WO2017216579A1 - Autophagy inducer compounds - Google Patents

Abstract

The invention relates to a heterocyclic compound 1,1'-(((propane-2,2-diylbis(4,1-phenylene))bis(oxy))bis(ethane-2,1-diyl))dipyrrolidine and its medical uses, for example as an autophagy inducer.

Description

AUTOPHAGY INDUCER COMPOUNDS

The invention relates to a heterocyclic compound and its medical uses.

Autophagy is a highly conserved homeostatic mechanism that involves lysosomal degradation of damaged and unwanted cellular components. It is believed to play an important role in inflammatory diseases such as atherosclerosis and plaque progression, and there is a known correlation between enhancing autophagy and protecting against heart, liver and other common age-related diseases. Autophagy may exert its beneficial effect in atherosclerosis and other diseases by degrading damaged intracellular organelles and thereby preventing oxidative injuries and cellular distresses.

Grainger et al. (1995, Nature Medicine 1: 1067-1073) and Reckless et al. (1997, Circulation 95: 1542-1548) have demonstrated that tamoxifen, a potent inducer of autophagy, inhibited atherosclerosis in mice models by suppressing the diet-induced formation of lipid lesions in the aorta by lowering of low-density lipoprotein (LDL) cholesterol.

Tamoxifen (prior art) Tamoxifen (2-[4-[(Z)-l,2-diphenylbut-l-enyl]phenoxy]-N,N-dimethylethan-amine) was originally a failed contraceptive that was redeveloped as a breast cancer drug. Tamoxifen has mixed agonist and antagonist activities that are species-, tissue- and cell- specific. In addition to its well-known antitumor properties derived from its anti-estrogenic activity in breast tissue, tamoxifen has also been found to increase the risk of endometrial cancer. Various analogues of tamoxifen have been developed as anti-cancer agents, including tesmilifene (N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine) which binds selectively to the high-affinity microsomal anti-oestrogen binding site but unlike tamoxifen has no affinity for oestrogen receptors. Other tamoxifen derivatives include ridaifen compounds such as Ridaifen A, Ridaifen B and Ridaifen D (as described in WO2014/061821).

The development of more selective autophagy inducers is needed if they are to become medicinally useful in the treatment and/or prevention of diseases where autophagy plays a role.

According to one aspect of the present invention, there is provided a compound of the formula (I):

(I).

The compound of formula (I) is: l,l'-(((propane-2,2-diylbis(4,l-phenylene))bis(oxy))bis(ethane-2,l-diyl))dipyrrolidine

(also referred to herein as "SKW128"). The novel compound described herein is shown surprisingly to be a highly effective autophagy inducer. In comparison with prior art compounds such as tamoxifen and also other proprietary compounds tested (including data not shown), an improvement of the present invention lies in the unexpected observation that SKW128 is a highly effective autophagy inducer (see Examples 2-4 below), with good pharmacokinetic and pharmacological properties (see Examples 5 and 6 below). By way of an exemplar medical application, SKW128 is shown to be surprisingly efficacious in a mouse model of non-alcoholic steatohepatitis (NASH) (see Examples 7 and 11 below).

A prior art search based on the structure of SKW128 was conducted after the invention was made. The closest prior art molecule identified ex post facto by the search was 3,3- bis[p-(2-pyrrolidinoethoxy)phenyl]butanol (Example 6 in US3718643A), a compound stated to inhibit cholesterol biosynthesis and aid in the regulation of cholesterol in the blood (although no biological data is provided in US3718643A). WO02/18334 describes heterocyclic compounds that are reported to bind to sodium channels and modulate their activity.

The compound of the invention may be in a pharmaceutically acceptable salt form.

The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the invention. This may include addition salts of inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, phosphate, diphosphate and nitrate or of organic acids such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulphonate, p-toluenesulphonate, palmoate and stearate. Exemplary salts also include oxalate, chloride, bromide, iodide, bisulphate, acid phosphate, isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate, bitartrate, ascorbate, gentisinate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, ethanesulfonate, and benzenesulfonate salts. For other examples of pharmaceutically acceptable salts, reference can be made to Gould (1986, Int J Pharm 33: 201-217). According to a further aspect of the invention, there is a provided a pharmaceutical composition comprising a compound of the invention as described herein and a pharmaceutically or therapeutically acceptable excipient or carrier.

The term "pharmaceutically or therapeutically acceptable excipient or carrier" refers to a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to the host, which may be either humans or animals, to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically-acceptable carriers such as those well known in the art may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

All suitable modes of administration are contemplated according to the invention. For example, administration of the medicament may be via oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, buccal, sublingual, transmucosal, inhalation, intra- atricular, intranasal, rectal or ocular routes. The medicament may be formulated in discrete dosage units and can be prepared by any of the methods well known in the art of pharmacy.

All suitable pharmaceutical dosage forms are contemplated. Administration of the medicament may for example be in the form of oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal products, oral mucoretentive products, topical creams, ointments, gels, films and patches, transdermal patches, abuse deterrent and abuse resistant formulations, sterile solutions suspensions and depots for parenteral use, and the like, administered as immediate release, sustained release, delayed release, controlled release, extended release and the like. Another aspect of the invention is the use of a compound of the invention as defined herein in the manufacture of a medicament for the treatment of a disease.

A further aspect of the invention is a compound of the invention for use as an autophagy inducer.

Further provided is a compound of the invention as defined herein for use in the treatment of a disease. The invention also encompasses a method of treating a disease, comprising the step of administering the compound or the pharmaceutical composition of the invention as defined herein to a patient in need of same.

The invention further encompasses the use of a compound of the invention as an autophagy inducer. The use may be in the treatment of a disease. Additionally or alternatively, the use may be in vitro, for example in an in vitro assay. A disease suitable for treatment according to the relevant aspects of the invention is one which is characterised by defective autophagy or which would benefit from modulation of autophagy.

Modified or altered autophagy has been shown to be relevant in neurodegenerative disease, as demonstrated by the accumulation of protein aggregates, for example in Alzheimer disease, Parkinson's disease, polyglutamine diseases, muscle diseases, and amyotrophic lateral sclerosis. Modified autophagy have also been implicated in other neurological diseases including epilepsies, neurometabolic and neurodevelopmental disorders such as schizophrenia. Autophagy inhibition plays a key role in the pathogenesis of inherited autophagic vacuolar myopathies (including Danon disease, X- linked myopathy with excessive autophagy, and infantile autophagic vacuolar myopathy), all of which are characterized by lysosomal defects and an accumulation of autophagic vacuoles. Autophagic vacuolar myopathies and cardiomyopathies can also be secondary to treatment with autophagy-inhibiting drugs (chloroquine, hydroxychloroquine and colchicine), which are used experimentally to interrogate autophagic flux and clinically to treat malaria, rheumatological diseases, and gout.

Autophagy impairment has also been implicated in the pathogenesis of inclusion body myositis, an age- associated inflammatory myopathy that is currently refractory to any form of treatment, along with other muscular dystrophies such as tibial muscular dystrophy. In all these striated muscle disorders, definitive tissue diagnosis used to require ultrastructural demonstration of accumulated autophagic vacuoles; more recently, it has been shown that IHC for LC3 and/or SQSTM1 can be used instead.

In addition, modified basal autophagy levels are seen in rheumatoid arthritis and osteoarthritis. Other aspects of the immune response associated with dysfunctional autophagy are seen in neutrophils from patients with familial Mediterranean fever and in monocytes from patients with TNF receptor-associated periodic syndrome, both of which are autoinflammatory disorders. Moreover, autophagy regulates an important neutrophil function, the generation of neutrophil extracellular traps (NETs). The important role of autophagy in the induction of NET formation has been studied in several neutrophil- associated disorders such as gout, sepsis, and lung fibrosis. Furthermore, there is a relationship between autophagy and the secretory pathway in mammalian macrophages for the release of IL1B, demonstrating a possible alternative role of autophagy for protein trafficking. This role has also been implied in neutrophils through exposure of protein epitopes on NETs by acidified LC3-positive vacuoles in sepsis and anti-neutrophil cytoplasmic antibody associated vasculitis. Patients with chronic kidney disease also have impaired autophagy activation in leukocytes, which is closely related to their cardiac abnormalities. There is also evidence for altered autophagy in pancreatic beta cells and in adipocytes of patients with type 2 diabetes.

A crucial role for therapy-induced autophagy in cancer cells has recently emerged, in modulating the interface of cancer cells and the immune system; primarily, by affecting the nature of danger signalling (i.e., the signalling cascade that facilitates the exposure and/or release of danger signals) associated with immunogenic cell death (ICD). Various observations have highlighted the important, context-dependent role of therapy-induced autophagy, in modulating the cancer cell immune cell interface by regulating the emission of ICD-associated danger signals. Recent studies also have implicated insufficient autophagy in the pathogenesis of nonresolving vital organ failure and muscle weakness during critical illness, leading causes of death in prolonged critically ill patients. A block of autophagy with consequent accumulation of autophagy substrates is detected in liver fibrosis and lysosomal storage diseases. Disease-associated autophagy defects are not restricted to macroautophagy but also concern other forms of autophagy. CMA impairment, for instance, is associated with several disease conditions, including neurodegenerative disorders, lysosomal storage diseases, nephropathies and diabetes.

The disease for treatment according to the present invention may be selected from any of the following as well as other diseases mentioned above: a neurodegenerative disorder (for example, Huntington's disease, Alzheimer's disease or Parkinson's disease), systemic lupus erythematosus ("lupus"), epilepsy, cancer, liver diseases including non- alcoholic fatty liver disease (NAFLD), including its extreme form non-alcoholic steatohepatitis (NASH), and al-antitrypsin deficiency (ATD), Niemann-Pick type C (NPC) disease, fibrinogen storage disease (FSB), inclusion body disease (IBD), lysosomal storage disease, muscular dystrophy (for example Duchenne muscular dystrophy or Limb-girdle muscular dystrophy), myopathy (for example myofibrillar myopathy, hereditary myopathy or diabetic cardiomyopathy), or an anti-inflammatory disorder selected from the group consisting of an autoimmune disease (for example multiple sclerosis, rheumatoid arthritis, lupus, irritable bowel syndrome, Crohn's disease), vascular disorders (including stroke, coronary artery diseases, myocardial infarction, unstable angina pectoris, atherosclerosis or vasculitis [such as Behcet's syndrome, giant cell arteritis, polymyalgia rheumatica, Wegener's granulomatosis, Churg-Strauss syndrome vasculitis, Henoch- Schonlein purpura or Kawasaki disease]), viral infection or replication (for example infections due to or replication of viruses including pox virus, herpes virus such as Herpesvirus samiri, cytomegalovirus [CMV], hepatitis viruses or lentiviruses [including HIV]), asthma and related respiratory disorders such as allergic rhinitis and COPD, osteoporosis (low bone mineral density), tumour growth, organ transplant rejection and/or delayed graft or organ function (for example in renal transplant patients), a disorder characterised by an elevated TNF-a level, psoriasis, skin wounds and other fibrotic disorders including hypertrophic scarring (keloid formation), adhesion formations following general or gynaecological surgery, lung fibrosis, liver fibrosis (including alcoholic liver disease) or kidney fibrosis, whether idiopathic or as a consequence of an underlying disease such as diabetes (diabetic nephropathy), disorders caused by intracellular parasites such as malaria or tuberculosis, neuropathic pain (such as post-operative phantom limb pain or postherpetic neuralgia), allergies, ALS, antigen induced recall response and immune response suppression.

The use of a numerical range in this description is intended unambiguously to include within the scope of the invention all individual integers within the range and all the combinations of upper and lower limit numbers within the broadest scope of the given range.

As used herein, the term "comprising" is to be read as meaning both comprising and consisting of. Consequently, where the invention relates to a "pharmaceutical composition comprising as active ingredient" a compound, this terminology is intended to cover both compositions in which other active ingredients may be present and also compositions which consist only of one active ingredient as defined. Unless otherwise defined, all the technical and scientific terms used here have the same meaning as that usually understood by an ordinary specialist in the field to which this invention belongs. Similarly, all the publications, patent applications, all the patents and all other references mentioned here are incorporated by way of reference in their entirety (where legally permissible).

Particular non-limiting examples of the present invention will now be described with reference to the following drawings, in which:

Fig. 1 is a graph showing the dose-dependent effect of SKW128 in an in vitro autophagy assay using human monocyte THP-1 cells. The x-axis shows concentration of SKW128, the right axis (or y-axis) shows fluorescence intensity (arbitrary units);

Fig. 2 is a graph showing results from a control plate with 5 μΜ tamoxifen in the assay used in Fig. 1. The x-axis shows the treatment used, the right axis (or y-axis) shows fluorescence intensity (arbitrary units); Fig. 3 is a graph showing the dose-dependent effect of SKW128 in an in vitro autophagy assay using human hepatocyte HepG2 cells. The x-axis shows concentration of SKW128, the right axis (or y-axis) shows fluorescence intensity (arbitrary units); Fig. 4 is a graph showing results from a control plate with 5 μΜ tamoxifen in the assay used in Fig. 3. The x-axis shows the treatment used, the right axis (or y-axis) shows fluorescence intensity (arbitrary units);

Fig. 5 is a Western blot showing LC3-II levels in HepG2 cells treated with SKW128 in the presence and absence of Bafilomycin A (a fusion blocker), shown as "+" and respectively. Treatments: A is Vehicle; B is 5 μΜ tamoxifen; C is 10 μΜ chloroquine; D is 10 μΜ SKW128;

Fig. 6 is a set of graphs A-C showing in vivo pharmacokinetics (PK) profiles for SKW128 administered intravenously into three individual rats at 3 mg/kg. The x-axis shows time in h, the right axis (or y-axis) shows SKW128 levels. In each graph, the dashed line represents terminal phase and the solid circles (·) indicate points included in Lambda_z;

Fig. 7 is a graph showing body weight at day 0 of mice in the experiment described in Example 11. The x-axis shows normal diet and various treatments under high-fat high- fructose diet, the y-axis shows body weight in g;

Fig. 8 is a graph showing body weight at day 84 (terminal body weight) of mice in the experiment described in Example 11. The x-axis shows normal diet and various treatments under high-fat high-fructose diet, the y-axis shows body weight in g;

Fig. 9 is a graph showing liver weight as a percentage of body weight at day 84 (terminal body weight) of mice in the experiment described in Example 11. The x-axis shows normal diet and various treatments under high-fat high-fructose diet, the y-axis shows liver weight as percentage of body weight; Fig. 10 is a histogram showing the steatosis maturity histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below;

Fig. 11 is a histogram showing the inflammation histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below;

Fig. 12 is a histogram showing the parenchymal fibrosis histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below; Fig. 13 is a histogram showing the necrotic foci histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below;

Fig. 14 is a histogram showing the autophagy foci histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below;

Fig. 15 is a histogram showing the Mallory Denk bodies histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below;

Fig. 16 is a histogram showing the biliary regeneration histopathology score of mice in the experiment described in Example 11. Histogram bars are as set out below; and

Fig. 17 is a graph showing alanine aminotransferase (ALT) activity in serum of mice in the experiment described in Example 11. The x-axis shows normal diet and various treatments under high-fat high-fructose diet, the y-axis shows ALT activity in U/L.

In each of Figs 10-16, the histograph bars represent the following:

Experimental

Example 1; l,l'-(((propane-2,2-diylbis(4,l-phenylene))bis(oxy))bis(ethane-2,l- diyl))dipyrrolidine ("SKW128")

SKW128 was prepared using synthesis procedures 1 or 2 as outlined below.

Synthesis procedure 1

To a solution of compound 1 (411 mg, 1.80 mmol, 1.0 eq.) in THF (10 mL) was added NaH (60%, 216 mg, 5.40 mmol, 3.0 eq.) at 0°C and stirred at 0°C for 30 minutes. Then to the reaction mixture was added compound 2 (1.45 g, 5.40 mmol, 3.0 eq.) and the reaction mixture was stirred at room temperature overnight. After completion, EtOAc (50 mL), was added and the organic layer was washed with water then brine, separated, dried (Na₂S0₄), filtered, concentrated under reduced pressure to give the residue. The residue was purified with silica gel column (2.5% MeOH in DCM) to give SKW128 as yellow solid (58 mg, Yield: 8%). ¹H NMR (400 MHz, CD₃OD) «57.16 (4H, d, J = 8.8 Hz, Ar-H), 6.88 (4Η, d, J = 8.8 Hz, Ar-H), 4.20 - 4.22 (4Η, m, OCH2CH2N), 3.28 - 3.33 (4H, m, NCH2CH2O), 3.09 (8H, m, NCH2CH2CH2), 1.99 (8H, m, NCH2CH2CH2), 1.64 (6H, s, 2CH3).

Synthesis procedure 2 (Steps A and B)

Step A: Synthesis of SKW128_2-1

NaOH (24.5 g, 613.3 mmol, 7.0 eq.) was added to a solution of 1 (3.50 g, 16.19 mmol, 1.0 eq.) and 2 (131.7 g, 700.86 mmol, 8.0 eq.) in acetone (50 mL). The resulting mixture was heated at 75 °C overnight. TLC showed SM 1 consumed not completely. The solvent was removed in vacuo. Then water was added to the mixture and extracted with EtOAC. The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and concentrated to give the crude product. The crude product was purified by silica chromatography (10% EtOAc in Petroleum ether) to give the product SKW128_2- 1 as a white oil (7.0 g, yield: 18.2%).

Step B: Synthesis of SKW128_2 (SKW128)

K₂C0₃ (3.77 g, 27.22 mmol, 6.0 eq.) was added to a solution of SKW128_2-1 (2.0 g, 4.55 mmol, 1.0 eq.) and 3 (1.29 g, 18.18 mmol, 4.0 eq.) in DMF (5.0 mL). The resulting mixture was stirred at rt overnight. TLC showed SM skwl28_2-l consumed

completely. Then water was added to the mixture and extracted with EtOAC. The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and

concentrated to give the crude product. The crude product was purified by silica chromatography (3.8% MeOH in DCM) to give the product SKW128_2 as a white oil (1.3 g, yield 67.7%). Example 2: Effect of SKW128 in an in vitro autophagy assay using human monocyte THP-1 cells

Methods

Human THP-1 cells, a myelo-monocytic cell line, were plated into 96 well plates (3.4 x 10⁵ cells/ml with 200μ1 of media/well) and differentiated into macrophages for 24 h by incubating with 200 nM phorbol-12-myristate (PMA) at 37°C in a humidified atmosphere containing 5% CO2. Following differentiation, the media was removed and replaced with treatments in triplicate using 5 μΜ tamoxifen ("TMX") or SKW128 (at concentrations of 0.1, 0.3, 1, 3, 10 and 20 μΜ) for 18 h (overnight) in a 37°C incubator in a final volume of 200μ1. At the end of the incubation step the cells were washed twice with fresh media (RPMI phenol red free/5% FBS) and 50μ1 RPMI phenol red free/5% FBS containing the Cyto-ID green staining dye provided in a commercially available autophagy kit (Abeam, abl39484) (final concentration IX) and Hoescht (1/1000), and were incubated for 45 minutes at 37°C in the dark. Lysosomal/autophagic vacuoles were detected using the Abeam kit which employs a proprietary dye, a cationic amphiphilic tracer which selectively labels autophagic vacuoles in the perinuclear region of the cell. Finally, cells were washed and fixed in 4% PFA for 10 minutes at RT. The cells were analysed using a SynergyHT plate reader (BioTek). Results

The extent of autophagy in this in vitro autophagy assay was measured using a fluorescent dye, which selectively labels autophagosomes. Tamoxifen ("TMX") was used as a positive control for all in vitro autophagy experiments to confirm the assay was functioning (see Fig. 2). TMX was used at 5 μΜ, as at higher concentrations the compound has a toxic effect on the cells.

The data in Fig. 1 show that SKW128 stimulates autophagy in THP-1 cells in a dose- dependent matter, with no cellular toxicity shown at the highest concentration used. SKW128 induces an increase of lysosomal/autophagic vacuoles in THP-1 cells, as measured by an increase in median fluorescence staining by flow cytometry techniques, compared to cells treated with vehicle. The calculated EC50 is 1.9 to 4 μΜ. SKW128 stimulates autophagy in THP-1 cells more effectively than TMX (see Fig. 2). Example 3: Effect of SKW128 in an in vitro autophagy assay using human HepG2 cells

The in vitro assay as described in Example 2 was repeated using liver hepatocyte HepG2 cells. HepG2 cells were harvested using trypsin/EDTA then diluted to 1 x 10⁵ cells/ml in EMEM (Eagles Minimal Essential Medium)/ 10%FBS, and adhered for 24 h. The data in Fig. 3 show that SKW128 also stimulates autophagy in HepG2 cells in a dose-dependent matter, with no cellular toxicity shown at the highest concentration used. The calculated EC50 is 0.5 to 1.4 μΜ. SKW128 stimulates autophagy in HepG2 cells more effectively than TMX (see Fig. 4).

Example 4: Measuring LC3-II (autophagic flux) using Western Blotting techniques

In Examples 2 and 3 above, we show that SKW128 induces a dose-dependent increase in autophagy signal, which we detected using a proprietary fluorescent dye/flow cytometer. Tamoxifen (TMX) has been shown to drive autophagic flux and was used as an internal positive control in our screening. Our assumption was that a fluorescent signal measured with our development compounds also reflected increased autophagic flux.

The proprietary fluorescent dye used in the screening assay in Examples 2 and 3 is a cationic amphiphilic tracer which selectively labels autophagic vacuoles in the perinuclear region of the cell. A population of the proprietary autophagy dye-labelled vesicles co-localise with the microtubule- associated protein 1A/1B light chain-3 (LC3, Mw ~17kDa,), a ubiquitous key autophagy protein. Changes in cellular LC3-II and the number of LC3-II vesicles correlate with autophagosome abundance, but this does not necessarily reflect autophagic flux (i.e. the rate of autophagosome delivery to the lysosome). This is because blockers of fusion between the autophagosome and the lysosome would result in an increase in the number of autophagosomes (but not flux) and would produce the same signal in this assay. Western blotting techniques are often used to attempt to assess the autophagy process and differentiate between flux enhancers and fusion blockers. Although the proprietary dye used in our assays in Examples 2 and 3 is a surrogate marker of autophagy, in the present example we have assessed LC3-II levels in HepG2 cells treated with SKW128 in the presence and absence of Bafilomycin A ("BafA", a fusion blocker). If levels of LC3-II were seen to increase synergistically following treatment of cells with SKW128 in the presence of optimal concentrations of BafA then we could assume that this signal was being driven by an alternative mechanism i.e. increased flux rather than increased blockade of fusion. Chloroquine (also known to block fusion) was used as a 'negative' control, as chloroquine and BafA both block fusion then there should be no increased LC3-II signal measured on administration of chloroquine. Using a Western blot technique based on that reported by David Rubinsztein (2012, Current Protocols in Cell Biology 54: 15.16.1 - 15.16.25), we assessed the expression levels of LC3-II in HepG2 cells (liver carcinoma cell line also used in the autophagy assay of Example 3). Briefly, cells were plated and cultured overnight and then washed with media and treated for 14-16 hours with Tamoxifen (5μΜ), SKW128 (ΙΟμΜ) or Chloroquine (ΙΟμΜ). During the last 4 hours of treatment, half the wells from each treatment group were treated with Bafilomycin A (400nM). At the end of the incubation, cells plates were plunged on ice and washed with ice cold PBS containing protease inhibitors prior to preparing cell lysates for evaluation by Western blot. Equal total protein was loaded onto 12% SDS-PAGE gels, separated by electrophoresis and transferred to PVDF membrane for probing with a rabbit anti-LC3 antibody (NB 100-2200, Novus Biologicals) and rabbit anti-actin (Sigma) as a loading control.

The results in Fig. 5 show that treatment with 10 μΜ SKW128 resulted in an increased level of LC3-II detected over those measured with BAF-A alone. Chloroquine did not induce levels of LC3-II over that of BafA (as detected by Western blot). Tamoxifen was synergistic with BafA in increasing LC3-II levels but not as effective as SKW128.

The experiment was repeated 3 times, and confirmed that SKW128 is not increasing the autophagy signal as shown in Examples 2 and 3 by blocking fusion of the autophagosome and the lysosome, but rather driving autophagic flux (as has been described for tamoxifen). Example 5: Inhibition of cytochrome P450 interactions (Drug-Drug interactions of SKW128)

Using E.coli CYPEX membranes in combination with specific probe substrates, we assessed the inhibition of individual CYPs by SKW128 (see Weaver et ah, 2003, Drug Metab Dispos 31:7, 955-966). The study was carried out with the introduction of a preincubation in the absence or presence of NADPH to distinguish between direct or time- dependent inhibition. We clearly demonstrated that SKW128 exhibited no significant drug-drug interactions, as set out in Table 1. Table 1. In vitro PK drug-drug interactions

Example 6: In vitro and in vivo properties of SKW128 which predict in vivo hepatic clearance

A. In vitro clearance of SKW128 in mouse and human hepatocytes

The intrinsic clearance (Clint) and half-life of SKW128 was measured in a mixed hepatocyte suspension of cryopreserved mouse or human hepatocytes. Briefly, compound is incubated with hepatocyte suspensions at 37°C over a time course and remaining compound at each time point is assessed by mass spectrometry (UPLC-MS/MS). Clint in mouse hepatocytes was <3 μΙ/min/lO⁶ cells and in human hepatocytes was 14.0 3 μΙ/min/lO⁶ cells. Half-life in mouse hepatocytes was >460 min and in human hepatocytes was 106 min. B. Plasma protein binding ("PPB"), lipophilicity and drug distribution of SKW128

The extent to which SKW 128 bound to plasma proteins such as albumin and alpha- 1 acid glycoprotein within human or mouse blood was determined by rapid equilibrium dialysis. Compounds were incubated at 5μΜ for 4 hours at 37°C. We found that PPB in mouse cells was 83.35% and in human cells was 78.87%. To understand whether SKW128 was highly bound to red blood cells the Blood: Plasma partitioning was assessed using parallel incubation of the compound in fresh blood and matched plasma. Compound (ΙμΜ) was incubated at 37°C for 30 min at pH7.4 before analysis by UPLC-MS/MS to determine bound vs unbound fractions. The Blood:Plasma ratio was 2.91 in mouse and 6.37 in human.

The partition coefficient (LogD) between buffer (PBS, pH 7.4) and n-octanol was measured to determine the lipophilicity of SKW128. The LogD at pH 7.4 of SKW128 was measured and shown to be 2.10.

C. In vivo pharmacokinetics of SKW128

SKW128 was administered to C57BI/6 male mice intravenously (lmg/kg) or orally (5mg/kg) by gavage. Whole blood diluted with water was prepared from these dosed animals over a time course up to 96 hours post dose to allow blood concentrations of drug to be estimated by UPLC-MS/MS. Analysis of the compound levels over the time course allows an estimation of pharmacokinetic properties of the drug. The measurements allowed calculation of the following parameters for SKW128:

Half-life in blood (T½) = 62 h

Observed clearance/F = 5 ml/min/kg

Volume of distribution/Vz/F = 29 1/kg

AUCall = 10366 ng.h/ml

AUCINF_obs = 15533 ng.h/ml

Bioavailability F(AUC) = >100%.

The distribution of drug into tissues from the study described above (following dosing with 5mg/kg PO) was measured 24 hours post dosing using UPLS-MS/MS and recorded in Table 2. Table 2. Tissue distribution of SKW128 in mouse

In summary, the pharmacokinetic and pharmacological studies shown in Examples 5 and 6 have demonstrated that SKW128 has improved functional activity in autophagy compared with tamoxifen, has good bioavailability with low in vivo clearance resulting in a relatively long half-life in blood. SKW128 does not appear to induce CYP P450s. Further pharmacokinetic and pharmacological test results are set out in Example 8 below. Example 7: SKW128 efficacy in a murine diet-induced non-alcoholic steatohepatitis ("NASH") model I

Introduction

NASH is a condition in which excess fat accumulates in the liver of patients with no history of alcohol abuse. It is regarded as an hepatic manifestation of metabolic syndrome, for which the incidence is increasing worldwide in line with the prevalence of obesity and type 2 diabetes. It is estimated that around 3% of adults worldwide have NASH (and around 20% have NAFLD). In NASH, not only steatosis but also intralobular inflammation and hepatocellular ballooning, often with progressive fibrosis. The dietary induced mouse model of non-alcoholic steatohepatitis (NASH) recapitulates many of the histopathological features of the human clinical syndrome (e.g. Clapper et al, 2013, Am J Physiol Gastrointest Liver Physiol 305: G483-G495). The clinical syndrome is quite heterogeneous and reflects a spectrum of disease severity from low grade steatosis, through to marked hepatic steatosis and cellular ballooning with varying degrees of inflammation, finally leading to parenchymal fibrosis. Clinically, a poorer prognostic outcome is associated with inflammation and fibrosis. The murine dietary model presents with characteristic histopathology - microvesicular and macrovesicular steatosis, ballooning degeneration of hepatocytes, inflammation and fibrosis - but, distinct from the human disease - shows a greater degree of spontaneous regeneration (such as biliary regeneration and hepatic regenerative micro-nodules) and variability in the inflammatory response to hepatocyte degeneration. It is an attractive model for delineating cellular sites of action of putative therapeutic agents due to the linear nature of the lesion in the relative absence of co-morbidity.

Methods

Histopathology assessment

Liver sections were provided from 59 animals from a study set of 64 animals (including animals used for studies not reported here). There were no slides from animals 15, 17, 38, 46 and 59. Three slides were provided from each animal - each slide stained with a different staining protocol - haematoxylin and eosin ("M&E"), Masson's trichome ("MT") and reticulin. In general, the quality of the slide processing and staining was good with no rejections on quality grounds.

All slides were asses in a blinded fashion - using a computer generated random number sequence (random.org) - in order to control for observer bias and diagnostic drift.

Histopathology grade criteria

Assignment of grade is based upon the most frequent lesion.

Steatosis maturity

0 - no significant pathology; 1 - few, scattered areas of small steatotic hepatocytes; 2 - confluent areas of steatotic hepatocytes showing variable vacuolation; 3 - confluent areas of steatotic hepatocytes with marked vacuolation - micro or macro; 4 - marked steatosis occupying most of liver zone with associated degeneration/ ballooning; 5 - marked zonal steatosis with associated degeneration/ ballooning and fibroplasia. Inflammation

0 - no significant pathology; 1 - occasional acute inflammatory foci, often peri-vascular; 2 - frequent acute inflammatory foci, peri-portal or peri-vascular; 3 - mixed inflammatory infiltrates, peri-vasculature and, often, peri-biliary; 4 - marked mixed inflammatory infiltrates associated with zones of hepatocyte degeneration; 5 - marked mixed inflammatory infiltrates, frequent, confluent, often associated with degeneration/ necrosis. Parenchymal fibrosis

0 - no significant pathology; 1 - low grade fibroplastic foci, often peri-vascular or peri- biliary; 2 - occasional expansion of fibroplastic expansion of parenchymal chords; 3 - confluent fibroplastic expansion of parenchymal chords; 4 - immature fibroplastic foci, with associated inflammation and hepatocyte degeneration; 5 - fibrosis foci, with marked inflammation and hepatocyte degeneration.

Necrotic foci

0 - no significant pathology; 1 - occasional, low grade, necrotic; 2 - frequent single necrotic foci; 3 - multiple, discrete necrotic foci in liver field; 4 - marked necrotic foci associated with zones of hepatocyte degeneration; 5 - marked, often confluent, necrotic foci, often associated with degeneration/ fibrosis.

Reticulin

0 - no significant pathology; 1 - sporadic, low grade, disorganisation of reticulin matrix; 2 - confluent, low grade, disorganisation; 3 - multiple zones showing reticulin fibrillation or partial loss; 4 - steatotic loss of reticulin network - multi-focal; 5 - complete loss of reticulin network due to hepatocyte ballooning/ degeneration/ fibrosis.

Autophagy foci

0 - occasional foci; 1 - < 20 foci per zone; 2 - 20-50 foci per zone; 3 - 50-70 per zone; 4 - 70-100 per zone; 5 - 100+ per zone. Mallory-Denk bodies

0 - occasional foci; 1 - < 20 foci per zone; 2 - 20-50 foci per zone; 3 - 50-70 per zone; 4 - 70-100 per zone; 5 - 100+ per zone. Biliary epithelial regeneration

0 - occasional biliary proliferation foci; 1- multi-focal small biliary regeneration; 2 - biliary regeneration involving multiple areas of biliary tree; 3 - occasional cell atypia; 4 - marked regeneration with multiple atypia; 5 - regeneration with occasional metaplasia. Results

As shown in Table 3, mice maintained on a normal diet showed no significant liver pathology. Autophagy foci were present and levels consistent with normal cell homeostasis. Mice maintained on a high fat/ fructose diet developed a mature steatohepatitis with a microvesicular or mixed microvesicular/ macrovesicular steatosis, parenchymal fibrosis, hepatocellular ballooning and necrosis and loss of the anatomical integrity of the reticulin network. Steatohepatitis was associated with a trend towards reduced autophagy foci, but elevated Mallory-Denk bodies - both consistent with reduced clearance of cell debris.

In terms of compound efficacy profiles, administration of 5 mg/kg dose of a related compound "SKW137" (4,4'-((((2,2-difluorocyclopropane-l,l-diyl)bis(4,l- phenylene))bis(oxy))bis(ethane-2,l-diyl))bis(l-methylpiperazine)) was associated with reduced steatosis maturity, parenchymal fibrosis and increased biliary regeneration. SKW128 (10 mg/kg dose) administration was not associated with a significant effect upon steatosis maturity, but was associated with reduced inflammation, parenchymal fibrosis, necrotic foci, loss of reticulin network and frequency of Mallory-Denk bodies. In addition, SKW128 (10 mg/kg) was associated with increased frequency of autophagy foci and regeneration of the biliary epithelium.

Overall steatosis severity and maturity are perhaps the most intractable aspect of this model - as severity will reflect systemic metabolic stress. It is encouraging that SKW128 showed significant efficacy on the consequential lesions of steatosis - and these data offer support to the concept of enhanced macroautophagy using SKW128 as a therapeutic approach to disconnection of the primary steatotic lesion from the resultant end- stage pathology.

The broadly good performance of the SKW128 on biliary regeneration suggests a general positive effect on reducing biochemical stress in the parenchyma.

Example 8: Selectivity of SKW128

Various further standard pharmacology tests were performed on SKW128 and the results are summarised below.

SKW128 was found to be devoid of tamoxifen-like polypharmacology, showing no binding to oestrogen receptor (even at ΙΟΟμΜ) and minimal inhibition of LTA4H (21% at 30μΜ).

SKW128 also exhibited an exceptionally clean CEREP profile testing of the molecular targets shown in Table 4 below.

Table 4. CEREP Profile for SKW128

Our studies have revealed that SKW128 is a powerful stimulator of flux through the autophagy pathway in many different cell types. SKW128 is much more potent and powerful than tamoxifen. SKW128 is highly specific, lacking the oestrogen receptor binding and P450 inhibition characteristics of tamoxifen. However, the molecular target responsible for the induction of autophagy by SKW128 (and tamoxifen) remains unknown.

Example 9: Further ADME properties of SKW128

As summarised in Table 5 below (and see also Example 6 above), our studies reveal that SKW128 shows significant protein binding (about 80% bound in human). The intrinsic clearance by hepatocytes in vitro varies according to species. We suggest that the principle metabolism of SKW128 involves FM02-mediated oxidation of pyrrolidine rings, which explains the lack of metabolism in mice. Table 5. Summary of ADME properties of SKW128

We consider that the importance of protein binding and intrinsic clearance on in vitro efficiency blunted by largely intracellular localisation of SKW128.

The in vivo pharmacokinetics (PK) study for SKW128 in mice shown in Example 6 above was repeated for rat, as shown in Fig. 6 and Table 6.

Table 6. Rat intravenous PK

More detailed information of the tissue distribution of SKW128 in mouse (compared to the results shown in Table 2) is provided in Table 7 below. Table 7. Detailed tissue distribution of SKW128 in mouse

SKW128 was found in all tissues (including brain) after a single oral dose. This suggests that, in principle, SKW128 may be able to treat pathology in all organ systems.

In summary, the in vivo PK for SKW128 shows allometric scaling between mouse and rat. PK is characterised by a high volume of distribution, resulting in low clearance and a long half-life. SKW128 is orally bioavailable in both mouse and rat (F = >80%), with Vss, clearance and half-life very similar to IV dosing. Despite high Vss, SKW128 shows good tissue distribution without obvious accumulation. We suggest the PK profile of SKW128 likely results from intracellular accumulation, perhaps in the lysosome or autosome. SKW 128 has an ADME profile consistent with, but not limited to, once-daily oral dosing. The unusual PK profile, with high Vss and long half-half, could be managed for example by adopting, in one embodiment of the invention, a loading dose and a maintenance dosing profile of SKW 128. For example, in one embodiment, SKW 128 in a lOOmg loading dose plus a 5mg daily maintenance dose is predicted to achieve continuous effective exposure in humans. Example 10: Tissue distribution of SKW128 in rat

SKW128 was orally administered by gavage to male Han Wistar rats at 5, 25, 100 and 250mg/kg daily for 14 days. Twenty four hours after the final dose, the animals were cardiac perfused with 20ml saline and tissues were excised, rinsed, blotted and weighed before snap freezing. The perfusion was carried out to allow us to determine levels of SKW128 in the tissue versus levels in the blood associated with tissue.

The distribution of drug into tissues from the study described above was measured using UPLS -MS/MS and recorded in Table 8.

Table 8. Tissue distribution of SKW 128 in mouse following perfusion

Liver 383793 7088

Lung 539139 9497

Kidney 434404 7874

Stomach 38821 643

Small intestine 128986 2341

Large intestine 129461 2011

Gastrocnemius 24405 392

muscle

Epididymal fat 13582 219

SKW128 was detected in all tissues at all dose levels with the exception of the 5mg/kg dose level where SKW128 could not be detected in brain tissue. The levels measured in tissues increased with dose and the highest levels were again seen in liver, lung and kidney. The lowest levels were measured in brain tissue suggesting that the material does not readily cross the blood brain barrier. The levels of SKW128 described earlier (Example 6, Table 2) are likely to reflect blood associated with brain tissue rather than the tissue itself. Example 11; SKW128 efficacy in a murine NASH model II

This example provides additional evidence for the efficacy of SKW128 in the murine NASH model as described in Example 7 above.

Methods

Mice acclimitised to diet were fed a high fat/fructose diet for 12 weeks and orally dosed daily by gavage with SKW128 (10, 3, lmg/kg), vehicle or Elafibrinor (lOmg/kg). Elafibrinor is a dual PPARa/δ agonist, which is currently being developed by Genfit for the treatment of NASH. Histopathology assessment

Liver sections were provided from 66 animals. Three slides were provided from each animal - each slide stained with a different staining protocol - haematoxylin and eosin ("M&E"), Masson's trichome ("MT") and reticulin. In general, the quality of the slide processing and staining was good with no rejections on quality grounds.

All slides were assessed in a blinded fashion - using a computer generated random number sequence (random.org) - in order to control for observer bias and diagnostic drift.

Histopathology grade criteria, steatosis maturity, inflammation, parenchymal fibrosis, necrotic foci, reticulin, autophagy foci, Mallory-Denk bodies, and biliary epithelial regeneration were scored exactly as described in Example 7 above.

Results

Mice were weighed pre-dose and at study end (12 weeks) and data is presented as body weight (g) on day 0 (Figure 7) and on day 84 (Figure 8). Livers were also weighed and have been expressed as a percentage of body weight at the end of the study (Figure 9). Only animals on a 'normal' diet gained weight over the 12 week study duration, all animals on the high-fat high-fructose diet remained stable. Elafibrinor dosed daily at lOmg/kg resulted in a significant increase in liver to body weight ratio when compared to vehicle treated mice on the same high-fructose high-fat diet. SKW128 had no impact on liver to body weight ratio compared to vehicle.

A histological analysis determined that mice maintained on a high fat/ fructose diet developed a mature steatohepatitis with a microvesicular or mixed microvesicular/ macrovesicular steatosis (Figure 10), inflammation (Figure 11), parenchymal fibrosis (Figure 12), hepatocellular ballooning and necrosis (Figure 13) and loss of the anatomical integrity of the reticulin network. Steatohepatitis was associated with a trend towards reduced autophagy foci (Figure 14), but elevated Mallory-Denk bodies (Figure 15) - both consistent with reduced clearance of cell debris.

In terms of compound efficacy profiles, administration of SKW128 (10 mg/kg) was associated with a significant effect upon steatosis maturity as was Elafibrinor (lOmg/kg). SKW128 at lower doses showed a trend towards a reduction in steatosis maturity (Figure 10). SKW128 demonstrated dose dependent reduction in inflammation at 10, 3 and lmg/kg. Elafibrinor at lOmg/kg also reduced the inflammation observed (Figure 11). Parenchymal fibrosis was reduced at all doses measured by both SKW128 and Elafibrinor (Figure 12). A dose dependent trend in reduction of necrotic foci was observed with SKW128 and a significant comparable reduction was seen following treatment with both Elafibrinor and SKW128 at lOmg/kg (Figure 13). The frequency of Mallory-Denk bodies was reduced with SKW128 at 3 and lOmg/kg and also observed to a lesser degree with Eafibrinor (lOmg/kg) (Figure 15). In addition, SKW128 (at 3 and 10 mg/kg) was associated with increased frequency of autophagy foci (Figure 14) and a marked regeneration of the biliary epithelium (Figure 16).

Overall steatosis severity and maturity are perhaps the most intractable aspect of this model - as severity will reflect systemic metabolic stress. It is encouraging that SKW128 showed significant efficacy on the consequential lesions of steatosis - and these data offer support to the concept of enhanced macroautophagy using SKW128 as a therapeutic approach to disconnection of the primary steatotic lesion from the resultant end-stage pathology.

The broadly good performance of the SKW128 on biliary regeneration suggests a general positive effect on reducing biochemical stress in the parenchyma. SKW128 at lOmg/kg and 3mg/kg generally behaved similarly to Elafibrinor at lOmg/kg although it should be noted that Elafibrinor did not significantly impact biliary regeneration. In addition it was noted that Elafibrinor also caused a very significant increase in Liver to body weigh ratio.

Alanine aminotransferase (ALT) is a pyridoxal-phosphate dependent enzyme that catalyses the reversible transfer of an amino group from alanine to a-keto-glutarate, generating pyruvate and glutamate. ALT is found predominantly in the liver and serum levels can be used as a marker of liver injury. Levels at the end of this 12 week study were measured in serum using a coupled enzyme assay. ALT activity is presented in Figure 17. SKW128 (at lOmg/kg) reduces levels of ALT to those observed in animals on a normal diet. Again there is a dose dependent trend in reduction at 10, 3 and lmg/kg SKW128. No reductions were observed following treatment with Elafibrinor (Figure 17). Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations to the same can be accomplished without departing from the spirit and scope of the present invention and that such modifications are clearly contemplated herein. No limitation with respect to the specific embodiments disclosed herein and set forth in the appended claims is intended nor should any be inferred.

Claims

Claims

1. A compound of the formula (I):

2. The compound according to claim 1 in a pharmaceutically acceptable salt form.

3. A pharmaceutical composition comprising a compound according to either of claim 1 or claim 2 and a pharmaceutically or therapeutically acceptable excipient or carrier.

4. Use of a compound according to either of claim 1 or claim 2 in the manufacture of a medicament for the treatment of a disease.

5. A compound according to either of claim 1 or claim 2 for use as an autophagy inducer.

6. A compound according to either of claim 1 or claim 2 for use in the treatment of a disease.

7. A method of treating a disease, comprising the step of administering a compound according to either of claim 1 or claim 2, or a pharmaceutical composition according to claim 3, to a patient in need of same.

8. Use of a compound according to either of claim 1 or claim 2 as an autophagy inducer.

9. The use according to claim 8 in the treatment of a disease.

10. The use according to either of claim 8 or claim 9 wherein the use is in vitro.

11. The use according to claim 4, the compound for use according to claim 6, the method of treatment according to claim 7, or the use according to claim 9, wherein the disease is selected from: a neurodegenerative disorder (for example, Huntington's disease, Alzheimer's disease or Parkinson's disease), systemic lupus erythematosus ("lupus"), epilepsy, cancer, liver diseases including non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and a 1 -antitrypsin deficiency (ATD), Niemann-Pick type C (NPC) disease, fibrinogen storage disease (FSB), inclusion body disease (IBD), lysosomal storage disease, muscular dystrophy (for example Duchenne muscular dystrophy or Limb-girdle muscular dystrophy), myopathy (for example myofibrillar myopathy, hereditary myopathy or diabetic cardiomyopathy), or an anti-inflammatory disorder selected from the group consisting of an autoimmune disease (for example multiple sclerosis, rheumatoid arthritis, lupus, irritable bowel syndrome, Crohn's disease), vascular disorders (including stroke, coronary artery diseases, myocardial infarction, unstable angina pectoris, atherosclerosis or vasculitis [such as Behcet's syndrome, giant cell arteritis, polymyalgia rheumatica, Wegener's granulomatosis, Churg-Strauss syndrome vasculitis, Henoch- Schonlein purpura or Kawasaki disease]), viral infection or replication (for example infections due to or replication of viruses including pox virus, herpes virus such as Herpesvirus samiri, cytomegalovirus [CMV], hepatitis viruses or lentiviruses [including HIV]), asthma and related respiratory disorders such as allergic rhinitis and COPD, osteoporosis (low bone mineral density), tumour growth, organ transplant rejection and/or delayed graft or organ function (for example in renal transplant patients), a disorder characterised by an elevated TNF-a level, psoriasis, skin wounds and other fibrotic disorders including hypertrophic scarring (keloid formation), adhesion formations following general or gynaecological surgery, lung fibrosis, liver fibrosis (including alcoholic liver disease) or kidney fibrosis, whether idiopathic or as a consequence of an underlying disease such as diabetes (diabetic nephropathy), disorders caused by intracellular parasites such as malaria or tuberculosis, neuropathic pain (such as post-operative phantom limb pain or postherpetic neuralgia), allergies, ALS, antigen induced recall response and immune response suppression.

12. The use according to claim 4, the compound for use according to claim 6, the method of treatment according to claim 7, or the use according to claim 9, wherein the disease is non-alcoholic steatohepatitis (NASH).