NZ730757B2 - Treatments for autoimmune diseases - Google Patents

Abstract

The invention relates to a novel approach to the treatment of autoimmune diseases, particularly multiple sclerosis.In a further embodiment of the invention there is provided a molecule capable of acting as substrate for the queuine-insertase enzyme complex and where said molecule is not a substrate for Hypoxanthine- guanine phosphoribosyltransferase and said molecule has the effect of lowering interferon gamma, for use in the treatment of auto-immune diseases. Preferably the autoimmune disease is multiple sclerosis. for Hypoxanthine- guanine phosphoribosyltransferase and said molecule has the effect of lowering interferon gamma, for use in the treatment of auto-immune diseases. Preferably the autoimmune disease is multiple sclerosis.

Description

/072488 Treatments for Autoimmune e Technical field The invention s to a novel approach to the treatment of autoimmune diseases, particularly multiple sclerosis.

Background art Multiple sclerosis (MS) is a debilitating disease of the central s system (CNS) in which the body’s own immune system attacks the white matter of the brain and spinal cord. This triggers inflammation-induced damage to the CNS protective myelin layer causing demyelination. Loss of myelin exposes neurons to further attack leading to formation of multiple sclerotic lesions. This damage disrupts the ability of parts of the nervous system to communicate, resulting in a wide range of problems including fatigue, blurred vision, cognitive impairment, and spasticity.

Many patients suffer from the development of rsible motor disability. Long term prognosis is poor, within 15 years of disease onset approximately 50% of patients are unable to walk unassisted (Polman and Uitdehaag, 2000).

MS takes several forms, with new symptoms either ing in isolated attacks (relapsing forms) or building up over time (progressive forms).

There is at present no known cure for multiple sclerosis. t treatments attempt to improve function after an attack and/0r prevent subsequent attacks.

Medications used to treat MS, while ly effective, can have adverse effects and be poorly tolerated.

There are a number of injectable front line therapies: . beta interferon 1a x) . beta interferon 1a (Rebif) . beta interferon 1b (Betaferon) . glatiramer acetate (Copaxone) erons may produce flu-like symptoms and some people taking glatiramer experience a post-injection reaction with flushing, chest tightness, heart palpitations, breathlessness, and anxiety, which usually lasts less than thirty minutes. More dangerous but much less common is liver damage. Glatiramer is associated with skin irritation at the site of injection.

WO 2016050806 Additional therapies include: . Natalizumab which s the relapse rate more than first-line agents; however, due to issues of adverse effects such as progressive multifocal leukoencephalopathy it is a second-line agent reserved for those who do not respond to other treatments or with severe e.

. Fingolimod (Gilenya) — licensed in March 2011 for people with rapidly evolving severe relapsing remitting MS (two or more relapses a year), and as a second line treatment for people whose MS remains active despite treatment with one of the beta interferon drugs.

. Dimethyl te (Tecfidera) was licenced by the FDA in 2013 and is an oral first line therapy for adults with relapsing remitting forms of MS.

. Teriflunomide (Aubagio) was approved by the FDA in September 2012, is an orally available modulatory drug for the treatment of relapsing forms of MS . Mitoxantrone, whose use is d by severe adverse effects, systolic dysfunction, infertility, and acute myeloid leukemia is a third-line option for those who do not respond to other medications. osteroids (or steroids) are sometimes given for a few days, either in the form of tablets or by intravenous drip. While there is no evidence that steroids have any effect on the long-term course of the disease, they can be effective at speeding up recovery from relapse.

No treatment has been shown to change the course of primary progressive MS and as of 2011 only one medication, mitoxantrone, has been approved for secondary ssive MS. In this population, tentative evidence supports mitoxantrone moderately slowing the ssion of the disease and decreasing rates of relapse over two years.

There is ongoing research g for more effective, convenient, and tolerable ents for relapsing-remitting MS.

Monoclonal antibodies have raised high levels of interest. The CD52 monoclonal antibody alemtuzumab, CD25 monoclonal dy daclizumab and CD20 monoclonal antibodies such as rituximab, ocrelizumab and ofatumumab have all shown some benefit and are under study as potential treatments. Their use has also been accompanied by the appearance of potentially dangerous adverse effects, most importantly opportunistic infections.

WO 2016050806 Accordingly, there is a huge unmet medical need for the treatment of MS.

Summary of the invention The present invention provides new medicaments for the treatment of autoimmune diseases, particularly multiple sis.

An imbalance of Th cell function and differentiation can lead to inflammation and autoimmune e. The activation of naive CD4+ T cells by antigen causes them to differentiate into specialized or T (Teff) cells (Th1, Th2, or Th17) and into regulatory T (Treg) cells, which ss the functions of Teff cells, thereby keeping immune responses in check. In particular, Th1 and Th17 cells and their signature nes IFN-y and IL-17 have been shown to play a critical role in the development of many autoimmune diseases, including multiple sclerosis (MS).

A novel mechanism for the regulation of this process and thus treatment of such es has been identified. Molecules which can exploit an enzyme x made of two proteins, tRNA guanine transglycosylase (TGT) also known as queuine tRNA-ribosyltransferase 1, and queuine tRNA—ribosyltransferase domain containing 1 (QTRTDl), subsequently referred to as the queuine-insertase enzyme complex, have been shown to have beneficial effects in models for autoimmune diseases.

Further, those les which are selective in avoiding competing pathways, such as not being substrate for Hypoxanthine-guanine phosphoribosyltransferase (HPRT) or, more positively lower interferon gamma levels are particularly efficacious.

The present invention provides new medicaments for the treatment of autoimmune diseases, particularly multiple sclerosis.

In a first embodiment it describes a molecule which is capable of acting as ate for the human queuine-insertase enzyme complex for the treatment of autoimmune disease.

More specifically it describes a le capable of acting as substrate for the queuine-insertase enzyme complex for use in the treatment of multiple sclerosis.

WO 2016050806 2015/072488 In a r embodiment of the invention there is provided a molecule capable of acting as substrate for the e—insertase enzyme complex and where said molecule is not a substrate for Hypoxanthine-guanine phosphoribosyltransferase (HPRT) for use in the treatment of autoimmune diseases. Preferably the autoimmune disease is multiple sclerosis.

Additionally described is a molecule capable of acting as substrate for the queuine— insertase enzyme complex, which also has the effect of lowering interferon gamma production in immune cell assays for use in the treatment of autoimmune diseases.

Preferably, the autoimmune disease is multiple sclerosis.

In a further embodiment of the invention there is provided a molecule capable of acting as substrate for the queuine-insertase enzyme x, where said le is not a substrate for HPRT and where said molecule has the effect of lowering interferon gamma, for use in the treatment of mmune diseases. Preferably the autoimmune e is multiple sclerosis.

For les that lower interferon gamma production in wild-type cells, preferred are those les that are able to lower it at a concentration of 100 uM Particularly preferred are those molecules which lower interferon gamma to $1750 ng/ml Most preferred are those molecules which lower interferon gamma to 51100 ng/ml.

Alternatively, can test for molecules that lower interferon gamma production by using cells from animals wherein the TGT gene has been knocked out, subsequently referred to as TGT-KO cells). Preferred are those molecules that at a concentration of 100 uM do not lower it $3,500 ng/ml.

Particularly preferred are those molecules which do not lower interferon gamma to 54250 ng/ml Most red are those molecules which do not lower it 35000 ng/ml.

The skilled reader will be aware of s for determining whether or not a particular molecule is a substrate for the queuine-insertase enzyme complex. For example, molecules capable of acting as a queuine-insertase enzyme complex WO 2016050806 substrate may be identified by use of a displacement assay as described below e 1): Production of [8-14C] Guanine labeled tRNA (tRNA*) Components added in the order listed in Table 1. Before adding the 8-[14C] guanine solution to the reaction the solution was lised with an equal volume (vol/vol) 0.01 M NaOH, as the [8-14C] Guanine is supplied in 0.01 M HCI aqueous solution.

A stock solution of yeast tRNA from Saccharomyces cerevisiae was made to a concentration of 2 ance units (260 nm) in ultrapure nuclease-free water.

The recombinant Escherichia coli (E. coli) tRNA guanine transglycosylase enzyme (E. coli TGT) ning an N-terminal polyhistidine tag was produced in BL21 BL21(DE3) tgt::Kmr cells as bed previously (Boland et al., 2009).

Table 1 Components of [8-14C] Guanine tRNA labeling reaction ent Volume (uL) Final conc. 1 M Tris-HCI pH 7.5 7.5 50 mM M NaCl 0.6 20 mM 1 M MgClz 0.75 5 mM 1 M D'I'l' 0.3 2 mM Yeast tRNA (Zabs/ul) 12.5 25 Abs total in rxn H20 to 130 pL E.co/i TGT 10 pL 10 pg [8-14C] guanine 10 pL The reaction was incubated for 2 h at 37 0C. After incubation the reaction was made up to 400 uL with buffer. The reaction e was extracted by addition of an of equal volume (400 uL), of Acid Phenol:ch|oroform (5:1; pH 4.5) and fuged at 16,000 x g for 5 min. The upper aqueous phase was transferred to a new 1.5 mL tube. The radiolabelled tRNA with [8-14C] guanine in the third position of the anticodon loop (tRNA*) was precipitated by the addition of 0.1 volume (40 uL) of 3 M sodium acetate (aq.) and 2 volumes of ethanol (800 uL) and incubated overnight at -20 oC. The next morning, the tRNA* was pelleted by centrifugation at 16,000 x g for 20 min at 4 °C. The pellet was washed with 1 mL of ice-cold 70 % ethanol, t disturbing the pellet. The tRNA* pellet was resuspended in 20 uL nuclease-free water and the concentration measured spectrophotometrically at A260.

WO 2016050806 Displacement assays Each reaction was set up in triplicate and incubated for 1 h at 37 oC. Each of the components in the reaction were added in the order shown in Table 2, with the tRNA* added last to initiate the on. ‘Compound’ refers to the molecules that are under investigation.

Table 2 Components of [8-14C] guanine cement assays Component Volume (pL) Final Concentration 1 M Tris-HCI pH 7.5 7.5 50 mM M NaCl 0.6 20 mM 1 M MgClz 0.75 5 mM 1 M Dithiothreitol 0.3 2 mM Compound (2 mM stock) 15 200 uM H20 to 140 Queuine-insertase enzyme 10 2 ug tRNA* 2 Abs units 1.8 uM Preparation of the DEAE columns: Approximately 25 grams of Whatmann DEAE 52— cellulose resin was weighted into a 50 ml sterile RNA free tube. A volume of 20 ml of 200 mM Tris—HCI pH7.5 was added and the tube inverted 5 times and centrifuged at 750 x g in a bench top centrifuge to nt the resin. The supernatant was removed by pouring and the pH checked. The washing of the resin was repeated another 4 times until the wash d a pH of 7.5. The resin was suspended in a 1:1 slurry with 200 mM Tris—HCI pH7.5 and loaded into a 1.5 ml spin column (containing a glass fibre filter) until a 1 ml final bed volume of resin was achieved.

After incubation the reaction was loaded onto the column and spun at 0.1 X g for 10 seconds. The hrough was collected and ed on the column. This step was repeated 5 times to allow maximum binding of tRNA. The column was then washed with 8 x 250 uL of Wash Buffer (20 mM Tris-HCI, pH7.5, 10 mM Mng, 200 mM NaCI) with 10 second spins at 0.1 x 9 between each wash. These loading and wash steps were collected into scintillation vials. The bound tRNA was then eluted in 4 x 250 uL aliquots in Elution Buffer (20 mM Tris—HCI, pH7.5, 10 mM MgClz, 1M NaCI) and collected into scintillation vials. 10 mL of Ecoscint A (scintillation cocktail) was added to all vials. The vials containing the flow-through and washes are counted for displaced [14C] guanine.

In this assay (see figure 1), maximum displacement by 200 uM queuine base, the natural ate of the queuine-insertase enzyme complex, is 240 pmol [14C] W0 2016;050806 guanine. Background values are S 10 pmol. Therefore, a displacement of 250 pmol is considered a positive substrate for TGT.

Assay for the ability of compounds to be used as substrates for the queuine-insertase enzyme complex—by displacing [14C] e from yeast tRNA (Figure 1). e, queuine, N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3—d]pyrimidin-5— yl)methy|)phenylpropan-1—aminium chloride (shown as compound I) and 7- methylguanine were assessed for their ability to replace guanine in yeast tRNA.

First, yeast tRNA was charged with [8-14C] guanine (tRNA*) by the E. coli TGT enzyme. In each reaction 2 A260 absorbance units of tRNA* was used along with 200 uM of specified le. ons were processed on 1 ml DEAE cellulose columns. Reaction flow-through and washes were collected and analysed by liquid scintillation counting for presence of displaced [14C] guanine.

Similarly, the skilled reader will be aware of methods le for determining whether a compound is a substrate or inhibitor of HPRT. For example, molecules that are neither a substrate nor an inhibitor for HPRT may be identified by use of the following assay: Assay to evaluate nds as substrates or tors of the Hypoxanthine-guanine phosphoribosyltransferase enzyme or 'HPRT Assay' (Figure 2).

Preparation and equilibration of : Dowex—1x8, mesh size 200-400, was weighed out in a sterile 50 ml tube and washed 3 times with aqueous 1 M HCI to charge the resin. The resin was then washed 5-6 times with (20 mls each) Milli-Q H20 until a neutral pH was reached. s (Bio-rad 10 mL plastic columns) were set up with 1 mL of wet Dowex (wet mix applied, allowed to settle and more applied 3O until a packed volume of 1 mL was reached). The packed resin was rinsed with 10 s (v/v) milli-Q H20 to ensure that a neutral pH and consistent packing was achieved.

HPRT Reactions are set up in 100 uL volume in triplicate with the following components: mM HEPES (pH 8) mM MgClz WO 2016050806 0.1 mM Phosphoribosyl pyrophosphate (PRPP — made fresh on day of ) pM [8-14C]-Guanine 400 pM Compound to be tested The reaction mix was preheated to 37 °C before 10 ng of recombinant human HPRT enzyme was added. Reactions are allowed to proceed for 1 h before being d by heating for 8 min at 100 °C. A l reaction comprising heat-inactivated enzyme (treated at 100 °C for 8 min prior to adding to the reaction) was also always included.

After vation, the reactions were cooled to room temperature (using ice), before being applied to a 1 mL packed resin column of Dowex-1x8, mesh size 200- 400. The column was washed with 5 mL milli-Q H20 and 10 mL 10 mM HCI, to remove unbound, unreacted guanine. GMP was eluted from the column with 5 mL of 50 mM HCI into scintillation vials. 15 mL of scintillation cocktail (Ecoscint A) was added and vials counted to evaluate the level of conversion of [8-14C]-guanine to GMP.

For molecules that are neither a HPRT substrate nor an inhibitor, the reaction will yield GMP product in amounts 260% of the control reaction containing only [8- 14C]-guanine.

Results for e, Queuine.HC| (2-Amino[[[(15,45,5R)-4,5-dihydroxy-2— cyclopenten-1—y|]amino]methyl]-1,7—dihydro-4H-pyrro|o[2,3-d]pyrimidin—4-one, drochloride) and N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3— midiny|)methy|)phenylpropan-1—aminium chloride (shown as Compound I on graph) are shown in Figure 2.

The ability of molecules to lower interferon gamma may be measured with 3O the following ex vivo assay (Figure 3): Chronic, monophasic EAE was induced in 8-10 week old female C57BL/6 mice as described by Stromnes and Goverman, (2006). Animals were transcardially perfused with ice-cold phosphate buffered saline (PBS) and the spleen was removed.

Single cell suspensions of spleen were made by extrusion through a 70 micron nylon mesh and the cell washed in cRPMI medium (Roswell Park Memorial Institute WO 2016050806 media containing 10% fetal bovine serum, 2 mM amine, 100 units penicillin, 1 mg/ml streptomycin) by centrifugation at 240 x g for 5 minutes, counted and seeded at a final density of 1 x 106/mL to omed 96 well plates containing medium alone (negative control) or 100 pl of 50 ug/mL myelin oligodendrocyte glycoprotein peptide (M06355). All stock concentrations of molecules were made up in sterile DMSO and tested in cells at 10 uM and 100 uM concentrations. Molecule administrations did not exceed 0.5% final DMSO concentration in cells. After 72 h, the plates were centrifuged at 240 X g for 3 minutes to allow the cells to settle down. Supernatants were lly removed and assayed for IFN gamma according to manufacturer’s ol supplied in the eBioscience ELISA kit.

This test can be used with both wild cells or TGT-KO cells as described herein Suitable molecules for use in the invention include: o(((3-phenylpropyl)amino)methyl)-3,7-dihydro-4H-pyrro|o[2,3— d]pyrimidinone N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin—5-yl)methyl) propan-l-aminium chloride; 2-amino((butylamino)methyl)-3,7-dihydro-4H—pyrrolo[2,3-d]pyrimidinone N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin—5-yl)methyl)butan aminium chloride 2-amino((hexy|amino)methy|)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidinone N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidinyl)methyl)hexan—1— aminium chloride Queuine; 2-amino((((1S,4S,5R)-4,5-dihydroxycyclopent-Z-en y|)amino)methy|)-3,7-dihydro-4H-pyrro|o[2,3-d]pyrimidinone Queuine HCI 2-Amino-5—[[[(15,45,5R)-4,5-dihydroxy-Z-cyclopenten—1- yl]amino]methyl]-1,7-dihydro—4H-pyrrolo[2,3-d]pyrimidinone, monohyd rochloride WO 2016050806 Without being bound by theory, it appears the compounds of the present invention operate via a new drug y. They t an enzyme complex made of two proteins: TGT (tRNA guanine transglycosylase) and QTRTDl (queuine tRNA transglycosylase domain containing 1) herein referred to as the queuine-insertase enzyme complex. The effect is to decrease populations of effector T cells (Teff) and/or increase the relative populations of regulatory T cells (Treg) cells. Treg cells are a part of the immune system that are intricately involved in the ination of self from non-self proteins, i.e., ting ns of the self from being attacked by the immune system The natural substrate for the queuine-insertase enzyme complex is queuine, a molecule that cannot be synthesised by eukaryotic cells. r, it is readily produced by most bacteria, and in humans queuine must be harvested either from injested food or the gut microflora. The queuine-insertase enzyme complex inserts queuine into the don loop of l-, histidinyl-, asparaginyl- and aspartyl— transfer RNA (tRNA of the GUN ; tRNAGUN) at the wobble on. The majority of tRNAGUN in the body is modified at the wobble position with queuine. It is notable that queuine is absent or depleted (hypomodified) in the wobble position of tRNAGUN isotypes in rapidly proliferating cells. Earlier work employing 6— thioguanine (6TG) has shown that use of this alternative substrate for the queuine- insertase enzyme complex can have a dramatic effect on the murine model of c MS (i.e. Experimental Autoimmune Encephalomyelitis; EAE). Unfortunately, from a therapeutic perspective, 6TG suffers from the problem that it is a more potent substrate for HPRT (approx. 10 fold higher than for the queuine-insertase enzyme complex). It is subsequently incorporated into DNA, making it genotoxic and unsuitable as a treatment for MS.

The present invention revolves around molecules able to t the beneficial aspects of 6TG ne-insertase dependent exchange into the wobble position of 3O tRNAGUN), but which circumvent the problem associated with 6TGs’ main biological activity (i.e. HPRT activity and subsequent insertion into DNA). s 4a -4d show that proliferation was reduced to less than 50% in wild-type T cells upon treatment with N—((2-aminooxo-4,7-dihydro—3H-pyrrolo[2,3- d]pyrimidinyl)methyl)phenylpropanaminium chloride at 100 pM concentration (shown as Compound I in Figure 4a-d). Lack of HPRT involvement in the observed effect was demonstrated by a similar reduction of proliferation of cells in which the HPRT gene had been knocked out (HPRT KO, Figure 4b). Lack of WO 2016050806 2015/072488 HPRT activity has also been confirmed by in vitro assay employing recombinant HPRT enzyme wherein the molecule is shown not to be a substrate or inhibitor (Figure 2). The requirement for the queuine-insertase enzyme complex is confirmed by the lack of effect of N—((2-aminooxo-4,7-dihydro—3H-pyrrolo[2,3- d]pyrimidinyl)methyl)phenylpropanaminium chloride (shown as Compound I) at 100 uM concentration on on proliferation in cells in which the TGT gene has been knocked out (TGT KO, Figure 4d). These data indicate that the mechanism of action is indeed mediated by the queuine-insertase enzyme complex, which is further confirmed (Figure 4c) by the fact that the double TGT:HRRT knockout is essentially unaffected when treated with N-((2-amino0xo-4,7- dihydro—3H-pyrrolo[2,3-d]pyrimidinyl)methyl)phenylpropanaminium de (Formula I) at 100 uM concentration. In vitro is of N-((2-amino oxo-4,7—dihydro-3H-pyrrolo[2,3-d]pyrimidinyl)methyl)-3—phenylpropan—1- aminium de (Compound I) using recombinant TGT enzyme has also shown that the compound is a substrate for TGT (Figure 1). The data demonstrates that said nd operates as a substrate for the queuine-insertase enzyme complex- but is not a substrate for HPRT.

The substrate of the queuine-insertase enzyme complex functions to suppress encepholitogenic effector and memory T cell tions without impacting the naive population. These same immune cell populations broadly contribute to the pathogenesis of all autoimmune diseases.

The transfer RNA (tRNA) of rapidly proliferating cells is ent (hypomodified) in queuine cation; examples include foetal liver, multiple tumour types and regenerating adult liver. By contrast, the tRNA of adult, fully differentiated cells contains high levels of queuine, which cannot be ced once incorporated.

It could be expected that the tRNA of rapidly expanding immune cells (as occurs in 3O an autoimmune response) are likewise ent in the queuine cation.

Incorporation of novel Queuine Insertase substrates selectively into the queuine— deficient tRNA of immune cells could disrupt proliferation and cytokine production thereby modulating the immune response.

Compounds of the present invention find utility in the ent of auto-immune conditions, including but not limited to multiple sclerosis, rheumatoid arthritis, WO 2016050806 ulcerative colitis, psoriasis, es and inflammatory bowel disease, including Crohn’s disease; and as agents to ss transplant rejection.

The invention also relates to a method of treating multiple sclerosis in a mammal, particularly a human, comprising administering to said mammal an amount of a molecule as defined , or a pharmaceutically acceptable salt or solvate thereof.

The term ‘treatment’ is intended to include curing, reversing, alleviating, palliative and prophylactic treatment of the condition.

The invention further relates to molecules of the invention in combination with other le agents, for use in the treatment multiple sis.

Patients suffering from multiple sclerosis are commonly co-administered additional therapeutic agents. For patients suffering a severe attack, intravenous corticosteroids, such as methylprednisolone or techniques such as or plasmapheresis may be coadministered with any treatment.

The effects of nerve cell damage caused by le sclerosis result in diverse forms of damage to the patient. Nerve damage can lead to pain, difficulty with l of bladder and many other issues. For this reason, additional medicaments are often prescribed patients with multiple sclerosis to help treat the effects of MS damage.

Suitable inistrants would include: For Bladder ms . botulinum toxin (Botox) . desmopressin spray, Desmotabs) . oxybutynin (Ditropan, Lyrinel) . tolterodine (Detrusitol) For Depression . amitriptyline (Triptafen) . fluoxetine (Prozac) . mine (Tofranil) . pa roxetine (Seroxat) WO 2016050806 2015/072488 For Erectile dysfunction alprostadil (Caverject, MUSE, Viridal Duo) sildenafil citrate (Viagra) tadalafil s) va rdenafil (Levitra) For Fatigue amantadine (Lysovir, rel) modafinil (Provigil) For Optic neuritis steroids For Pain amitriptyline (Triptafen) carbamazepine (Tegretol) gabapentin (Neurontin) ibuprofen imipramine (Tofranil) Iamotrigine tal) phenytoin (Epanutim) pregabalin a) For Problems with walking fampridine (Fampyra) For Psuedobulbar affect Nuedexta For Spasticity and spasms baclofen (Lioresal) botulinum toxin (Botox) carbamazepine (Tegretol) clonazepam (Rivotril) WO 2016050806 . dantrolene (Dantrium) . diazepam (Valium) . gabapentin (Neurontin) . phenol . Tetrahydrocannabinol and cannabidiol (Sativex) . tizanidine (Zanaflex) For Tremor . clonazepam (Rivotril) . thalamotomy For Trigeminal neuralgia . carbamazepine (Tegretol) . gabapentin (Neurontin) . oxcarbazepine (Trileptal) . phenytoin (Epanutim) . pregabalin (Lyrica) Other therapeutic agents are commonly administered to patients with MS. Other such medicaments are well known to physicians and others skilled in therapy.

Such agents may be administered sequentially, simultaneously or concomitantly.

The invention also relates to a pharmaceutical composition comprising a le of the present invention and a pharmaceutically able diluent or carrier.

Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Compounds of formula (I) may be stered orally. Oral administration may involve swallowing, so that the compound enters the intestinal tract, or buccal or sublingual administration may be ed by which the compound enters the blood stream ly from the mouth. Formulations suitable for oral WO 2016050806 administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multiand nano-particulates, gels, solid solution, me, films, ovules, sprays and liquid formulations.

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

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

For tablet dosage forms, depending on close, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight °/o to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl ose, calcium carboxymethyl ose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, rystalline cellulose, lower alkyl-substituted hydroxypropyl ose, , pregelatinised starch and sodium alginate. lly, the disintegrant will se from 1 weight % to 25 weight %. In one embodiment of the present invention, the disintegrant will comprise from 5 weight % to 20 weight % of the dosage form. Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline ose, gelatin, sugars, hylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl 3O cellulose and hydroxypropyl cellulose. Tablets may also n diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, rystalline cellulose, starch and dibasic calcium phosphate dihydrate. Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and rbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight °/o to 5 weight °/o of the tablet, and glidants may se from 0.2 weight % to 1 weight % of the tablet. Tablets also generally contain lubricants such as magnesium stearate, calcium te, zinc stearate, sodium stearyl WO 2016050806 te, and mixtures of magnesium te with sodium lauryl sulphate.

Lubricants lly comprise from 0.25 weight % to 10 weight %. In one embodiment of the present invention, lubricants comprise from 0.5 weight % to 3 weight % of the tablet. Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste-masking agents.

Exemplary tablets n up to about 80% drug, from about 10 weight % to about 90 weight °/o binder, from about 0 weight °/o to about 85 weight % diluent, from about 2 weight °/o to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.

Tablet blends may be ssed ly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt led, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated.

Formulations of tablets are discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

Consumable oral films for human or veterinary use are typically pliable water— soluble 0r water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise a compound of formula (I), a film-forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser or fier, a viscosity-modifying agent and a solvent. Some components of the formulation may perform more than one function. The film-forming polymer may be selected from natural polysaccharides, proteins, or synthetic hydrocolloids and is typically present in the range 0.01 to 99 weight %, more typically in the range 30 to 80 weight %. Other possible ingredients include anti-oxidants, colorants, flavourings and flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, 3O surfactants and taste-masking agents. Films in accordance with the invention are lly prepared by evaporative drying of thin aqueous films coated onto a le backing support or paper. This may be done in a drying oven or tunnel, lly a combined coater dryer, or by freeze-drying or ing.

Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release includes delayed, sustained, pulsed, lled, ed and programmed release. Suitable modified release formulations for the purposes of the invention are described in US Patent No.

WO 2016050806 6,106,864. s of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in ceutical Technology On—line, 25(2), 1-14, by Verma et al (2001). The use of chewing gum to achieve controlled release is described in WO-A-OO/35298.

Compounds of formula (I) may also be stered directly into the blood stream, into muscle, or into an internal organ. Such parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous administration. le devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

Compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally.

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

The pressurised container, pump, spray, er, or nebuliser contains a solution or suspension of the compound of formula (I) comprising, for example, ethanol, 3O aqueous ethanol, or a suitable alternative agent for sing, solubilising, or extending release of the compound, a propellant as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or sion formulation, the drug product is micronised to a size le for delivery by inhalation (typically less than 5 microns). This may be ed by any riate comminuting , such as spiral jet milling, fluid bed jet milling, supercritical fluid sing to form nanoparticles, high pressure nisation, or spray drying.

WO 2016050806 Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and dges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or ium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include n, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution ation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from lug to 20mg of the compound of the invention per actuation and the actuation volume may vary from lul to 100ul. A typical formulation may comprise a compound of formula (I), propylene glycol, sterile water, ethanol and sodium de. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.

Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or rin sodium, may be added to those formulations of the invention ed for intranasal administration. ations for intranasal administration may be formulated to be immediate and/or modified release using, for example, PGLA. Modified release includes delayed, sustained, pulsed, controlled, targeted and programmed release.

Compounds of formula (I) may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH- adjusted, sterile saline.

Compounds of formula (I) may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives f or polyethylene glycol— 3O containing polymers, in order to e their solubility, dissolution rate, taste, bioavailability and/or stability when using any of the aforementioned modes of administration. Drug-cyclodextrin xes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion xes may be used. As an alternative to direct complexation with the drug, the extrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha- beta- and gamma-cyclodextrins, examples of which may be found in international patent publications 1/11172, WO-A—94/02518 and WO-A-98/55148 WO 2016050806 Experimental The synthesis of various molecules suitable for use in the present invention are described below.

All ng materials and reagents are commercially available and were obtained from Aldrich with exception of 2-amino-3H-pyrro|o[2,3-d]pyrimidin-4(7H)-one which was purchased from Fluorochem.

Preparation 1: 2-octanoylamino-pyrrolo[2,3-d] dinone I \ MNMH A 50 cm3 round-bottomed flask ning a stirring bar was charged with 2-amino- 3H-pyrrolo[2,3—d]pyrimidin-4(7H)-one (2.00 g, 13.33 mmol). The flask was fitted with a septum and placed under an Ar here. Freshly distilled pyridine (20.00 cm3) was added via e and the resulting sion cooled on ice. The on was allowed to equilibrate at this temperature (ca. 5 min) and then octanoyl chloride (6.80 cm3, 39.99 mmol) was added dropwise. The resulting suspension was heated at 85 °C for 30 min. After cooling to room temperature 6.5% ethanolic ammonia (60 cm3) was added and the resulting suspension stirred at room temperature overnight. The precipitate of product was removed via vacuum filtration and washed with ethanol followed by diethyl ether to yield the desired product (2.56 g, 70%) pure as a yellow solid, m.p. > 300 °C (decomposition).

Procedure based on Akimoto et al. 1986 and Akimoto et al. 1988 6H (400MHz, DMSO-de): 0.86 (3H, t, J 5.1), 1.26 (8H, m), 1.58 (2H, app. quintet), 2.01 (1H, br 5, NH), 2.43 (2H, t, J 5.1), 6.40 (1H, cl, J 2.0), 7.01 (1H, cl, J 2.0), 11.43 (1H, br 5, NH), 11.67 (1H, br 5, NH) HRMS(m/z ESI'): Found: 275.1517 ([M-H]' C14H19N402; Requires: 275.1508) WO 0806 pPreparation 2: 2-octanoylamino((dibenzyl)amino)methyl)- pyrrolo[2,3-d]pyrimidinone A 50 cm3 reaction vessel containing a stirring bar was charged with 2- octanoylamino-pyrrolo[2,3-d]pyrimidinone (1.00 g, 3.60 mmol), ylamine (2.00 cm3, 10.80 mmol), formalin (349.00 pL, 12.60 mmol) and 80% aqueous acetic acid (36 cm3). The resulting suspension was heated at 60 °C for 20 h, cooled to room temperature, diluted with 0.5 M HCl (36 cm3) and stirred at room temperature for 30 min. The mixture was neutralised with conc. aq. ammonia (36 cm3) and extracted with chloroform (3 x 50 cm3), The organic ts were combined, dried (MgSO4) and evaporated to s. The crude residue was purified by column chromatography (9:1 dichloromethane-MeOH — 7:3 dichloromethane-MeOH) to give the desired compound (1.45 g, 84%) as a yellow powder m.p. > 300 °C (decomposition). Procedure based on Akimoto et al. 1986 and Akimoto et al. 1988 (m (400 MHz, DMSO-de): 0.86 (3H, t, J 7.1), 1.25 (8H, m), 1.57 (2H, m) 2.42 (2H, t, J 7.1), 3.57 (4H, s), 3.76 (2H, s), 6.88 (1H, s), 7.23 (2H, t, J 7.3), 7.31 (4H, app. t), 7.41 (4H, d, J 7.3), 11.34 (1H, s, NH), 11.57 (1H, 5, NH), 11.68 (1H, s, NH) HRMS(m/zESI+): Found: 486.2863 ([M+H]+ C29H35N502; requires: 486.2869) Example 1: o(((3-phenylpropyl)amino)methyl)pyrrolo[2,3- d]pyrimidinone )\\ I H A large carousel tube containing a stirring bar was charged with 2-octanoylamino- —((dibenzyl)amino)methyl)-pyrrolo[2,3—d]pyrimidin—4—one (100.0 mg, 0.21 WO 2016050806 mmol), 3-phenylpropylamine (146.00 uL, 1.03 mmol) and 1:1 THF-methanol (2.00 cm3). The suspension was ed and the reaction vessel sealed. The sion was heated at 75 °C for 24 h, cooled to room temperature and treated with 5 M KOH (146.00 uL) and stirred at room temperature for 65 h. The solution was concentrated in vacuo and the crude residue purified by column chromatography (9:0.9:0.1 dichloromethane-MeOH-NH4OH). The resulting solid was washed with HPLC grade hexane followed by diethyl ether in order to remove trace impurities.

This gave the desired compound (28 mg, 46%) as an orange powder, m.p. > 300 0C position). Procedure based on Akimoto et al. 1986 and Akimoto et al. 1988 6H (600 MHz, DMSO-de): 1.24 (1H, br s, NH), 1.66 (2H, app. quintet,), 1.91 (1H, 5, NH), 2.44 (2H, t, J 6.9,), 2.57 (2H, t, J 6.9,), 3.59 (2H, s,), 6.15 (2H, br s,), 6.45 (1H, s,), 7.15 (1H, t, J 7.4,), 7.16 (3H, m), 7.25 (2H, app. t,), 10.70 (1H, br s) 5c (600 MHZ, DMSO-de): 30.6, 32.9, 45.2, 47.5, 48.6, 79.2, 98.7 (q), 113.6 (q), 125.5, 128.2, 128.3, 142.3 (q), 152.2 (q), 160.5 (C=O) Vmax (film)/cm'1: 697, 748, 749, 1080, 1420, 1596, 2927 HRMS (m/z ESI+): Found: 298.1662 ([M+H]+ C16H20N50; Requires: Preparation 3: 2-chlorooxopropanenitrile CI CN In a dry round bottomed flask under a positive re of argon, a suspension of NaOMe , 0.13mol) in dry THF (90mL) was cooled to -5 0C. Methyl formate (9mL, 0.15 mol) was added dropwise over 1 min by syringe and stirring was 3O continued at -5 0C for 20 min. Then chloroacetonitrile (8.33mL, 0.13 mol) was added dropwise via a dropping funnel over 45 min. The mixture turned from white to yellow and was d for a further 2 h at -5 0C at which point the reaction mixture was orange. The bath was removed and the reaction was allowed to warm up to room temperature. An aliquot of the reaction mixture was treated with a drop of concentrated HCI and analysed by TLC which indicated the presence of the WO 2016050806 desired product with an Rf = 0.45, eluting with 100% EtOAc. The mixture was cooled to 0 0C and concentrated HCI (12 mL) was added dropwise during which time the mixture reaction became cherry-red. The resultant suspension was filtered through a pad of celite, and the celite was washed with EtOAc until the filtrate became colourless. The collected filtrates were concentrated at reduced pressure with the water bath at a temperature no higher than 40 0C to afford (formyl)acetonitrile1 as a black oil, in tative yield, which was used without further purification. Procedure based on Brooks 2012. 6H (400 MHz, CDCI3) 9.38 (s, 1H). 5c NMR (400 MHZ, DMSO-de) 5 168.2, 126.6, 67.8.

Preparation 4: o-4,7—dihydrooxo-3H—pyrrolo[2,3-d] pyrimidine- -carbonitrile “1H\ H2NNM 2,4-Diaminohydroxypyrimidine (3.00 g, 24 mmol) was added to a solution of sodium acetate (6.4g, 76 mmol) in millipore water (90 mL) and stirred at 50 °C for 1 hour. While still at 50 °C a solution of crude chloro(formy|)acetonitrile (3.00 g, 32 mmol) in mQ water (44 mL) was added dropwise with a dropping funnel, during which time the reaction turned beigeand heating continued for 18 h at 50 °C, after which time the reaction was heated to 100 °C for 3 h. The on mixture was allowed to cool to room temperature and the solid removed by tion. The solid was suspended in EtOH and 5M aqueous KOH solution was added until the solid dissolved. Charcoal was added to the solution and the mixture d for 30 minutes before removal of the solid by filtration. The pH of the filtrate was adjusted to pH=6 with trated aqueous HCI solution during which time a precipitate formed and was collected by filtration. In order to remove the final traces of water from the solid it was dissolved in a mixture of toluene/methanol 1/1 and then trated at reduced pressure. The resultant solid was dried over P205 to afford the desired compound (1.68 g, 9.6 mmol, 40% yield) as beige solid. Procedure based on Brooks 2012. 6H (400 MHz, DMSO-de) 6 11.98 (br s, 1H) 10.74 (br s, 1H), 7.59 (s, 1H), 6.43 (s, 2H).

W0 2016;050806 5c(100 MHZ, DMSO-de) 5 158.0, 154.3, 152.1, 128.2, 116.4, 99.2, 86.0.

HRMS (m/z ESI'): C7H5N50 [M-H]' Found 174.0415 Requires: 174.0416.

Preparation 5: 4,7-Dihydrooxo[(triphenylmethyl)amino]-3H- pyrrolo[2,3-d]pyrimidinecarbonitrile 0 O C.

O “1 I 3 H N In a dry round bottomed flask under an atmosphere of argon, trityl chloride (1.20 g, 4.28 mmol) was added to a solution of 2-amino—4,7-dihydrooxo-3H- pyrrolo[2,3-d]pyrimidine—5-carbonitrile (0.50 g, 2.85 mmol) in dry pyridine (29 mL). The e reaction was heated at 90 °C for 48 h. The reaction mixture was concentrated under reduced pressure then absorbed on silica gel and purified by flash chromatography on silica gel eluting with dichloromethane/MeOH with a gradient ng at 2% of MeOH and rising to 10%. The desired compound was obtained as a brown solid (0.63 g, 1.5 mmol, 53% yield).

Procedure based on Olgen 2008. 6H (400 MHz, DMSO-de) 6 11.80 (br s,1H); 10.64 (br s, 1H), 7.56 (s, 1H), 7.41(s, 1H), 7.29-7.28 (m, 12H), 7.23-7.17 (m, 3H), 5.73 (s, 1H).

HRMS (m/Z ESI+): N50 [M-H]Jr Found 416.1514 es: 416.1511.

Preparation 6: 4,7-Dihydrooxo[(triphenylmethyl)amino]-3H- pyrrolo[2,3-d]pyrimidinecarboxaldehyde WO 2016050806 HMDS , 1.3mL) was added to a mixture of 4,7-dihydrooxo-2— [(triphenylmethy|)amino]-3H-pyrrolo[2,3-d]pyrimidinecarbonitrile (1.30 g, 3 mmol) with ammonium sulphate (397 mg, 0.3mmol) in dry toluene (8mL) in a round bottomed flask. A reflux condenser was fitted, and the flask was heated at reflux temperature overnight. The mixture was cooled to room temperature and concentrated under reduced pressure. Under a positive pressure of argon, the crude reaction mixture was solubilised in dry dichloromethane (8mL) and cooled to -78 °C. At this temperature, DiBAL-H (4.5 mL, 1 M in dichloromethane, 4.5 mmol) was added dropwise. After 2 hours, analysis by TLC (EtOAc 100%) indicated that some starting material remained. So, a further 2mL DiBAL—H solution was added dropwise. After 1 hour, the reaction was complete and a mixture of H20/AcOH (9/1, 3.5 mL) was added at -78 °C. The reaction mixture was allowed to warm to room temperature slowly. A mixture of EtOAc/H20 (1/1, 300 mL) was added to the reaction mixture and stirring continued at room ature for 2 hours. The layers were separated and the organic layer was washed with brine and the aqueous layers were extracted with EtOAc. The combined c fractions were dried over MgSO4, filtered and trated at reduced pressure. The crude reaction t was filtered through a pad of silica gel eluting with EtOAc to afford a yellow solid (1.01 g, 2.38 mmol, 76 %). ure based on Brooks 2010 and Brooks 2012. 5H(4oo Mrb,[N%SO-d5)611.82(s,1H),10.63(s,1H),9.99(s,1H),7.54(s,1H), 7.31-7.27 (nu 13H),7.23-7.19 (nL 3H).

HRMS (m/Z ESI'): C26H18N402 [M-H]' Found 419.1508 Requires: 419.1508.

Preparation 7: 5-((3-phenylpropylamino)methyl)(tritylamino)-3H- pyrrolo[2,3-d] pyrimidin-4(7H)-one O NH /L§ | \ THHN N N H General procedure A: To a suspension of N—((4-oxo—2-(tritylamino)-4,7-dihydro— rolo[2,3-d]pyrimidinyl)methyl)formamide (200.0 mg, 0.48 mmol) and sodium sulphate (5.0 mg) in methanol (5 cm3) under an argon here was added 3-phenylpropylamine (74.00 ”L, 0.52 mmol) and the resulting suspension stirred at room temperature for 2 h. Sodium borohydride (55.00 mg, 1.43 mmol) was then added and the reaction mixture d at room temperature for a further WO 2016050806 1 h. Water (5 cm3) was added and the resulting suspension stirred for 10 min before being extracted with dichloromethane (3 x 5 cm3). The combined organic layers were dried (M9804) and trated in vacuo to yield the crude product which was purified by flash chromatography (9:1 romethane-MeOH) to yield the desired compound as a white solid (210 mg, 41.8 %), m.p. > 300 °C (decomposition). Procedure based on Brooks 2010 and Brooks 2012. 1H (400 MHz, DMSO-ds): 1.73 (2H, quintet, J 7.8), 2.56 (4H, m), 3.74 (1H, s), 6.42 (1H, s, H-6), 7.19 (20H, m), 7.45 (1H, bs, NH), 10.78 (1H, bs, NH) 13C (400 MHz, DMSO-de): 31.7, 32.8, 45.4, 47.8, 70.4 (q), 99.7 (q), 114.9, 117.6 (q), 125.9, 126.0, 126.9, 128.0, 128.6, 129.0, 142.6 (q), 145.4 (q), 150.0 (q), 150.4 (q), 159.7 (c=0) HRMS (m/z - 551+): Found: 540.2757 [M+H]+ C35H34N50 Requires: 540.2765) vmaX/cm-l: 1542, 1611, 1670, 2868, 2951 Example 2: aminooxo-4,7—dihydro-3H-pyrrolo[2,3-d]pyrimidin- -yl)methyl)phenylpropanaminium chloride H2NNHN General ure B: A 5 cm3 reaction vessel containing a stirring bar was charged with 5-((3-phenylpropylamino)methyl)-2—(trity|amino)-3H-pyrrolo[2,3— d]pyrimidin-4(7H)-one (210.0 mg, 0.39 mmol) and 1.25 M methanolic HCI (3 cm3).

The resulting solution was stirred at room temperature for 16 h. The precipitated product was removed by vacuum filtration and washed with dichloromethane to yield the desired compound as a white powder (84 mg, 68 %), m.p. > 300 °C position). Procedure based on Brooks 2010 and Brooks 2012. an (600 MHz, DMSO-de): 1.90 (2H, app. quintet), 2.63 (2H, t, J 7.8), 2.90 (2H, m), 4.13 (2H, t, J 5.2), 6.57 (2H, bs), 6.80 (1H, d, J WO 0806 2.3), 7.16 (3H, m), 7.26 (2H, t, J 7.0), 9.11 (2H, bs), 11.05 (1H, m, NH), 11.31 (1H, broad doublet, NH) 5c (125 MHZ, e): 27.6, 32.1, 42.9, 45.6, 48.9, 98.6, 108.7 (q), 117.9 6.4, 128.6, 128.7, 140.9 (q), 152.9 (q), 160.5 (C=O) HRMS (m/Z ESI+): Found: 298.1662 (MJr C16H20N50 Requires: 298.1664) vmax (film)/cm’1: 1456, 1625, 2443, 2713, 2756, 2873, 2933, 3184 Preparation 8 5-((3-butylamino)methyl)(tritylamino)-3H—pyrrolo[2,3- d] pyrimidin-4(7H)-one O NH I \ TrtHN N N Prepared as per general procedure A using N—((4-oxo(tritylamino)-4,7-dihydro— 3H-pyrro|o[2,3-d]pyrimidiny|)methyl)formamide (200.00 mg, 0.48 mmol), n- butylamine (95.00 0L, 0.95 mmol) and NaBH4 (55 mg, 1.43 mmol) to yield the desired product as a white powder (200 mg, 88%), mp > 300 °C (decomposition). 5H (400 MHZ, DMSO-de): 0.84 (3H, t, J 7.3), 1.30 (2H, app. sextet), 1.51 (2H, app. quintet), 2.82 (2H, t,J 7.3), 4.01 (2H, s), 6.62 (1H, s), 7.24 (15H, m), 7.57 (1H, bs), 11.07 (1H, bs) 6c (400 MHz, DMSO-de): 13.9, 19.6, 28.0, 42.9, 46.0, 70.6 (q), 99.4 (q), 108.9, 118.0 (q), 127.0, 128.1, 129.0, 145.2 (q), 150.5 (q), 150.6 (q), 160.3 (C=O) Vmax (film)/Cm_1: 1545, 1613, 1672, 2870, 2956 HRMS (m/Z ESI+): Found: 478.2600 ([M+H]+ C30H32N50; Requires: 478.2607) WO 2016050806 Example 3 N-((2-aminooxo-4,7-dihydro-3H—pyrrolo[2,3-d] pyrimidin- -yl)methyl)butanaminium chloride 0 [:1H2 ‘ \ H2NJ\\N fl Prepared as per general procedure B using 5-((buty|amino)methyI) (tritylamino)-3H—pyrrolo[2,3-d]pyrimidin-4(7H)-one (108 mg, 0.39 mmol) and 1.25 M methanolic HCI (3 cm3) to yield the d product as a white powder (72 mg, 67 %), m.p. > 300 0C position). 6H (400 MHz, DMSO-de): 0.86 (3H, t, J 7.4), 1.31 (2H, app. sextet), 1.57 (2H, app. quintet), 2.90 (2H, m), 4.12 (2H, t, J 5.3), 6.49 (2H, bs), 6.81 (1H, s), 9.01 (2H, bs), 10.98 (2H, bs), 11.29 (1H, bs) 6c (400 MHz, e): 18.7, 24.7, 32.7, 47.4, 50.6, 103.6 (q), 114.0, 123.2 (q), 153.3 (q), 157.6 (q), 164.7 (C=O) HRMS (m/z ESI+): Found: 236.1518 (M+ C11H18N50 Requires: 236.1511) Vrnax (film)/cm'1: 1456, 1625, 1668, 2443, 2713, 2756, 2873, 2933, 3184 Preparation 9 5-((3-hexylamino)methyl)(tritylamino)-3H—pyrrolo[2,3- d]pyrimidin-4(7H)-one O NH l \ TrtHN N N Prepared as per general procedure A using N-((4-oxo(trity|amino)-4,7-dihydro- 3H-pyrrolo[2,3-d]pyrimidiny|)methy|)formamide (200.0 mg, 0.48 mmol), n- WO 2016050806 hexylamine (125.0011L, 0.95 mmol) and sodium borohydride (55 mg, 1.43 mmol) to yield the desired product as a white powder (200 mg, 83.0 %), m.p. > 300 °C (decomposition). 6H (400MHz, DMSO-de): 0.82 (3H, t, J 7.4), 1.20 (6H, m), 1.34 (2H, app. quintet), 2.40 (2H, t, J 7.4), 3.60 (2H, s), 6.30 (1H, s), 7.23 (15H, m), 7.37 (1H, bs), 10.62 (1H, bs) 66 (400 MHz, DMSO-de): 14.4, 22.5, 26.8, 31.6, 29.6, 45.2, 48.3, 70.4 (q), 99.7 (q), 115.2, 116.7 (q), 127.0, 128.1, 129.0, 145.4 (q), 150.1 (q), 150.5 (q), 159.7 (C=O) Vmax (film)/cm’1: 1552, 1648, 1734, 2856, 2928 HRMS (m/Z ESI‘): Found: 504.2769 ([M-H]' C32H34N50; Requires: 504.2763) Example 4 N-((2-aminooxo-4,7-dihydro-3H—pyrrolo[2,3-d] pyrimidin- 5-yl)methyl)hexanaminium chloride HN%5leatx l H2NNHN Prepared as per general procedure B using 5-((hexylamino)methyl)-2— lamino)-3H-pyrrolo[2,3-0‘]pyrimidin-4(7H)-one (190 mg, 0.39 mmol) and 1.25 M olic HCI (3 cm3) to yield the desired product as a white powder (70 mg, 64.8 %), m.p. > 300 oC. 5H (400 MHz, DMSO-de): 0.83 (3H, t, J 7.2), 1.25 (6H, m), 1.59 (2H, app. , J 7.2), 2.87 (2H, m), 4.11 (2H, t,J 5.3), 6.71 (2H, bs), 6.84 (1H, cl, J 3.6), 9.11 (2H, bs), 11.25 (1H, bs), 11.46 (1H, bs) 66 (400 MHz, DMSO-da): 14.3, 22.3, 25.8, 25.9, 31.1, 42.6, 46.1, 98.9 (q), 109.3 (q), 118.5, 148.1 (q), 152.8 (g), 159.8 (c=0) Vmax (film)/cm’1: 1578, 1625, 1669, 2429, 2712, 2861, 2930, 2957, 3266 WO 2016050806 HRMS (m/Z ESI+): Found: 264.1830 ([M+H]‘r C13H22N50 Requires: 264.1824) All the examples described herein are TGT substrates.

All the examples described herein are not inhibitors or substrates for HPRT To assess the potential of these compounds in vivo, a chronic monophasic EAE disease in mice was induced before treatment with the new chemical entity (NCE).

EAE Disease was induced in 8-10 week old female mice (C57BL/6) by sub- cutaneous (s.c.) injection of 200 pl emulsion containing 150 pg MOG33—55 e (Genscript) in Complete ’s Adjuvant (CFA; containing 5mg/ml heat- vated Mycobacterium tuberculosis). On the same day, mice were administered 500 ng Pertussis Toxin suken, Japan) intraperitoneally (i.p.) and again two days later. Disease severity was recorded every 24 hours: O-Normal; 1-flaccid tail; 2-imparied/wobbly gait; 3-complete hind limb weakness; 4-hind limb and forelimb paralysis; 5-moribund state/dead. Protocol is based on the Nature Protocols for Active induction of experimental allergic alomyelitis, which es the scoring methodology: Stromnes IM, an JM (2006) Active induction of experimental allergic encephalomyelitis. Nat Protoc. 1(4):1810-9.

Figure 5 shows the s of the in vivo testing for molecule N-((2-aminooxo-4,7- dihydro—3H—pyrrolo[2,3-d]pyrimidinyl)methyl)phenylpropan-l-aminium chloride (shown as Compound I) . The EAE score relates to an ment of disease ssion with t to issues like tail paralysis and limb paralysis, a higher score is a worse condition. A score of 1 indicates decreased tail tone, a score of 2 indicates hind leg weakness (paraparesis), a score of 3 indicates hind limb paralysis and/or incontinence. Note that the untreated animals have a continuous and progressive worsening of disease. In contrast, the animals treated with N-((2-amino-4—oxo-4,7- dihydro-3H-pyrrolo[2,3-d]pyrimidin-5—yl)methyl)pheny|propan-l-aminium chloride (shown as Compound I) exhibit rapid (within 24 hrs) reversal of symptoms at the highest dosage given (30 mg/kg, i.p.) and animals were scored as disease free after 4 daily WO 2016050806 treatments. At lower doses animals were slower to respond but in all cases disease progression was halted and reversed.

Figure 6 shows that in agreement with the observed disability scores, treated animals showed a dramatic return to normal weight gain, ng a weight level 96.6% that of controls (19.9 grams versus 20.6 grams), at 21 days post inoculation (dpi). This contrasts with the sustained drop in body weight of non-treated EAE diseased s to a level 86.4% that of controls at 21 dpi (17.8 grams versus .6 grams). Motor coordination and hind-limb strength were also evaluated by the ability of mice to cross a ntal bar (Figure 7). Non-diseased animals crossed the bar with an average time of 4.3 i 0.57 seconds. From 9 dpi, EAE mice showed a rapid deterioration in performance and were subsequently unable to maintain a grip on the apparatus. Strikingly, N-((2-aminooxo-4,7-dihydro-3H— pyrro|o[2,3-d]pyrimidin—5-yl)methy|)—3-phenylpropan—1-aminium chloride (shown as nd I) treatment fully restored the performance of EAE diseased mice to control levels within 4 treatments.

In addition to the data shown in Figures 5, 6 and 7, all the molecules described herein were tested in the c EAE model and found to show substantial improvement up to and including al of symptoms to a disease free state in subjects.

Claims (13)

The Claims Defining the ion are as Follows:

1. A use of a molecule capable of acting as a substrate for the queuineinsertase enzyme complex and wherein said molecule is not a substrate for 5 Hypoxanthine-guanine oribosyltransferase (HPRT) in the manufacture of a medicament for treatment of an autoimmune disease.

2. The use ing to claim 1, wherein the molecule capable of acting as a substrate for the queuine-insertase enzyme complex has the effect of lowering interferon gamma production. 10

3. The use according to claim 1 or claim 2, n the molecule capable of acting as a substrate for the queuine-insertase enzyme complex has a displacement of ≥50 pmol in a guanine displacement assay.

4. The use ing to any one of claims 1 to 3, n the molecule yields guanosine monophosphate product in amounts ≥60% when compared to a 15 control in an HPRT assay.

5. The use according to any one of claims 1 to 4, wherein the le lowers interferon gamma production in wild-type cells at a concentration of 100

6. The use according to any one of claims 1 to 5, wherein the autoimmune 20 disease is selected from rheumatoid arthritis, ulcerative colitis, sis, diabetes and inflammatory bowel disease.

7. The use according to claim 6, wherein the inflammatory bowel disease is Crohn’s disease.

8. The use ing to any one of claims 1 to 5, wherein the autoimmune 25 disease is transplant rejection.

9. The use according to any one of claims 1 to 5, wherein the autoimmune disease is multiple sclerosis.

10. The use ing to any one of claims 1 to 5, wherein the autoimmune disease is rheumatoid arthritis. 30

11. The use according to any one of claims 1 to 10, in combination with an additional therapeutic agent.

12. The use according to claim 11, wherein the therapeutic agent is a medicament for the treatment of the effects of multiple sclerosis damage in a patient. 35

13. The use according to any one of claims 1 to 12, wherein the molecule is: 2-amino(((3-phenylpropyl)amino)methyl)-3,7-dihydro-4H-pyrrolo[2,3- d]pyrimidinone; aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidinyl)methyl)- 3-phenylpropanaminium chloride; 2-amino((butylamino)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin- 4-one; 5 N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin yl)methyl)butanaminium chloride; 2-amino((hexylamino)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin- 4-one; N-((2-aminooxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin 10 yl)methyl)hexanaminium chloride; 2-amino((((1S,4S,5R)-4,5-dihydroxycyclopenten yl)amino)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidinone (Queuine); or 2-Amino[[[(1S,4S,5R)-4,5-dihydroxycyclopenten 15 yl]amino]methyl]-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidinone, monohydrochloride (Queuine HCl); or a ceutically acceptable salt or solvate of any one thereof. 000000000000 guanine 0 200 150 0 50 0 (|0qu) *VNal 1399A n 2 mm; peoeldsgp euguenfi [Om] SUBSTITUTE SHEET (RULE 26) |(400 Compound (400uM) Queuine (4000M) 2 Guanine FIG. §Enzyme ’77 Active VIIIIA nactivated Enzyme 120 100 O O O O O 00 to V N U0!1OBGJ eseJe;sueJu/(soquoquoqd euguenfi ;o lug/woe % SUBSTITUTE SHEET (RULE 26) Effect of a | on the proliferation of wild - type spleenocytes I3 % Non-proliferative Cells 120% % Proliferative Cells (100%) A O‘2o\ Total of 60% Percentage 40% OuM ’louM 100pM 500nm Concentration Compound I Effect of Formula | on the proliferation of HPRT Knockout spleenocytes I3 % oliferative Cells 120% % Proliferative Cells A 100% (100% 80% Total of 60% Percentage 40% OuM 10uM 100uM 500uM Concentration Compound I SUBSTITUTE SHEET (RULE 26) Effect of Formula | on the proliferation of GT Double Knockout spleenocytes El % Non-proliferative Cells 120% % Proliferative Cells. . Proliferation 100% Total 60% Percentage 40% OuM 10uM 100uM 500uM Concentration Compound I Effect of Formula | on the Proliferation of Wild-type and Single TGT Knockout spleenocytes El % Non-proliferative Cells 120% % Proliferative Cells. . Proliferation 100% Total 60% tage 40% 0% VA 100uM ZOOuM Concentration Compound I SUBSTITUTE SHEET (RULE 26) ‘ O _‘N ‘ O ‘ O 9‘ ‘ O ‘ O t ‘ O ‘ O m_\ ‘ O ‘ O m_‘ ‘ O ‘ O _\_\ CezmmEsEEEmoa mGE ‘ O mama €558 SEED: O m _ \\ 2:858 2:858 852-82 O N Banacom O m ma mxBEm:chanoOfiommomE .3883 .Bmmoma commomfiéolel O E958; O m mil? m_<m_III m<m|4| mil? O O _\ eJoos ueoguno SUBSTITUTE SHEET (RULE 26) { rm { o 2 { o { o‘u‘l'l‘l o C { I 4‘ 9 { o‘n‘ll { Ioil..." 2 { r r cozmmEsEEEwoq { "In.” 980 mxBEom csoquofiommowEmzmlll 9:392 9:088 a 958600 8:858 882-82 958600 .‘l...'O N EnumcomEmEHmofi O m .8835 .8885 8335 commwmfiéoz m m<m|§l m<m|¢r m_<m_IOI .vommmm__o-cozln_l IOI d (sweJ6)1q6!e/V\ SUBSTITUTE SHEET (RULE 26) 4444444444444 om cozmmEzEEEmoa m NF or m>mo mxBEom 9:083? mvtmEmN meEom w _ _ _ 950qu0 950qu0 950qu0 863-82 w vcsoquo Enumcow v 68320 .8385 .38829 .8385 N m_<m_+ m_<m_|l| m_<m_+ ED... wfiéozlnl 8886-826: Egg; 4 o (spuooes) Jeg |e1uongoH uo engi SUBSTITUTE SHEET (RULE 26)