WO2017216576A1 - Autophagy inducer compounds - Google Patents

Abstract

The invention relates to difluoro heterocyclic compounds and their medical uses. Compounds of the invention have the general formula (I): wherein each X is identical and forms part of an ethoxyamine basic side chain which includes a 5- or 6-membered heterocyclic ring, and the heterocyclic ring X is selected from the group consisting of: formula (II).

Description

AUTOPHAGY INDUCER COMPOUNDS

The invention relates to difluoro heterocyclic compounds and their 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.

WO2013/062079 describes l,l'-difluoro-2,2'-diphenylcycloproprane derivatives as modulators of metabotropic glutamate receptor subtype 2 (mGlu2 receptor) and their uses in the treatment of mental disorders. Miyashita et al. (2005; In: "Environmental Fate and Safety Management of Agrochemicals", American Chemical Society, Vol. 899: 159-166) describes a l,l '-difluoro-2,2'-diphenylcycloproprane derivative (compound 34 in Fig. 2) as a potential oestrogen receptor inhibitor. US5015666, US5658914, US5658951, Cheng et al. (2004, Mol Pharmacol 66: 970-977), Singh et al. (1996, Bioorganic Chem 24: 81- 94), and Day et al. (1991, J Med Chem 34: 842-851) describe l,l'-dichloro diphenylcycloproprane derivatives and their potential medical uses. WO02/18334 describes heterocyclic compounds that are reported to bind to sodium channels and modulate their activity.

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 general formula (I):

(I)

wherein: each X is identical and forms part of an ethoxyamine basic side chain which includes a 5- or 6-membered heterocyclic ring; and

the heterocyclic ring X is selected from the group consisting of:

As elaborated below, we have found that compounds of general formula (I) are shown surprisingly to be a highly effective autophagy inducers. In comparison with prior art compounds such as tamoxifen and also structurally similar proprietary compounds tested (including "SKW166" [see below] and others not shown), an improvement of the present invention lies in the unexpected observation that compounds of the invention are highly effective autophagy inducers, with good pharmacokinetic and

pharmacological properties. 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).

The compound of the invention may be l,l'-((((2,2-difluorocyclopropane-l,l- diyl)bis(4,l-phenylene))bis(oxy))bis(ethane-2,l-diyl))dipyrrolidine ("SKW120"). The chemical structure of SKW120 is:

Alternatively, the compound of the invention may be 4,4'-((((2,2-difluorocyclopropane- 1,1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 -diyl))bis( 1 -methylpiperazine) ("SKW137"). The chemical structure of SKW137 is:

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 SKW120 in an in vitro autophagy assay using human monocyte THP-1 cells. The x-axis shows concentration of SKW120, the right 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 shows fluorescence intensity (arbitrary units);

Fig. 3 is a graph showing the dose-dependent effect of SKW120 in an in vitro autophagy assay using human hepatocyte HepG2 cells. The x-axis shows concentration of SKW120, the right axis shows fluorescence intensity (arbitrary units);

Fig. 4 is a graph showing results from a control plate using tamoxifen in the assay used in Fig. 3. The x-axis shows concentration of TMX used, the right axis shows fluorescence intensity (arbitrary units); Fig. 5 is a graph showing the dose-dependent effect of SKW137 in an in vitro autophagy assay using human monocyte THP-1 cells. The x-axis shows concentration of SKW137, the right axis shows fluorescence intensity (arbitrary units); Fig. 6 is a graph showing results from a control plate with 5 μΜ tamoxifen in the assay used in Fig. 5. The x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);

Fig. 7 is a graph showing the dose-dependent effect of SKW137 in an in vitro autophagy assay using human hepatocyte HepG2 cells. The x-axis shows concentration of SKW137, the right axis shows fluorescence intensity (arbitrary units);

Fig. 8 is a graph showing results from a control plate with 5 μΜ tamoxifen in the assay used in Fig. 7. The x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);

Fig. 9 is a graph showing the dose-dependent effect of reference compound 4,4'((((2,2- difluorocyclopropane- 1 , 1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2.1 - diyl))bis(piperazine) ("SKW166") in an in vitro autophagy assay using human monocyte THP-1 cells. The x-axis shows concentration of SKW166, the right axis shows fluorescence intensity (arbitrary units);

Fig. 10 is a graph showing results from a control plate with 5 μΜ tamoxifen in the assay used in Fig. 9. The x-axis shows the treatment used, the right axis shows fluorescence intensity (arbitrary units);

Fig. 11 is a Western blot showing LC3-II levels in HepG2 cells treated with SKW120 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 SKW120; and Fig. 12 is a Western blot showing LC3-II levels in HepG2 cells treated with SKW137 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 SKW137.

Experimental

Example I], l,l'-((((2,2-difluorocyclopropane-l,l-diyl)bis(4,l- phenylene))bis(oxy))bis(ethane-2,l-diyl))dipyrrolidine ("SKW120")

SKW120 was prepared using the following sequential synthesis procedures (a)-(f).

(a) Synthesis of SKW120-1

To the solution of 1 (850 mg, 4.98 mmol, 1.0 eq.) and 2 (540 mg, 4.99 mmol, 1.0 eq.) in DCE (30 mL) was added AlCb (999 mg, 7.49 mmol, 1.5 eq.) and the reaction mixture was stirred at rt for 2 hours. Diluted with DCM (100 mL), and the organic layer was washed with water then brine. The organic layer was separated, dried (MgS0₄), filtered and concentrated under reduced pressure. The residue was purified with F.C.C. (5% EtOAc in Petroleum ether) to give the product as a white solid (650 mg, yield 54%).

(b) Synthesis of SKW120-2

MeMgBr (3 M, 1.28 mL, 3.84 mmol, 1.5 eq.) was added dropwise to the solution of SKW120-1 (620 mg, 2.56 mmol, 1.0 eq.) in THF (15 mL) at 0°C under N₂ atmosphere Then the resulting mixture was allowed to warm up to rt and stirred at rt for another 4 hours. After the reaction was completed, water was added to quench the reaction. The reaction mixture was exacted with EtOAc (50 mL). The organic layer was washed by brine, separated, dried (Na₂S0₄), filtered and removed under reduced pressure to give the residue. The residue was purified by silica chromatography (10% EtOAc in Petroleum ether) to give the product SKW120-2 as a white solid (620 mg, yield 94%).

(c) Synthesis of SKW120-3

p-TsOH (83 mg, 0.48 mmol, 0.2 eq.) was added to the solution of SKW 120-2 (620 mg, 2.40 mmol, 1.0 eq.) in toluene (60 mL). The resulting reaction mixture was heated at reflux for 1 hour. After the reaction was complete, water was added to quench the reaction. The reaction mixture was extracted with EtOAC (100 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and removed under reduced pressure to give the residue. The residue was purified by silica chromatography (20% EtOAc in Petroleum ether) to give the product SKW120-3 as a white solid (460 mg, yield 80%).

To a sealed tube containing SKW120-3 (460 mg, 1.91 mmol, 1.0 eq.) in ACN (15 mL) was added TMSCF3 (1.36 g, 9.57 mmol, 5.0 eq.) and Nal (1.43 g, 9.57 mmol, 5.0 eq.). The resulting reaction mixture was heated at 80°C for 2 hours. After the reaction was complete, the resulting reaction mixture was directly purified by silica chromatography (10% EtOAc in Petroleum ether) to give SKW120-4 as a white solid (473 mg, yield

To the reaction solution of SKW120-4 (473 mg, 1.63 mmol, 1.0 eq.) in DCM (15 mL) was added BBr₃ (1 M, 8.15 mL, 8.15 mmol, 5.0 eq.) at -70°C. Then the reaction solution was stirred at room temperature for 5 hours. After the reaction was completed, DCM (50 mL) was added, quenched with sat. NaHC0₃ solution. The organic layer was separated, washed with brine, dried with MgS0₄, filtered and concentrated to give the crude residue. The residue was purified by silica chromatography (20% EtOAc in Petroleum ether) to give SKW120-5 as a yellow solid (380 mg, yield 89%).

(f) Synthesis of SKW120

The reaction solution of SKW120-5 (130 mg, 0.50 mmol, 1.0 eq.), 3 (288 mg, 2.50 mmol, 5.0 eq.), PPh (525 mg, 2.00 mmol, 4.0 eq.) and DEAD (348 mg, 2.00 mmol, 4.0 eq.) in THF (2 mL) was stirred at rt for 18 hours. After the reaction was completed, water was added and the reaction mixture was extracted with EtOAc (50 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and concentrated under reduced pressure to give the residue. The residue was purified by Prep. HPLC (0.1% HCOOH in CAN/water) to give SKW120 as a white solid (19 mg, yield 8%). ^lH NMR (400 MHz, CD₃OD) (57.38 (4H, d, J = 8.8 Hz, Ar-H), 6.96 (4Η, d, = 8.8 Hz, Ar-H), 4.29 - 4.31 (4Η, t, = 4.8 Hz, OCH2CH2), 3.59 - 3.62 (4H, t, = 4.8 Hz, OCH2CH2), 3.43 (8Η, m, N(CH₂)₂), 2.09 (2Η, m, CH2CF2C), 2.08 (8Η, m,

CH2CH2CH2).

Example 2: 4,4X(((2,2-difluorocyclopropane-l,l-diyl)bis(4,l- phenylene))bis(oxy))bis(ethane-2,l-diyl))bis(l-methylpiperazine) ("SKW137")

SKW137 was prepared using the following sequential synthesis procedures (a)-(b).

(a) Synthesis of SKW137-1

SKW137-1

CS2CO3 (1.55 g, 4.75 mmol, 5.0 eq.) was added to a solution of 1 (0.25 g, 0.95 mmol, 1.0 eq.) and 2 (3.58 g, 19.06 mmol, 20.0 eq.) in acetonitrile (20 mL). The resulting mixture was heated at 80 °C overnight. TLC showed SM 1 consumed completely. The solvent was removed in vacuo. Then water (250 mL) was added to the mixture and extracted with EtOAC (100 mL). 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 SKW137-1 (Pi,112 mg, yield 24.8%). (b) Synthesis of SKW137

The reaction mixture of SKW137-1 (100 mg, 0.21 mmol, 1.0 eq.), 3 (126 mg, 1.26 mmol, 6.0 eq.) and CS2CO3 (410 mg, 1.26 mmol, 6.0 eq.) in THF (20 mL) was stirred at 60°C for overnight. After the reaction was completed, water was added and the reaction mixture was extracted with EtOAc (50 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and concentrated under reduced pressure to give the residue. The residue was purified by silica chromatography (2% MeOH in DCM) to give SKW137 as a yellow solid (P₂, 26 mg, yield 24%). ^lH NMR (400 MHz, CD3OD) 31.33 (4H, d, 7 = 8.8 Hz, Ar-H), 6.89 (4Η, = 8.4 Hz, Ar-H), 4.12 (4Η, t, = 5.2 Hz, OCH2CH2), 2.64 - 2.85 (20H, m, NCH2CH2) 2.37 (6H, s, NCH3), 2.01 - 2.06 (2Η, m, CF2CH2C).

Example 3 (Reference): 4,4 (((2,2-difluorocyclopropane-l,l-diyl)bis(4,l- phenylene))bis(oxy))bis(ethane-2.1-diyl))bis(piperazine) ("SKW166")

SKW166 was repared using the following sequential synthesis procedures (a)-(h).

To a solution of a mixture of 1 (5.00 g, 23.34 mmol, 1.0 eq.) in DMF (100 mL) was added NaH (3.72 g, 93.00 mmol, 4.0 eq.) slowly at 0°C and stirred at 0°C for 30 minutes. Then Mel (10.00 g, 70.47 mmol, 3.0 eq.) was added to the mixture, and the reaction mixture was stirred at rt for 7 h. After completion, water was added to the mixture, and the resulting reaction mixture was extracted with EtOAc (3 X 200 mL). The organic layer was separated and washed with brine, dried (Na₂S0₄), filtered, concentrated under reduced pressure to give the crude SKW166-1 as yellow solid (5.66 g, Yield 100%).

(b) Synthesis of SKW166-2

MeMgBr (3 M, 15.6 mL, 46.80 mmol, 1.8 eq.) was added dropwise to the solution of SKW166-1 (6.40 g, 26.41 mmol, 1.0 eq.) in THF (50 mL) at 0°C under N₂ atmosphere. Then the resulting mixture was allowed to warm up to rt and stirred at rt for another 3 hours. After the reaction was completed, water was added to quench the reaction. The reaction mixture was exacted with EtOAC (200 mL). The organic layer was washed by brine, separated, dried (Na₂S0₄), filtered and removed under reduced pressure to give the residue. The residue was purified by silica chromatography (10% EtOAc in Petroleum ether) to give the product SKW166-2 as a white solid (5.60 g, yield 82%).

(c) Synthesis of SKW166-3

p-TsOH (375 mg, 1.97 mmol, 0.1 eq.) was added to the solution of SKW166-2 (5.60 g, 21.68 mmol, 1.0 eq.) in toluene (200 mL). The resulting reaction mixture was heated at reflux for 1 hour. After the reaction was complete, water was added to quench the reaction. The reaction mixture was extracted with EtOAC (200 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and removed under reduced pressure to give the residue. The residue was purified by silica chromatography (10% EtOAc in Petroleum ether) to give the product SKW166-3 as a white solid (5.00 g, yield 96%).

(d) Synthesis of SKW166-4

SKW166-3 SKW166-4

To a sealed tube containing SKW166-3 (500 mg, 2.08 mmol, 1.0 eq.) in ACN (15 mL) was added TMSCF3 (1.48 g, 10.40 mmol, 5.0 eq.) and Nal (1.56 g, 10.40 mmol, 5.0 eq.). The resulting reaction mixture was heated at 80°C for 2 hours. After the reaction was complete, the resulting reaction mixture was directly purified by silica

chromatography (10% EtOAc in Petroleum ether) to give SKW 166-4 as a white solid (470 mg, yield 78%).

(e) Synthesis of SKW166-5

SKW166-4 SKW166-5

To the solution of SKW166-4 (470 mg, 1.62 mmol, 1.0 eq.) in DCM (10 mL) was added BBr₃ (1.22 g, 4.87 mmol, 3.0 eq.) at 0°C and the reaction was stirred at room temperature for 0.5 hour. After the reaction was completed, water was carefully added to quench the reaction. The resulting reaction mixture was extracted with DCM (100 mL) and the organic layer was washed with brine, separated, dried (MgS0₄), filtered and removed under reduced pressure to give the residue. The residue was purified by silica chromatography (30% EtOAc in Petroleum ether) to give the product SKW166-5 as a pale yellow solid (380 mg, Yield 89%).

(f) Synthesis of SKW166-6

The reaction mixture of SKW166-5 (210 mg, 0.80 mmol, 1.0 eq.), 2 (3.01 g, 16.00 mmol, 20 eq.) and NaOH (320 mg, 8.00 mmol, 10.0 eq.) in Acetone (10 mL) was stirred at reflux for 72 hours. After the reaction was completed, water was added and the reaction mixture was extracted with EtOAc (50 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and concentrated under reduced pressure to give the residue. The residue was purified by silica chromatography (5% EtOAc in Petroleum ether) to give SKW 166-6 as a pale yellow solid (200 mg, yield 52%).

(g) Synthesis of SKW166-7

The reaction mixture of SKW166-6 (200 mg, 0.42 mmol, 1.0 eq.), 3 (782 mg, 4.20 mmol, 10 eq.) and K2CO3 (580 mg, 4.20 mmol, 10 eq.) in DMF (5 mL) was stirred at rt overnight. After the reaction was completed, water was added and the reaction mixture was extracted with EtOAc (100 mL). The organic layer was washed with brine, separated, dried (Na₂S0₄), filtered and concentrated under reduced pressure to give the crude SKW166-7 as a yellow oil (200 mg, yield 69%).

(h) Synthesis of SKW166

To the solution of SKW166-7 (200 mg, 0.29 mmol, 1.0 eq.) in DCM (10 mL) was added TFA (3 mL) and the reaction solution was stirred at rt for 2 hours. After the reaction was completed, 1 M NaOH solution was added to quench the reaction. The reaction was extracted with DCM (50 mL), and the organic layer was washed with brine, separated, dried (MgS0₄), filtered and concentrated under reduced pressure to give the residue. The residue was purified by HPLC (0-30% MeOH in water, contained 0.01% HCOOH) to give SKW166 as a pale yellow gum (20 mg, yield 14%). Example 4: Effect of SKW120 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% C0₂. Following differentiation, the media was removed and replaced with treatments in triplicate using 5 μΜ tamoxifen ("TMX") or SKW120 (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 SKW120 stimulates autophagy in THP-1 cells in a dose- dependent matter, with no cellular toxicity shown at the highest concentration used. SKW120 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 1.1 to 2.3 μΜ. SKW120 stimulates autophagy in THP-1 cells more effectively than TMX (see Fig. 2).

Example 5: Effect of SKW120 in an in vitro autophagy assay using human HepG2 cells

The in vitro assay as described in Example 4 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 SKW120 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 0.3 to 0.8 μΜ. SKW120 stimulates autophagy in HepG2 cells more effectively than TMX (see Fig. 4).

Example 6: Effect of SKW137 in an in vitro autophagy assay using human monocyte THP-1 cells

The in vitro assay as described in Example 4 was repeated for SKW137. The data in Fig. 5 show that SKW137 stimulates autophagy in THP-1 cells in a dose-dependent matter at the lower concentrations used. SKW137 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 0.9 to 1.9 μΜ. SKW137 stimulates autophagy in THP-1 cells more effectively than TMX (see Fig. 6).

Example 7: Effect of SKW137 in an in vitro autophagy assay using human HepG2 cells

The in vitro assay as described in Example 5 was repeated for SKW137. The data in Fig. 7 show that SKW137 also stimulates autophagy in HepG2 cells in a dose-dependent matter at the lower concentrations used. The calculated EC50 is 0.4 to 1.3 μΜ. SKW137 stimulates autophagy in HepG2 cells more effectively than TMX (see Fig. 8).

Example 8 (Reference): Effect of SKW166 in an in vitro autophagy assay using human monocyte THP-1 cells

The in vitro assay as described in Example 4 was repeated for SKW166 at concentrations of 1 μΜ, 3 μΜ and 10 μΜ. SKW166 is structurally similar to the compounds of general formula (I) of the present invention, except that each of the symmetric ethoxyamine basic side chains comprises a 6-membered heterocyclic ring which is an unmethylated piperazine group (compared to the corresponding 1-methylpiperazine rings in SKW137).

The data in Fig. 9 show that SKW166, unlike SKW137 and SKW120, did not stimulate autophagy in THP-1 cells, except slightly at the highest concentration used. SKW166 was less effective at stimulating autophagy in THP-1 cells than 5 μΜ TMX (see Fig. 10). Example 9: Measuring LC3-II (autophagic flux) using Western Blotting techniques

In Examples 4-7 above, we show that SKW120 and SKW137 induce 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 4-7 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 4-7 is a surrogate marker of autophagy, in the present example we have assessed LC3-II levels in HepG2 cells treated with SKW120 or SKW137 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 SKW120 or SKW137 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 Examples 5 and 7). Briefly, cells were plated and cultured overnight and then washed with media and treated for 14-16 hours with Tamoxifen (5μΜ), SKW120 (ΙΟμΜ), SKW137 (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. 11 and Fig. 12 show that treatment with 10 μΜ SKW120 and SKW137, respectively, 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 Baf A (as detected by Western blot). Tamoxifen was synergistic with Baf A in increasing LC3-II levels but not as effective as SKW120 or SKW137.

The experiments were repeated 3 times, and confirmed that SKW120 and SKW137 are not increasing the autophagy signal as shown in Examples 4-5 and 6-7, respectively, by blocking fusion of the autophagosome and the lysosome, but rather driving autophagic flux (as has been described for tamoxifen).

Example 10: Inhibition of cytochrome P450 interactions (Drug-Drug interactions of SKW137)

Using E.coli CYPEX membranes in combination with specific probe substrates, we assessed the inhibition of individual CYPs by SKW137 (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 SKW137 exhibited no significant drug-drug interactions, as set out in Table 1. Table 1. In vitro PK drug-drug interactions

Example 11; In vitro and in vivo properties of SKW137 which predict in vivo hepatic clearance

A. In vitro clearance of SKW137 in mouse, rat and human hepatocytes

The intrinsic clearance (Clint) and half-life of SKW137 was measured in a mixed hepatocyte suspension of cryopreserved mouse, rat 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 20.0 μΙ/min/lO⁶ cells, in rat hepatocytes was 63 μΙ/min/lO⁶ cells, and in human hepatocytes was 10 μΙ/min/lO⁶ cells. Half-life in mouse hepatocytes was >70 min, in rat hepatocytes was 24.3 min and in human hepatocytes was 139 min.

B. Plasma protein binding ("PPB"), lipophilicity and drug distribution of SKW137

The extent to which SKW 137 bound to plasma proteins such as albumin and alpha-1 acid glycoprotein within human, rat 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 85.35%, in rat cells was 90.68% and in human cells was 75.70%. To understand whether SKW137 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 3.06 in mouse and 5.28 in human. The partition coefficient (LogD) between buffer (PBS, pH 7.4) and n-octanol was measured to determine the lipophilicity of SKW137. The LogD at pH 7.4 of SKW137 was measured and shown to be 2.07. C. In vivo pharmacokinetics of SKW137

SKW137 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 SKW137:

Half life in blood (T½) = 48 h

Observed clearance/F = 6 ml/min/kg

Volume of distribution/Vz/F = 27 1/kg

AUCall = 3816 ng.h/ml

AUCINF_obs = 13018 ng.h/ml

Bioavailability F(AUC) = 37 (127 inf)%.

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 SKW137 in mouse

Tissue Concentration (ng/ml) Tissue/blood ratio

Blood 108 NA

Lung 17205 159

Liver 26714 247

Heart 3509 32.5

Brain 433 4 Small intestine 28058 260

Large intestine 1718 15.9

Stomach 1357 12.6

In summary, the pharmacokinetic and pharmacological studies described above have demonstrated that SKW137 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. SKW137 does not appear to induce CYP P450s.

Example 12: SKW137 efficacy in a murine diet-induced non-alcoholic steatohepatitis ("NASH") model

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 ("R"). 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.

As shown in Table 3, administration of 5 mg/kg dose of SKW137 was associated with reduced steatosis maturity, parenchymal fibrosis and increased biliary regeneration.

Table 3. Histopathology results

Abbreviations: M&E is haematoxylin and eosin; MT is Masson' s trichome; and R is reticulin.

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 general formula (I):

(I)

wherein:

each X is identical and forms part of an ethoxyamine basic side chain which includes a 5- or 6-membered heterocyclic ring; and

the heterocyclic ring X is selected from the group consisting of:

and

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

3. The compound according to either of claim 1 or claim 2 wherein the compound is 1,1'- ((((2,2-difluorocyclopropane- 1 , 1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 - diyl))dipyrrolidine ("SKW120").

4. The compound according to either of claim 1 or claim 2 wherein the compound is 4,4'- ((((2,2-difluorocyclopropane- 1 , 1 -diyl)bis(4, 1 -phenylene))bis(oxy))bis(ethane-2, 1 - diyl))bis( 1 -methylpiperazine) ("S KW 137") .

5. A pharmaceutical composition comprising a compound according to any of claims 1 to 4 and a pharmaceutically or therapeutically acceptable excipient or carrier.

6. Use of a compound according to any of claims 1 to 4 in the manufacture of a medicament for the treatment of a disease.

7. A compound according to any of claims 1 to 4 for use as an autophagy inducer.

8. A compound according to any of claims 1 to 4 for use in the treatment of a disease.

9. A method of treating a disease, comprising the step of administering a compound according to any of claims 1 to 4, or a pharmaceutical composition according to claim 5, to a patient in need of same.

10. Use of a compound according to any of claims 1 to 4 as an autophagy inducer.

11. The use according to claim 10 in the treatment of a disease.

12. The use according to either of claim 10 or claim 11 wherein the use is in vitro.

13. The use according to claim 6, the compound for use according to claim 8, the method of treatment according to claim 9, or the use according to claim 11, 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.

14. The use according to claim 6, the compound for use according to claim 8, the method of treatment according to claim 9, or the use according to claim 11, wherein the disease is non-alcoholic steatohepatitis (NASH).