WO2017216507A1 - Compounds - Google Patents

Abstract

The invention relates to difluorocyclopropane compounds, methods for their manufacture, and their uses for example as medicaments. According to one aspect, there is provided a compound of general formula (I): wherein X and Y are phenyl groups, one of which is substituted by an ethoxyamine basic side chain; R₁ and R₂ are independently selected from H or a C₁-C₆ alkyl group; and wherein one or each of X and Y, and the or each C₁-C₆ alkyl group, are optionally substituted or further substituted by one or more alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radicals of 1 to 20 carbon atoms, and/or by one or more fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyloraminodialkyl radicals.

Description

Compounds

The invention relates to difluorocyclopropane compounds, methods for their manufacture, and their uses for example as medicaments.

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 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 (IUPAC name: 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. The development of more selective autophagy inducers is needed if they are to become medicinally useful in the treatment and/or prevention of diseases such as cardiovascular diseases. WO2013/062079 describes 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, -difluoro-2,2'-diphenylcycloproprane derivative (compound 34 in Fig. 2) as a potential oestrogen receptor inhibitor. US5,015,666, US5,658,914 and US5,658,951 describe triarylcyclopropane derivatives and diphenylcyclopropyl analogues stated to be useful as anti-oestrogens and anti-tumour agents. 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 Ι, -dichloro diphenylcycloproprane derivatives and their potential medical uses.

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

wherein

X and Y are phenyl groups, one of which is substituted by an ethoxyamine basic side chain; Ri and R₂ are independently selected from H or a Ci-C₆ alkyl group; and

wherein one or each of X and Y, and the or each Ci-C₆ alkyl group, are optionally substituted or further substituted by one or more alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radicals of 1 to 20 carbon atoms, and/or by one or more fluoro, chloro, bromo, iodo, hydroxy, oxyalkyl, amino, aminoalkyl or aminodialkyl radicals.

According to the invention, one of the phenyl groups (X or Y) is substituted by the ethoxyamine basic side chain, and consequently the other phenyl group is un- substituted by the ethoxyamine basic side chain.

Initially, we developed a range of dichlorocyclopropane analogues of tamoxifen (data not shown). However, we found these to be unstable, and thus turned our attention to difluorocyclopropane analogues. Known methods for generating relevant difluorocyclopropane compounds were limited. We describe herein methods to improve the efficiency of generating such compounds. Novel compounds described herein are shown using in vitro data to be effective autophagy inducers (see below).

The compound described above may have a structure of general formula (II):

wherein Ri and R₂ are as described above; and R-3 is an ethoxyamine basic side chain.

Alternatively, the compound described above may have a structure of general formula (III):

wherein R4 is an amine group. Optionally, the amine group is other than a morpholine group. For example, the amine group may be selected from aminodimethyl, aminodiethyl, pyrrolidin-l-yl and piperidin-l-yl.

Exemplar compounds within the scope of formulae (I), (II) and (III) include:

(±)- 1 , 1 -difluoro-2-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenyl cyclopropane;

(±)- 1 , 1 -difluoro-2-(4-(2-(diethylamino)ethoxy)phenyl)-2-phenyl cyclopropane;

(±)- 1 , 1 -difluoro-2-(4-(2-(pyrrolidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane; and (±)- 1 , 1 -difluoro-2-(4-(2-(piperidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane.

A further exemplar compound is (±)-l, l-difluoro-2-(4-(2-(morpholin-l- yl)ethoxy)phenyl)-2-phenylcyclopropane.

According to a further aspect of the invention, there is a provided a pharmaceutical composition comprising a compound 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, transmucosal, buccal, sublingual, 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 as defined herein in the manufacture of a medicament for the treatment of a disease.

Further provided is a compound 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 as defined herein to a patient in need of same.

A disease suitable for treatment according to these aspects of the invention is one which is characterised by defective autophagy or which would benefit from modulation of autophagy. The disease may be 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) and a 1 -antitrypsin deficiency (ATD), Niemann-Pick type C ( PC) 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 T F-α 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.

Numerous methods have been used in the prior art for the synthesis of difluorocyclopropyl stilbene analogues including reaction of CH₂Br₂ and CBr₂F₂ with a strong aqueous base in the presence of tetrabutylammonium hydrogensulphate as a PTC catalyst, thermal decomposition of sodium trifluoroacetate, chlorodifluoroacetate and bromodifluoroacetate, and NaF induced thermal decomposition of TFDA. They all failed to affect the difluorocyclopropanation on the relatively nucleophilic-poor stilbenes.

We found previously that the difluorocyclopropanation of stilbenes with Me₃SiCF₃ using microwave irradiation was more effective than known reactions (see Wong & Fox, 2012, 20^th International Symposium of Fluorine Chemistry, Kyoto, 22-27 July 2012). For example, the stilbenes transformed into difluorocyclopropanes after heating the reaction mixture in a sealed microwave tube for just 20 minutes at 80°C (Scheme 1, below), at a conversion rate of - 22%.

~ 22%

conversion

Scheme 1

Through optimisation of various parameters, conversion of up to 30 % was achieved

(see Scheme 2 below, where "temp" refers to microwave heating). Replacement of Nal with a different salt: NaF, NaCl, NaBr, KF, KCl, KBr, KI and CsF resulted in no or only trace amount of product being formed. When TUF, toluene, diglyme, DMF and DMSO were used as solvents instead of CH₃CN, only trace amount of difluorocyclopropanes were detected. If reaction temperature was below 80°C, little difluorocyclopropane was formed in the 20 minutes timescale, indicating that a sustainable high temperature (> 80°C) is required to overcome the energy barrier for the addition of :CF₂ to the olefins. Conversion of trans-stilbe to the desired trans- difluorocyclopropane increased with increasing reaction temperature, but only up to ~ 30% at 130°C. At 140°C, isomerisation of tram'-difluorocyclopropane to czs-difluorocyclopropane was observed in a small percentage (~ 2%) of compounds. With cz^'s-stilbene, the situation is a little bit more complicated. Table 1 provides a summary of selected reactions when cz^'s-stilbene was used as the model substrate.

<

(±) (±) B C D

Scheme 2

Microwave % of crude reaction mixture

Catalyst heating

Entry Solvent

(equiv.) conditions: A B C D

°C Mins

1 THF Nal (2.5) 80 20 - 99 Trace 0 0

2 Toluene Nal (2.5) 80 20 - 99 Trace 0 0

3 Diglyme Nal (2.5) 80 20 - 99 Trace 0 0

4 CH₃CN Nal (2.5) 60 20 - 99 Trace 0 0

5 CH₃CN Nal (2.5) 80 20 - 36 - 41 - 22 - 1

6 CH₃CN Nal (2.5) 100 20 - 34 - 50 - 15 - 1

7 CH₃CN Nal (2.5) 120 20 - 26 - 57 - 1 - 16

8 CH₃CN Nal (2.5) 80 10 - 63 - 20 - 17 0

9 CH₃CN KF (2.5) 80 20 - 99 Trace 0 0

10 CH₃CN CsF (0.2) 60 20 - 99 Trace Trace 0

11 THF CsF (0.2) 60 20 - 99 Trace Trace 0

12 Toluene CsF (0.2) 60 20 - 95 Trace - 5 0

13 Toluene CsF (0.5) 60 20 - 98 Trace - 2 0

Table 1 : Difluorocyclopropanation using Me3SiCF₃ and cz^'s-stilbene as a model substrate.

At 80 °C, about 22% of cz^'s-stilbene was converted to difluorocyclopropane, while a significant amount of starting material (~ 41%>) was isomerized into the tra/w-stilbene which was also accompanied by the formation of trans- difluorocyclopropane (~ 1 %>) (see Entry 5). Interestingly, at temperature > 120°C, tram'-difluorocyclopropane (16 %>) was detected as the major cyclopropane product (see Entry 7). The cis- and trans- isomerisation was increased from 41%> to 57%>, whereas the overall formation of difluorocyclopropane was reduced from 23%> to 17%>.

Difluorocyclopropanation using microwave radiation provides a fast and convenient way to form difluorocyclopropanes in just 20 minutes. The conversion shown in Entry 5 of

Table 1 may seem low in the first instance, but when the method is applied for more reactive alkenes, the conversion is more remarkable. This is shown in Table 2:

Table 2: Conversion of more reactive olefins using microwave radiation.

We have found the above and other prior art methods for producing dichlorocyclopropanes, using for example the relatively nucleophilic-poor stilbenes as starting material, to be lacking. The present disclosure provides improved methods. In another aspect of the present invention, there is provided a method of making a compound as defined herein, wherein the method comprises a step of conducting a chemical reaction in a sealed tube in the presence of a reactive compound, a fluorination reagent, a catalyst and solvent, thereby making an intermediate compound for making the compound of the invention as defined herein.

The intermediate compound may be a difluorocyclopropane compound, for example a diphenyl difluorocyclopropane compound (such as (±)-l, l-Difluoro-2-(4-hydroxyphenyl)- 2-phenylcyclo-propane). The difluorocyclopropane compound may be further modified such as by substitution, for example by substituting an hydroxyl (-OH) group with an ethoxyamine basic side chain, to form a compound of the invention as defined herein.

The chemical reaction may be facilitated by heating. Additionally or alternatively, the chemical reaction may be facilitated by microwave radiation. In the method of the invention, the reactive compound may be 4-hydroxy-a- phenylstyrene, the fluorination reagent may be trifluoromethyltrimethylsilane (TMSCF₃), the catalyst may be Nal and the solvent may be CH₃CN. The chemical reaction may be conducted at or above 80°C (for example, at not more than 120°C), for example by heating for 2 hours and/or by microwave radiation for 20 min.

The very high conversion obtained with other alkenes further provides evidence that stilbenes are unreactive toward difluorocarbene. The improvements in the methods noted above are therefore significant. As used herein, the term "about" refers to an interval around the considered value. As used in this patent application, "about X" means an interval from X minus 10% of X to X plus 10% of X, and preferably an interval from X minus 5% of X to X plus 5% of X.

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 (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 histogram showing the effect of compounds (±)-l, l-difluoro-2-(4- hydroxyphenyl)-2-phenylcyclopropane ("SKW35") and (±)-l, l-difluoro-2-(4-(2- (dimethylamino)ethoxy)phenyl)-2-phenylcyclopropane ("SKW36") in a THP-1 autophagy assay (see Example 9);

Fig. 2 is a histogram showing the effect of compound (±)-l, l-difluoro-2-(4-(2- (diethylamino)ethoxy)phenyl)-2-phenylcyclopropane ("SKW46") in a TFIP-1 autophagy assay (see Example 9);

Fig. 3 is a histogram showing the effect of compound (±)-l, l-difluoro-2-(4-(2- (pyrroli din- l-yl)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW48") in a TFIP-1 autophagy assay (see Example 9); and Fig. 4 is a histogram showing the effect of compounds (±)-l, l-difluoro-2-(4-(2- (morpholin-l-yl)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW47") and (±)-l, l- difluoro-2-(4-(2-(piperi din- l-yl)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW49") in a TFIP-1 autophagy assay (see Example 9).

Experimental Example 1 (Reference): 4-Hydroxy-a-phenylstyrene

This compound was prepared according to the method of Fischer & Wan (1999, J. Am. Chem. Soc. 121 : 4555-4562). A solution of 4-hydroxyacetophenone (2.72 g, 20 mmol, 1.0 eq.) in THF (150 ml) was added dropwise to PhMgBr [62 mmol, 3.1 eq., prepared by adding PhBr (6.53 ml, 62 mmol, 3.1 eq.) in THF (30 ml) to Mg turning (1.60 g, 66 mmol, 3.3 eq.) in THF (30 ml) with a few grains of I₂ crystals] cooled on in ice bath. After the reaction was stirred for 30 minutes and the reaction was completed as monitored by TLC. The reaction was quenched by water (100 ml) and the organic material was exacted by diethyl ether (3 x 50 ml), dried over MgS0₄ to give the tertiary alcohol. A little of ^-toluenesulfonic acid were added to the tertiary alcohol in toluene (100 ml) and the resulting solution was heated at reflux for 1 hour before the solvent was reduced in vacuo. Water was added and the organic material was extracted with diethyl ether (3 x 50 ml), dried over MgS0₄ to afford an oil. The crude oil was purified by standard silica chromatography to give the title compound (3.49 g, 89 %) as a light yellowish oil. ¾ NMR (300 MHz, CDC1₃): δ 7.30-7.21 (5H, m, Ar-H), 7.17-7.11 (2H, m, HOCCHCH), 6.75-6.67 (2H, m, HOCCH), 6.20-5.80 (1H, m, OH), 5.35 (1H, d, J = 1.2, CH₂), 5.31 (1H, d, J = 1.2, CH₂); ¹³C NMR (75 MHz, CDC1₃) δ 155.78 (C-OH), 149.77, 142.09, 134.30 (ipso Ar-C and CCH₂) 129.90, 128.62, 128.43, 127.96, 115.35 (Ar-C), 113.22 (C¾)- These data are consistent with that previously reported (see Ojha & Prabhu, 2012, J. Organic Chem. 77: 11027-11033). Example 2 ( Preparation of Intermediate): (±)- 1 , l-Difluoro-2-(4-h droxyphenyl)-2- phenylcyclo-propane ("SKW35")

To a 35 ml pressure tube containing 4-hydroxy-a-phenylstyrene (1.05 g, 5 mmol, 1.0 eq.) in CH₃CN (20 ml) was added Nal (3.75 g, 25 mmol, 5.0 eq.) and TMSCF₃ (3.7 ml, 25 mmol, 5.0 eq.) and the pressure tube was securely sealed. The resulting mixture was heated at 80° C for 2 hours. After that, the mixture was filtered and the solid washed with CH₃CN (2 x 10 ml). The filtrate and washings were reduced in vacuo and re- dissolved in methanol (15 ml), followed by addition of N-bromosuccinimide (0.18 g, 1 mmol, 0.2 eq.). The mixture was then stirred at room temperature for 2 hours. After that, the methanol was removed in vacuo and the crude was purified using standard silica chromatography to give 0.82 g (67 %) of the title compound as an oil which solidified to a low-melting waxy solid on standing. ¾ NMR (300 MHz, CDC1₃): δ 7.40-7.18 (7H, m, Ar-H), 6.77-6.72 (2H, m, HOCCH), 5.26 (1H, br s, OH), 2.10-1.94 (2H, m, CH₂); ¹⁹F NMR (282 MHz, CDC1₃) δ -130.62 - -130.74 (m). ¾ NMR spectrometric data are consistent with those reported in WO 2013062079 Al (no synthetic method given). Example 3: (±)-l,l-Difluoro-2-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenylcyclo- propane ("SKW36")

A mixture of 2-chloro-N,N-dimethylethylamine hydrochloride (0.43 g, 3 mmol, 0.75 eq.), l, l-difluoro-2-(4-hydroxyphenyl)-2-phenyl cyclopropane (0.98 g, 4 mmol, 1.0 eq.) and anhydrous K₂C0₃ (1.93 g, 14 mmol, 3.5 eq.) in acetone (50 ml ) was heated at reflux overnight. After cooling to ambient temperature, the solid was filtered and the filtrate was reduced in vacuo to give a crude product. The crude was purified using silica chromatography (silica gel, acetone: petroleum ether, 20: 80) to give 0.74 g (77 %) of the title compound as an oil which solidified upon stored at 0°C. ¾ NMR (300 MHz, CDCI₃) δ 7.23 - 6.99 (7H, m, Ar-H), 6.71 - 6.64 (2H, m, CH₂OCCH), 3.83 (2H, t, J = 6, OG¾CH₂), 2.50 (2H, t, J = 6, OCH₂G¾), 2.12 (6H, s, N(G¾)₂), 1.91 - 1.78 (2H, m,CF₂G¾). ¹⁹F NMR (282 MHz, CDC1₃) δ -130.55- -130.76 (m).HRMS (ESI): m/z for (Ci₉H₂₂F₂NO) [M+H] calculated 318.1669 found 318.1668.

Example 4: (±)-l,l-Difl oro-2-(4-(2-(diethylamino)ethoxy)phenyl)-2-phenylcyclo- propane ("SKW46")

This compound was made by the method of Example 3 using 2-chloro-N,N- diethylethylamine hydrochloride (0.52 g, 3 mmol, 0.75 eq.), l, l-difluoro-2-(4- hydroxyphenyl)-2-phenylcyclopropane (0.98 g, 4 mmol, 1.0 eq.) and anhydrous K₂C0₃ (1.93 g, 14 mmol, 3.5 eq.) to give 0.71 g (69 %) of the title compound as an oil. ¾ NMR (300 MHz, CDC1₃) δ 7.52 - 7.00 (7H, m, Ar-H), 6.81 (2Η, m, CH₂OCCH), 4.13 (2Η, t, J = 5.5, OG¾CH₂), 3.01 (2H, t, J = 5.5 OCH₂CH₂), 2.79 (4Η, q, J = 7.0, CH₂CH₃), 2.05 - 1.87 (2H, m, CF₂G¾), 1.14 (6H, t, J = 7.0, CH₂CH₅). ¹⁹F NMR (282 MHz, CDCI3) δ -128.98 - -132.05 (m). HRMS (ESI): m/z for (C₂iH₂₆F₂NO) [M+H] calculated 346.1982 found 346.1976.

Example 5: (±)- 1 , l-Difluoro-2-(4-(2-(pyrrolidin- l-yl)ethoxy)phenyl)-2-phenylcyclo- propane ("SKW48")

This compound was made by the method of Example 3 using l-(2- chloroethyl)pyrrolidine hydrochloride (0.51 g, 3 mmol, 0.75 eq.), l, l-difluoro-2-(4- hydroxyphenyl)-2-phenylcyclopropane (0.98 g, 4 mmol, 1.0 eq.) and anhydrous K₂C0₃ (1.93 g, 14 mmol, 3.5 eq.) to give 0.78 g (76 %) of the title compound as an oil which solidified upon stored at 0°C. ¾ NMR (300 MHz, CDC1₃) δ 7.38 - 7.1 1 (7H, m, Ar-H), 6.85 - 6.76 (2H, m, CH₂OCCH), 4.04 (2H, t, J = 6.0, OG¾CH₂), 2.84 (2H, t, J = 6.0 Hz, OCH₂G¾), 2.67 - 2.45 (4H, m, N(CH₂)₂), 2.04 - 1.90 (2H, m, CF₂G¾), 1.82 - 1.68 (4H, m, NCH₂G¾G¾). ¹⁹F NMR (282 MHz, CDC1₃) δ -129.46 - -130.61 (m). HRMS (ESI): m/z for (C₂iH₂₄F₂NO) [M+H] calculated 344.1826 found 344.1819.

Example 6: (±)-l,l-Difluoro-2-(4-(2-(piperidin-l-yl)ethoxy)phenyl)-2-phenylcyclo- propane ("SKW49")

This compound was made by the method of Example 3 using l-(2-chloroethyl)piperidine hydrochloride (0.55 g, 3 mmol, 0.75 eq.), l, l-difluoro-2-(4-hydroxyphenyl)-2- phenylcyclopropane (0.98 g, 4 mmol, 1.0 eq.) and anhydrous K₂C0₃ (1.93 g, 14 mmol, 3.5 eq.) to give 0.70 g (65 %) of the title compound as an oil which solidified upon stored at 0°C. ¾ NMR (300 MHz, CDC1₃) δ 7.42 - 7.08 (7H, m, Ar-H), 6.88 - 6.71 (2Η, m, CH₂OCCH), 4.00 (2Η, t, J = 6.0, OG¾CH₂), 2.69 (2H, t, J = 6.0 Hz, OCH₂CH₂), 2.51 - 2.31 (4Η, m, NG¾CH₂CH₂), 2.06 - 1.86 (2H, m, CF₂G¾), 1.62 - 1.47 (4H, NCH₂G¾CH₂), 1.44 - 1.30 (2H, m, NCH₂CH₂G¾). ¹⁹F NMR (282 MHz, CDCI₃) δ -130.05 - -13 1.16 (m). HRMS (ESI): m/z for (C₂₂H₂₆F₂NO) [M+H] calculated 358.1982 found 358.1982.

Example 7 (Reference): (±)-l,l-Difl oro-2-(4-(2-(morpholin-l-yl)ethoxy)phenyl)-2- phenylcyclopropane ("SKW47")

■Θ

HCI

A mixture of 4-(2-Chloroethyl)morpholine hydrochloride (0.55 g, 3 mmol, 0.75 eq.), l, l-difluoro-2-(4-hydroxyphenyl)-2-phenyl cyclopropane (0.98 g, 4 mmol, 1.0 eq.) and anhydrous K₂C0₃ (1.93 g, 14 mmol, 3.5 eq.) in acetone (50 ml ) was heated at reflux overnight. After cooling to ambient temperature, the solid was filtered and the filtrate was reduced in vacuo to give a crude oil. The crude oil was purified using silica chromatography (silica gel, acetone: petroleum ether, 20: 80) before 2 ml of 3N HC1 solution was added and the solvent was reduced in vacuo. The solid was recrystallised in acetone to give 0.52 g (44 %) of the title compound as a white solid. ¾ MR (400 MHz, CD₂C1₂) δ 7.48 - 7.04 (7H, m, Ar-H ), 6.82 (2H, m, CH₂OCCH), 4.47 - 4.42 (2H, m, ArOG¾CH₂), 4.26 - 3.70 (4H, m, N(CH₂G¾)₂0), 3.57 - 3.16 (4H, m, ArOCH₂G¾ and N(G¾CH₂)₂0), 2.98 (2H, br s, N(G¾CH₂)₂0), 1.98 (2H, dq, J = 15.5, 8.0, G¾CF₂). HRMS (ESI): m/z for (C₂iH₂₄F₂N0₂) [M-Cl] calculated 360.1775 found 360.1770.

Example 8: Effect of difluorocyclopropane compounds in an in vitro autophagy assay

Methods

Human THP-1 cells, a myelo-monocytic cell line, were plated into 24 well plates (8 x 10⁵ cells/ml with 500 μΐ of media/well) and differentiated into macrophages for 24 hr by incubating with 200 nM phorbol-12-myristate (PMA) at 37°C in a humidified atmosphere containing 5% C0₂. Following differentiation TFIP-1 cells were washed with fresh media and treated in triplicate with vehicle (0.8% DMSO in RPMI-1640 media), 3 μΜ tamoxifen (TMX, positive control) or different difluorocyclopropane compounds (final concentration 10μΜ) for 18 hr (overnight) in a 37°C incubator in a final volume of 500μ1. At the end of the incubation step the cells were washed and removed from each well using trypsin/EDTA and placed in to glass tubes before being processed for the detection of lysosomal/autophagic vacuoles using a commercially available autophagy kit (Abeam, ab 139484).

The Abeam kit uses a proprietary dye, which is a cationic amphiphilic tracer, which selectively labels autophagic vacuoles in the perinuclear region of the cell. The procedure of staining was carried out per the manufacturer's instructions. Briefly, the cells were centrifuged (100 x g for 5 minutes at RT), washed in fresh media (RPMI phenol red free/5% FBS) and resuspended in 500 μΐ media containing the Cyto-ID green staining dye provided in the kit (final concentration IX) and were incubated for 30 minutes at 37°C in the dark. Finally, cells were washed and fixed in 2% PFA. The cells were analysed in the FITC channel on a flow cytometer (BD Facs Calibur) with at least 15,000 events collected per sample. The cell population was gated in the forward and side scatter channels and the median fluorescence intensity (MFI) in the FITC channel was reported for each sample.

The following compounds were tested: 1. Tamoxifen ("TMX"; positive control)

2. (±)-l, l-Difluoro-2-(4-hydroxyphenyl)-2-phenylcyclopropane ("SKW35"; see Example 2)

3. (±)- 1 , 1 -Difluoro-2-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW36"; see Example 3)

4. (±)-l, l-Difluoro-2-(4-(2-(diethylamino)ethoxy)phenyl)-2-phenyl cyclopropane

("SKW46"; see Example 4) 5. (±)- 1 , 1 -Difluoro-2-(4-(2-(pyrrolidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW48"; see Example 5)

6. (±)- 1 , 1 -Difluoro-2-(4-(2-(piperidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane ("SKW49"; see Example 6)

7. (±)- 1 , 1 -Difluoro-2-(4-(2-(morpholin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane

("SKW47"; see Example 7).

Results

We have tested a number of difluorocyclopropane compounds as well as tamoxifen (positive control) in an in vitro autophagy assay using THP-1 macrophages. The extent of autophagy was measured using a fluorescent dye, which selectively labels autophagosomes. All experimental compounds were tested at 10 μΜ. Tamoxifen ("TMX") was used as a positive control for all in vitro autophagy experiments. It was tested at 3 μΜ as at higher concentrations the compound has a toxic effect on the cells.

SKW35 has no amine side chain and does not stimulate autophagy (see Fig. 1). In contrast, all compounds tested containing an amine side chain (including SKW36, SKW46, SKW48 and SKW49) stimulate autophagy in THP-1 cells, which demonstrates the presence of an amine side chain is important for induction of autophagy (see Figs 1- 4). In all cases, SKW36, SKW46, SKW48 and SKW49 induce 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 (see Figs 1 - 4). The presence of the oxygen in the morpholine group of SKW47 abolishes the induction of autophagy in THP-1 cells compared to SKW49 (contains a piperidine group, see Fig. 4). Later testing of the hydrochloride salt of SKW49 showed this had no effect on induction of autophagy, confirming that it is the morpholine group in SKW47 which is responsible for the lack of autophagic activity in THP-1 cells. Conclusions

Several of the difluorocyclopropane compounds tested here can induce autophagy in human THP-1 cells. We have found the presence of an amine group is important for this activity, but where this amine is part of a morpholine group the activity is abolished by the presence of the oxygen.

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. All documents cited herein are incorporated by reference in their entirety.

Claims

Claims

1. A compound of general formula (I):

wherein

X and Y are phenyl groups, one of which is substituted by an ethoxyamine basic side chain;

Ri and R₂ are independently selected from H or a Ci-C₆ alkyl group; and

wherein one or each of X and Y, and the or each Ci-C₆ alkyl group, are optionally substituted or further substituted by one or more alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radicals of 1 to 20 carbon atoms, and/or by one or more fluoro, chloro, bromo, iodo, hydroxy, oxy alkyl, amino, aminoalkyl or aminodialkyl radicals.

2. The compound according to claim 1, having a structure of general formula (II):

(Π) wherein Ri and R₂ are as described above; and

R₃ is an ethoxyamine basic side chain.

3. The compound according to either of claim 1 or claim 2, having a structure of general formula (III):

wherein R4 is an amine group (optionally other than a morpholine group).

4. The compound according to claim 3, wherein the amine group is selected from aminodimethyl, aminodi ethyl, pyrrolidin-l-yl and piperidin-l-yl.

5. The compound according to any preceding claim, wherein the compound is selected from:

(±)- 1 , 1 -difluoro-2-(4-(2-(dimethylamino)ethoxy)phenyl)-2-phenyl cyclopropane;

(±)- 1 , 1 -difluoro-2-(4-(2-(diethylamino)ethoxy)phenyl)-2-phenyl cyclopropane;

(±)- 1 , 1 -difluoro-2-(4-(2-(pyrrolidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane; and (±)- 1 , 1 -difluoro-2-(4-(2-(piperidin- 1 -yl)ethoxy)phenyl)-2-phenyl cyclopropane.

6. The compound according to any of claims 1 to 4, wherein the compound is (±)- 1, 1- difluoro-2-(4-(2-(morpholin-l-yl)ethoxy)phenyl)-2-phenylcyclopropane.

7. A pharmaceutical composition comprising a compound according to any of claims 1 to 6 and a pharmaceutically or therapeutically acceptable excipient or carrier.

8. Use of a compound according to any of claims 1 to 5 in the manufacture of a medicament for the treatment of a disease.

9. A compound according to any of claims 1 to 5 for use in the treatment of a disease.

10. A method of treating a disease, comprising the step of administering a compound according to any of claims 1 to 5 or a pharmaceutical composition according to claim 7 to a patient in need of same.

11. The use according to claim 8, the compound for use according to claim 9, or the method of treatment according to claim 10, wherein the disease is a neurodegenerative disorder (for example, Huntington's disease, Alzheimer's disease or Parkinson's disease), systemic lupus erythematosus ("lupus"), epilepsy, cancer, liver diseases including nonalcoholic fatty liver disease (NAFLD) 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 T F-α 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. A method of making a compound as defined in any of claims 1 to 6, wherein the method comprises a step of conducting a chemical reaction in a sealed tube in the presence of a reactive compound, a fluorination reagent, a catalyst and solvent, thereby making an intermediate compound for making the compound as defined in any of claims 1 to 6.

13. The method according to claim 12, wherein the intermediate compound is a difluorocyclopropane compound, for example a diphenyldifluorocyclopropane compound (such as (±)-l, l-Difluoro-2-(4-hydroxyphenyl)-2-phenylcyclo-propane). 14. The method according to claim 13, wherein the difluorocyclopropane compound is modified by substitution, for example by substituting an hydroxyl (-OH) group with an ethoxyamine basic side chain, to form a compound as defined in any of claims 1 to 6.

15. The method according to any of claims 12 to 14, wherein the chemical reaction is facilitated by heating. 16. The method according to any of claims 12 to 15, wherein the chemical reaction is facilitated by microwave radiation.

17. The method according to any of claims 12 to 16, wherein the reactive compound is 4-hydroxy-a-phenylstyrene, the fluorination reagent is trifluoromethyltrimethylsilane (TMSCF₃), the catalyst is Nal and the solvent is CH₃CN, and wherein the chemical reaction is optionally conducted at 80°C for example by heating for 2 hours and/or by microwave radiation for 20 min.