then wrapped in aluminium foil and incubated for 30 min. After 30 min 50 μΙ Stop solution was added to each well and read at 490 nm using a SpectraMax Pius 384-weil plate reader (Molecular Devices LLC, Sunnyvale, California, USA). Blank subtracted data was analyzed and graphed using GraphPad PRISM v6.0 (GraphPad Software, San Diego, California, USA). Treatment of cells with 100 μΜ 19 caused a significant increase in LDH release relative to lower concentrations of 19 and vehicle-treated control cells (Figure 2C). Treatment with compound 26 did not increase LDH release at concentrations up to 100 μΜ (Figure 2D).

Taken together, these results show that compounds 19 and 26 do not possess potent cytotoxic effects against a mammalian cancer cell line. These findings are significant as the compounds of formula (I) will be useful as non-cytotoxic antimetastatic/anticancer drugs for invasive cancers in which uPA-mediated plasminogen activation is upregulated. Example 5 - Characterization of apoptotic effects of compound 19 on human embryonic kidney cells

In order to determine whether the cytotoxic effects of compound 19 were due to the initiation of apoptosis, pro-apoptotic caspase 3 and 7 activity was quantitated in HEK- 293 cells post-treatment using the Apo-ONE ^® Homogeneous Caspase-3/7 Assay (Figure 3, Cat # G7790, Promega Corporation, Fitchburg, Wisconsin, USA). Sub- confluent HEK-293 cells were harvested and seeded into a black Greiner CELLSTAR ^® 96 well plate (Cat # 655079, Greiner Bio-One GmBH, Kremsmijnster, Austria) at a density of 5000 cells/well in a volume of 40 μΙ RPMI 1640 media with incubation for 18h. On day of treatment 5x compound stocks in 5%DMSO/media were prepared in a separate 96-well plate under sterile conditions. 10 μΙ of each 5x compound stock was added to 40 μΙ of cell suspension on assay plate to give 1 X compound dilutions in 1 % v/v DMSO (1 -30 μΜ) and the plate incubated for 5 h (time sufficient to trigger detectable induction of apoptosis according to manufacturers instructions). Using the Apo-ONE® Homogeneous Caspase-3/7 Assay Kit 30 μΙ of Z-DEVD-R1 10 Caspase substrate was diluted into 2970 μΙ Assay buffer and 50 μΙ of the mixture added to each assay well and the plate shaken using a plate shaker at 300 rpm for 2 h followed by reading using a POLARstar OMEGA Fluorescence Plate reader (BMG-Labtech GmBH, Ortenberg, Germany) ex. 480 nm, em 520 nm. Blank subtracted data was analyzed and graphed using GraphPad PRISM v6.0 (GraphPad Software, San Diego, California, USA). Caspase 3/7 activity was significantly increased in HEK-293 cells treated with 30 μΜ compound 19 relative to cells treated with lower concentrations of 19, HMA or vehicle (Figure 3). The result shows that the cytotoxic effects of compound 19 at high doses are due, at least in part, to the initiation of apoptosis.

Example 6- Evaluation of selectivity of selected compounds of formula (I) against representative trypsin-like serine proteases

In order to the measure the on-target selectivity of compounds of formula (I) for uPA, certain compounds were screened against a variety of related trypsin-like serine proteases using a chromogenic in vitro assay (Figure 4 and Table 2). For experiments using uPA (Cat # U4010, Sigma-Aldrich, St. Louis, Ml, USA), trypsin, tPA, thrombin and plasmin (all from Marker Gene Technologies Inc., Eugene, OR, USA) concentrated stocks were prepared in dH ₂ 0 and stored at -80° C until thawed for use. Thawed enzyme stocks were maintained at -20° C between each respective assay using a Nalgene Labtop Cooler Jr (Cat# 51 15-0012, Thermo-Fisher Scientific, Waltham, Massachusetts, USA). A 312.5 μΜ stock solution of S-2288 chromogenic substrate 2288 (D-lle-L-Pro-l-Arg-p-nitoraniline, Chromogenix, Massachusettes, USA) was prepared in dH ₂ 0 and aliquoted with storage at -20° C. Assay buffer (10 mM HEPES, 150 mM NaCI, 0.01 % v/v Tween-20, pH 7.4) was prepared and used within 7 days of preparation with storage at 4° C. On the day of experimentation compound stocks (20-50 mM in anhydrous DMSO) were serially diluted in assay buffer in a clear Greiner CELLSTAR ^® 96 well plate on ice (Greiner Bio-One GmBH, Kremsmunster, Austria) to give each dilution at 10X the final assayed concentration. In a separate black Greiner CELLSTAR ^® 96 well plate (Greiner Bio-One GmBH, Kremsmunster, Austria) residual volumes of assay buffer were added via multichannel pipette to compound fluorescence blanks and inhibited enzyme control wells as appropriate. Next 80 μΙ of dilute fluor (250 μΜ final) was added to all wells, followed by 10 μΙ of 10x compound dilution to appropriate wells. Immediately prior to assay concentrated enzyme stocks were diluted to 100 nM and 10 μΙ of this dilution were added to all relevant wells, initiating reaction. DMSO was present at a final concentration of 1 % v/v. Change in absorbance overtime at 405 nm was measured at 37°C using a Molecular Devices SpectraMax Plus 384-well plate reader (Molecular Devices LLC, Sunnyvale, California, USA). IC ₅₀ values were determined by plotting percentage of residual activity (V0) versus log drug concentration and fitted to a sigmoidal dose response curve using GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA). For experiments using Human Plasma Kallikrein, Factor Xa, Factor Xia or Activated Protein C the chromogenic substrates S-2366 (pyroGlu-Pro- Arg-pNA) Human Plasma Kallikrein, Factor Xla and Activated Protein C), S-2288 (H-D- lle-Pro-Arg-pNA, Factor Xia) or S-2444 (pyroGlu-Gly-Arg-pNA, uPA H99Y) were used (all substrates sourced from Chromogenix, Massachusettes, USA). All enzymes were soruced from Marker Gene Technologies, Inc., Eugene, OR, USA. Enzymes were used at the following final cocnentrations: Human Plasma Kallikrein 23.5 nM, Factor Xa 10 nM, Activated Protein C 10 nM or Factor Xia 2 nM. Concentrated enzyme stocks were prepared in dH ₂ 0 and stored at -80° C until thawed for use. Experiments were conducted as described above for experiments using other trypsin-like serine proteases. DMSO was present at a final concentration of 1 % v/v. Change in absorbance overtime at 405 nm was measured at 37°C using a BioTek Synergy 4 384-well plate reader (BioTek Instruments Inc., Winooski, Vermont, USA). IC ₅₀ values were determined by plotting percentage of residual activity (V0) versus log drug concentration and fitted to a sigmoidal dose response curve using GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA).

The results indicate that, in general, compounds of the formula (I) show a high degree of selectivity for uPA over other related enzymes from the trypsin-like serine protease class (Table 2 and Figure 4). Across those compounds tested, compounds of formula (I) exhibited selectivities in the 20-1 OO's of fold range. Based on these results the compounds described would not be expected to cause toxicities through the inhibition of these related off-targets.

Table 2: Evaluation of compound selectivity against a variety of trypsin-like serine proteases via a chromogenic enzyme activity assay

Example 7 - Inhibition of Epithelial Sodium Channel (ENaC) activity by amiloride, HMA and compounds 5, 19, 23 and 24

Inhibition of epithelial sodium channels (ENaC) is responsible for the diuretic and K ⁺ - sparing effects of amiloride in mammals. The major and most common side-effect associated with amiloride treatment are cardiac arrhythmias caused by amiloride- induced hyperkalemia. For this reason, ENaC inhibition is not desirable for amiloride- derived anticancer therapeutics. In order to determine whether 6-substitution of HMA increased activity against ENaCs, certain compounds were screened against HEK-293 cells transiently expressing the α, β and γ subunits of Human ENaC using a fluorescent membrane potential dye kit and an automated fluorescence plate reader (Figure 5, FLIPR ^TETRA , Molecular Devices, Sunnyvale, California, USA). HEK-293 Cells were be cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and appropriate selection antibiotics. Semiconfluent cells were transfected with cDNA for ENaC α, β and γ subunits (genes SCNN1 A, SCNN1 B and SCNN1 G) prior to experimentation. Cells were plated at a density of 20,000 cells/well in a 384-well black wall, flat clear bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine Multiwell Cell Culture Plate) and incubated at 37 ^e C overnight. Following manufacturer instructions, cells were loaded with 20 μί of membrane potential dye solution for 30 min at 37 °C from the FLIPR Membrane Potential Red Dye Fluorescence Assay Kit (Molecular Devices, Sunnyvale, California, USA). This assay allows the fluorometric detection of changes in membrane potential following modulation of transmembrane ion channel/exchanger activity, namely antagonism of stimulated ENaC activity in this case. Drug, vehicle and control solutions were prepared from frozen stocks in DMSO on the day of experimentation through dilution into HEPES-buffered physiological salt solution (NaCI 137 mM, KCI 4 mM, CaCI ₂ 1 .8 mM, MgCI ₂ 1 mM, HEPES 10 mM, Glucose 10 mM, DMSO 0.3% v/v, pH 7.4) in a 384-well microtiter plate ready for addition to the assay plate by the FLIPR ^TETRA instrument. All amiloride derivatives were tested at 8 concentrations (n = 4 wells at each concentration). Positive control cells were stimulated with 1 μΜ S-3969 (ENaC agonist, n = 4 wells). Negative control cells were first stimulated with 1 μΜ S-3989 and then treated with 30 μΜ benzamil (potent ENaC antagonist, n = 4 wells). Following dye loading cells were preincubated with 5 μΙ drug, vehicle or control solutions for 5 min. Next 5 μΙ of S-3939 solution (final [S-3969] = 1 μΜ) was added to stimulate ENaC activity. All data was normalized to agonist-treated positive and antagonist-treated negative controls such that agonist-treated signal equaled 100% activity and antagonist-treated signal equaled 0% activity. Normalized data was used to determine IC ₅₀ values for each amiloride derivative using the log[inhibitor]-noramlized response variable slope algorithm in GraphPad PRISM v6.0 (Graphpad Software, La Jolla, California, USA). No significant difference was found between any compound of the formula (I) and HMA, indicating that 6-substitution of HMA does not effect the low affinity of the parent structure for human ENaC (Figure 5). In comparison, amiloride showed significant inhibition of agonist-stimulated ENaC activity under these conditions. As such, compounds of the formula (I) would not be expected to possess ENaC-mediated diuretic and K ⁺ -sparing effects in vivo.

Example 8 - Effects of Amiloride, HMA, 19 and 24 on diuresis and urinary K ⁺ /Na ⁺ levels in Sprague Dawley Rats

In order to confirm that the absence of ENaC inhibitory potency (Example 7) corresponded to decreased effects on diuresis and urinary K7Na ⁺ levels, amiloride, HMA, 19 and 24 were tested in male Sprague Dawley rats (n = 8 per group). With the exception of the vehicle control group all animals received a 25 mg/kg IP injection (1 ml/kg) of acetazolamide, a carbonic anhydrase inhibitor with diuretic and naturetic properties. Following acetazolamide administration animals were dosed with 1 .5 mg/kg of either amiloride, HMA, 19 or 24 via IV injection (1 ml/kg). Animals were housed in metabolic cages and urine collected for 6 h. In line with expectation amiloride massively decreased urinary K ⁺ , concomitantly increasing urinary Na ⁺ (Figure 6). Amiloride increased urine flow by 51 %. In comparison, HMA, 19 and 24 caused very modest decreases in urinary K ⁺ relative to animals treated with acetazolamide alone and no significant effects on urinary Na ⁺ or urine flow. These results suggest the abrogation of ENaC activity by 5 or 5,6-disubstitution of amiloride removes the potent diuretic and antikaliuretic effects of this drug. This result provides additional evidence for the assertion that compounds of formula (I) are unlikely to suffer from hyperkalemic dose limiting toxicities in humans as are observed with amiloride.

Example 9 - Inhbition of cell migration in 2D scratch wound healing assay

Experiments were undertaken to determine the effect of compounds 19 and 24 on breast cancer cell migration using a 2D scratch wound healing assay (Figure 7). Collagen I (Rat tail, Gibco, #A1048301 -01 ) was diluted from a 3 mg/ml stock to 300 μg/mi in 17.4 rnM acetic acid on ice under aseptic conditions and 50 μΙ of the diluted collagen was aliquoted into a 96-well irnagelock plate (Essen Bioscience, #4379). The plate was gently rocked by hand to distribute the collagen evenly across the wells and then incubated for 3 h at 37 °C, 5% C0 ₂ . The wells were washed with 1 xPBS and seeded with MDA-MB-231 cells at a density of 250,000 cells/ml in a volume of 100 μΙ of DM EM/10% FCS followed by incubation overnight at 37 °C, 5% C0 ₂ . After * 18h, culture media was removed via multichannel pipette and replaced with 100 μΙ of fresh sreum-free DMEM. On a separate 96-weli plate drugs were diluted to 5 μΜ serum-free DMEM (Final concentration of vehicle 0.2% DMSO v/v). Using the IncuCyte 96-pin WoundMaker™ (Essen Bioscience, #4493) a scratch was made in the confluent cell monolayer followed by washing of all wells with 100 μΙ 1 X Sterile PBS. Next 100 μΙ of each drug dilution or vehicle control solution was added to the appropriate wells via multichannel pipette. The plate was then incubated for 24 h, 37 °C, 5% C0 ₂ inside the IncuCyte ZOOM Live Cell Imaging System with image acquisition every 2 h using a 10X phase contrast objective. Degree of wound healing caused by the migration of ceil into the scratch wound was quantitated by measuring the confluency of the masked wound area over time. Data was analysed using GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA), with statistical significance calculated using the one-way, parametric, unpaired ANOVA algorithm. HMA, 19 and 24 at 5 μΜ significantly decreased migratory wound healing in MDA-MB-231 over 2-24h post-treatment, decreasing wound confluency by over 25% after 24h, (Figure 7, one-way ANOVA, p = 0.0206). The compounds did not differ in their ability to inhibit migration despite their differing uPA inhibitory potencies (uPA IC ₅₀ HMA 2206 nM, 19 297 nM, 24 86 nM). Example 10- Evaluation of anti-invasive effects of compounds 19 and 24 on ex vivo cancer cell invasion.

The anti-invasive effects of compounds 19 and 24 were detrmined using ex vivo organotypic model of cancer cell invasion (Figures 8 and 9). In this model, invasive cancerous epithelial cells are seeded on top of a dermal-equivalent fibroblast contracted Collagen I matrix and induced to invade downwards into the plug via the establishment of an air-liquid interface between the growth medium and the bottom of the seeded plug. Diffusion of growth factors and nutrients from the media into the plug creates a chemotactic and trophic gradient which attracts epithelial cell invasion. After sufficient time in culture, invasion in terms of number of invaded cells and invasion depth is quantified and invasion indices calculated for cells receiving different treatments. In this experiment human SKOV-3 ovarian adenocarcinoma cells were used due to their overexpression of uPA/uPAR and high invasive and metastatic potential. Tendons from excess deceased laboratory rats were surgically removed after washing in 70% v/v ethanol/water solution. Isolated tendons were frozen at -8G°C and mashed using a glass rod prior to dissolution of collagen I in 1500 ml 0.5 M Acetic acid at 4°C for 48 h with magnetic stirring. Extracted collagen was filtered through wet household paper towel to remove any remaining sheath. The extract was centrifuged at 10,000 rpm at 4°C for 1 h and the pellet discarded. Sufficient NaCI was added to the supernatant to form a 10% w/v NaCI solution in a 2 L conical flask with excess NaCI added until the solution exhibited a persisting white colour and no longer separated upon standing. The solution was centrifuged at 10,00 rpm at 4°C for 30 min and supernatant discarded. The precipitate was dissolved in 0.25 M acetic acid at a 1 :1 ratio for 24 h at 4°C with magnetic stirring. The resulting collagen solution was dialyzed against 6-8 changes of 5 L 0. 17.4 mfvl acetic acid with changes occurring twice daily. Dialyzed collagen was centrifuged at 20,000 rpm at 4°C for 1 .5 h. Isolated collagen I was adjusted to a concentration of 2.0 mg/ml for use. Purity of extracted collagen I was verified by SDS- PAGE. Human skin-derived telomerase -immortalized fibroblasts were maintained in culture for 4-14 days post-confluency without changing of media prior to harvesting to ensure quiescence. On day of collagen I seeding a collagen I matrix solution was prepared by adding 3 ml 10x MEM solution to 25 ml collagen I solution along with 3 ml 0.22 M NaOH and subsequent dropwise addition of NaOH until a pH of « 7.4 was achieved as determined by colour change of internal Phenol Red indicator. Harvested Fibroblasts were resuspended in 3 mi FBS and seeded at a density of 1 .0x10 ⁶ ceils/12 matrices into the neutralized collagen I matrix solution and 2.5 mi aiiquoted into separate sterile 35 mm sterile cell culture dishes, followed by. After « 30 min « 2 mi fibroblast growth medium (D EM/10% FBS/1 xPenstrep) was added to and nascent collagen l/fibroblast matricies were manually detached from the sides of each dish using a sterile pipette tip with rotation of each dish. Matrices were allowed to contract for 8-12 days until they had reached a diameter of « 20 mm. Following contraction matrices were transferred using tweezers to a sterile 24-weil ceil culture plate. Harvested SKOV-3 ovarian adenocarcinoma cells were seeded with a volume of 100 μΙ of cell suspension at a density of 1 x10 ^s cells/mi. Seeded plugs were transferred to sterile steel mesh grids in 60 mm ceil culture dishes (3 plugs per grid, 1 grid/treatment group per 60 mm dish) and w 10 ml media added such that an air liquid interface was established with the bottom of the plug (as growth media diffuses from the bottom of the plug towards the top a chemotactic gradient is established, inducing epithelial ceils seeded atop to invade downwards through the collagen matrix towards the bulk growth media). On day of initial drug treatment, culture media was removed from each dish and ¾ 10 ml of the appropriate drug treatment (ail drugs present at 2 μΜ) /vehicle control was added to establish an air-liquid interface as described above. Media was refreshed with drug or vehicle containing media solutions every 2 days. Dilutions of drug or vehicle in media were prepared from DMSO stocks every 7 days and stored at 4 °C until used. After 21 days plugs were fixed in 4% neutral buffered formalin for 24-48h. Fixed samples were processed using a Peioris Dual Retort tissue processor (Leica, Germany). Histological staining was conducted on 4 μηι sections deparfinized in xylene and rehydrated via graded washing in ethanol (70% v/v ethanol/water-100% ethanol). Cytokeratin staining was performed using an Autostainer XL (Leica, Germany). Ten images per treatment group were acquired using a DM4000 bright field microscope (Leica, Germany) and analyzed for epitheiai ceil invasive index where invasive index is defined as: (# epithelial ceils invaded into collagen plug/ # epithelial cells forming contiguous monolayer along top of the piug)x100. Scores for each image were collated and statistical analysis performed using Microsoft Excel for Mac 201 1 v.14.1 .0. Graphs were generated using GraphPad PRISM v6.0 (GraphPad Software, San Diego, California, USA). Representative photomicrographs from distinct replicate plugs for each treatment group are shown in Figure 8. Compound 19 significantly inhibited SKOV-3 cell invasion relative to vehicle treated cells after 21 days (p = 0.0287), decreasing invasion by 5.8% (Figure 9). HMA failed to significantly inhibit invasion (1 .4% decrease, p = 0.1099). No significant decrease in invasion was observed for amiloride. This result demonstrates the anti-invasive activity of compound 19 in the ex vivo context and further highlights the potental of compounds of formula (I) as antimetastatic cancer therapeutics. Example 11 - Effects of compound 19 and 24 administration on weight loss in AlbPLGI mice

Compounds 19 and 24 were selected for experiments in mice aimed at determining their dose-tolerability and acute toxicity profiles (Figure 10). Cohorts of four female age- matched C57AlbPLG1 mice (transgenic mice engineered to stably express the human gene for plasminogen) were treated with 2.5 mg/kg, 5 mg/kg and 10 mg/kg of HMA, 19 or 24 via intraperitoneal bolus injection (100 μΙ injection volume, 20% DMSO v/v in saline). Mice were weighed and monitored twice daily for signs of moribundity. The toxicity endpoint for the study was a greater than 10% decrease in initial body. Studies using higher dose levels of each treatment were not undertaken until studies at the next lowest dose were completed in the absence of death of having reached toxicity or animal welfare endpoints. All compounds were well tolerated up to 10 mg/kg/day, with no significant differences in mean body weigth relative to vehicle control animals or deaths at any dose level tested. No signs of acute toxicity were observed for any individual receiving any treatment, including animals treated with compound 19, the compound of formula (I) found to have the highest cytotoxic activity in in vitro cell viability and apoptosis assays. Representative data from the highest dose groups (10 mg/kg/day) is presented in Figure 10. These results show the general tolerability of compounds of formula (I) in mice and validate their use in mouse models of cancer metastasis at doses up to 10 mg/kg/day.

Example 12 - Maximum tolerated single dose of HMA, 19 and 24 in male Balb/c mice via IV bolus injection

In order to determine the dose tolerability of compounds in a relevant model, 9-10 week- old male Balb/c mice were administered a single IV bolus injection of amiloride, HMA, 19 or 24 and monitored for any signs of acute toxicity or distress over 72 h. Amiloride, HMA and 24 were formulated in 50 mM acetate buffer, 5% v/v DMSO. 0.5% v/v Tween- 80 (pH 5.5). Due to lower solubility 19 was forumulated in in 50 mM acetate buffer, 7.5% v/v DMSO. 0.75% v/v Tween-80 (pH 5.5). All treatment groups consisted of n = 3 mice and compounds dosed at 10 ml/kg. Dose limiting toxicity was observed for amiloride at the higest dose tested (40 mg/kg), with significant weight loss over 72 h (Table 3). Table 3: Maximum tolerated single IV bolus doses of Amiloride, HMA, 19 and 24 in male Balb/c mice.

Maximum Tolerated Dose of Drug Administered as

a sin le IV Bolus

HMA was well tolerated at 40 mg/kg with no signs of ill effects over 72 h. Both 19 and 24 caused death of animals immediately after dosing at 40 mg/kg. 19 at 20 mg/kg caused mild lethargy 2 h following dosing with recovery to normal activity by 24 h. Similar to the 40 mg/kg dose, 24 at 20 mg/kg caused immediate death following dosing. 24 at 10 mg/kg was well tolerated with no signs of ill effects over 72 h. Necropsy and histopathological analysis revealed no distinct cause of death with no evidence of major organ toxicity.

Example 13- Pharmacokinetic analysis of amiloride, HMA, 19 and 24 in male Sprague Dawley rats following I. V. administration

In order to determine the effects of differing 6-substituions on the pharmacokinetic parameters of HMA full pharmacokinetic profiles were determined for Amiloride, HMA, compounds 19 and 24 in male Sprague Dawley rats over a 24h period (Table 4). All animals were fasted overnight prior to infusion. Three animals per treatment cohort received a nominal dose of 3 mg/kg via I.V. infusion (10 min infusion, indwelling jugular vein catheter, 1 ml infusion volume). Arterial blood was sampled and blood plasma extracted pre-dose, 5 min and 10 min during infusion and 5 min, 20 min, 1 h, 2.5h, 5h, 7h, 10h, 16h and 24h post-infusion. Total urine was collected pre-dose, 0-7h and 7-24h post-dose. Amiloride and HMA were formulated in 50 mM acetate buffer/5% DMSO v/v, pH 5.5. Compounds 19 and 24 were 50 mM acetate buffer/5% (v/v) DMSO /0.5% (v/v) Tween-80, pH 5.5. Table 4: Pharmacokinetic parameters for Amiloride, HMA, compounds 19 and 24 in male Sprague Dawley rats via IV administration. Values equal mean (n = 3 animals per treatment group)

Amiloride behaved in a relatively polar manner when intrinsic hydrophobicity was determined by comparative analytical HPLC (Amiloride gLog D _{7 4} 0.8). Compound 19 was considerably more hydrophobic, whereas compound 24 showed decreased hydrophobicity relative to HMA, increases attributable to the inclusion of the hexamethylene group at the 5-position and heteroaryl substituents at the 6-position respectively (Table 4, 19 gLog D _{7 4} 5.1 , 24 gLog D _{7 4} 3.4, HMA gLog D _{7 4} 3.8). Compounds 19 and 24 showed significantly longer plasma half lifes relative to HMA (HMA t _1/2 7.3 h, 19 t _1/2 6.8 h, 24 t _1/2 7.0 h), with t _1/2 s comparable to that of amiloride (Amiloride ti _/2 7.3 h 19 p = 0.01 , 24 p = 0.0001 ). HMA, compounds 19 and 24 showed large increases in plasma clearance rates relative to amiloride, attributable to their prevalence for non-renal metabolism (>7.5-fold increase relative to Amiloride). Amiloride was primarily eliminated via renal excretion with 72.6% of the administered dose recovered unchanged in the urine. Despite its much much more hydrophobic nature, 13.4% of the administered dose was recovered unchanged in the urine, indicating considerable renal extraction along with a moderate propensity for hepatic metabolism as indicated by the apparent renal and non-renal blood clearance rates respectively. In contrast, elimination of the more hydrophobic HMA and compound 19 relied almost entirely on non-renal clearance, with <1 % of the initial dose recovered from the urine. To summarize, these results demonstrate that derivatization of HMA at the 6-position can illicit large favourable increases in key pharmacokinetic parameters (e.g. plasma t _1/2, plasma V _ss ), relative to the parent structure. Despite large differences in their intrinsic hydrophobicities and in vivo clearance profiles compounds 19 and 24, two potent 6-HMA analogues featuring structurally diverse groups at the 6-position, possess plasma half lives similar to that of Amiloride, the safe clinically used drug (Table 3). These results demonstrate that compounds of formula (I) can improve the overall druglikeness of the parent structure alongside optimization for uPA potency.

Example 14 - Pharmacokinetic analysis of amiloride, HMA, 19 and 24 in male Balb/c mice following I. V. administration

Pharmacokinetic profiles were determined for Amiloride, HMA, 19 and 24 in male Balb/c mice over a 24h period (Table 5). All animals were fasted overnight prior to infusion. Three animals per treatment cohort received a nominal dose of 3 mg/kg via I.V. infusion (10 min infusion, indwelling jugular vein catheter, 1 ml infusion volume). Arterial blood was sampled and blood plasma extracted pre-dose, 5 min and 10 min during infusion and 5 min, 20 min, 1 h, 2.5h, 5h, 7h, 10h, 16h and 24h post-infusion. All compounds were formulated in 50 mM acetate buffer/10 % (v/v) DMSO/5% Kolliphor® HS-15.

Table 5: Pharmacokinetic parameters for Amiloride, HMA, 19 and 24 in male Balb/c mice via IV administration. Values equal mean (n = 3 animals per treatment group).

Similar plasma t _1/2 s were observed for both amiloride and 24 in Balb/c mice relative to those seen in rats. In contrast, 19 and HMA exhibited substantially shorter halflives in mice. Moderate clearance patterns were observed in both species.

Example 15 - Efficacy of amiloride, 19 and 24 in a xenograft mouse model of metastatsis

uPA overexpression is a well-characterized determinant of cell invasiveness and metastatic potential in a variety of aggressive malignancies. Enhancement of invasive capacity is conferred by increased plasminogen activation and consequent downstream protease activation which results in broad scale remodelling of the surrounding stroma necessary for metastatic spread. In order to determine the effects of pharmacological blockade of uPA activity amiloride and 24 were evaluated in a late-stage mouse model of experimental metastasis. Luciferase-tagged HT-1080 human fibrosarcoma cells (BioWare Brite HT-1080-Red-FLuc) were used due to their high endogenous expression of uPA/uPAR and high metastatic potential. 7-8 week old male and female NOD.Cg- Prkdc<scid>IL2rg<tm 1 Wjl>SzJAusb mice were sourced from Australian BioResources, Moss Vale, NSW, Australia and allowed to acclimatize for 4 days prior to beginning experimentation. Males and females were randomized into 6 treatment groups of 6 mice each, equal numbers male and female. Animals were dosed daily from day -1 to day 20 with 7.5 mg/kg amiloride, 19, 24 or vehicle (50 mM acetate buffer (pH 5.5), 7.5% DMSO v/v, 0.75% Tween-80) via IP injections not exceeding 1 00 uL total volume. On Day 0 all animals except sham control (n = 1 ) received 2.5x10 ⁵ cells via the lateral tail vein in an injection volume of 100 uL I xDPBS. Animals were weighed and assessed for clinical signs daily. Humane endpoints of >1 5% acute weight loss (from previous 7 day maximum individual weight) or >20% chronic weight loss (from all-time individual maximum weight) or a body condition score >3 were selected. On day 21 all remaining animals were euthanized via slow C0 ₂ asphyxiation, necropsied and lungs removed and weight. Lungs were snap frozen in liquid N ₂ and stored at -80°C until homogenization. Lungs were homogenized on ice using a PRO Scientific Bio-Gen PRO200 Homogenizer in cell lysis buffer (1 50 mM NaCI, 1 0 mM Tris Base, 1 % Triton X100 v/v, pH 8.0) and centrifuged twice at 4 °C, 1 ,000 rpm, 15 min to remove cell debris. Homogenates were prepared to approximately 50 mg/ml protein based on individual wet lung weights. Luciferase activity was quantified according to manufacturers instructions using the Molecular Diagnostics SpectraMax Glo Steady-Luc Report Assay Kit. Luciferase assays were conducted in clear-bottomed white walled 96 well plates (Nunc) using a BMG Labtech PolarSTAR multimode plate reader. Data was analysed using Graph Pad PRISM v7.0 and statistical significance determined using the unpaired parametric t-test. Amiloride did not decrease lung tumour burden relative to vehicle control-treated mice (p = 0.5373, Figure 1 1 ). 19 decreased lung tumour burden by 21 % (p < 0.0001 ) compared to control mice but was not significantly different to amiloride. 24 showed greater efficacy, decreasing lung tumour burden by 33% compared to control (p <0.0001 ) and 26% relative to amiloride (p = 0.0003). No luciferase activity was detected in the lungs of the sham control animal. These results demonstrate the in vivo antimetastatic effects of 6-HMA analogues and, in the case of 24, superior efficacy relative to that of the parent drug amiloride.

Example 16- X-ray co-crystal structures of 19 and 24 bound to the active site of human uPA

X-ray crystallographic studies were undertaken to determine how compounds of formula (I) interact with uPA. Co-cyrstals from a variety of compounds of formula (I) were obtained using a 'soak-in' method similar to that described for huPA-H99Y in Jiang et al., 2015, The International Journal of Biochemistry and Cell Biology, 62, 88-92. Analysis of the resulting structures (structures for 19 and 24 are presented in Figure 12) revealed that specific interactions between the 2-acylguanidine moiety and 3-NH ₂ and uPA observed in amiloride and HMA co-crystal sturctures are preserved for all compounds of formula (I) with all compounds adopting superposable orientations within the uPA active site when relative to each other and the parent compounds. As such it can be inferred that the increased potency observed for the majority of compounds of formula (I), and compounds 19 and 24, specifically is due to the formation of favourable interactions between the substituents in the 6-position and the S1 β subsite which they project towards. For compounds 19 and 24 no specific H-bonding or ionic interactions were observed between the 6-substitutent and protein, indicating that increased potency results from new non-specific polar and hydrophobic interactions formed at this position.

Example 17 - Screening of amiloride, HMA, 19 and 24 against a panel of human cardiac ion channels

A significant obstacle in the development of small molecule drugs is the cardiotoxic risk often encountered with hydrophobic compounds. Cardiotoxic risk arises through the blockade of a variety of pro-torsadogenic cardiac ion channels (e.g. hERG) which when inhibitied to a high enough extent can result in lethal cardiac episode (e.g. torsades des pointes). Understanding hERG inhibitory potential has become a common triage point in the development of novel therapeutics with potent hERG inhibition needing to be addressed prior to further pre-clinical or clinical evaluation. Recent reports have found that cardiotoxic risk via hERG inhibition maybe counteracted by concomitant inhibition of other caridiac ion channels, namely NaV1 .5 and CaV1 .2 (Crumb et al., 2016, Journal of Pharmacological and Toicological Methods, 81 , 251 -262J. In order to evaluate the cardiotoxic risk profile of 6-HMA analogues, amiloride, HMA, 19 and 24 were screened against hERG, NaV1 .5 and CaV1 .2 using automated patch clamp assays (see Table 6). Amiloride showed relatively high potency (IC ₅₀ 5.6 μΜ) against NaV1 .5, although essentially no inhibition of hERG or CaV1 .2. HMA in contrast showed potent inhibition of hERG (IC ₅₀ 3.3 μΜ) and NaV1 .2 (IC ₅₀ 8.3 μΜ) and moderate inhibition of CaV1 .2 (IC ₅₀ 30 μΜ). 19 showed highly potent activity against all channels, with IC ₅₀ values extrapolated to be below the lowest concentration tested (<3 μΜ). 24 in comparison showed decreased activity relative to all other compounds with only moderate activity against NaV1 .5 observed (12 μΜ). These results suggest that 19 may have cardiotoxic potential in humans due to its potent inhibition of hERG, however this risk may be ameliorated by it's similarly potent activity against both NaV1.5 and CaV1.2. 24 is unlikely to exhibit cardiotoxic potential via cardiac ion channel blockade due to its absence of activity against hERG.

Table 6: Inhibitory potencies of amiloride, HMA, 19 and 24 against a panel of human cardiac ion channels.

The citation of any reference herein should not be construed as an admission that such reference is available as prior art to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endevour to which this specification relates. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features, compositions and compounds.