Compounds for use in senotherapy

Abstract

The present invention relates to compounds of formula I: in which: each of A, B and D is independently N or CH; each of X and Y is independently N, CH or CR9 in which R9 is F, Br, Cl, I or -OH, with the proviso that when one of X or Y is N or CR9, the other is CH; and R1, R2, R7 and R8 are CH3, and each of R3, R4, R5 and R6 is H; or one of R1 or R2 is CH3 and the other, together with R3 or R4 represents a bond and one of R7 and R8 is CH3 and the other, together with R5 or R6, represents a bond; and stereoisomers or geometric isomers thereof; in free or salt form; for use as an anti-senescence medicament B D A R7 X Y CO2H R1 R8 R4 R5 R6 R2 R3

Description

[0001] Compounds for use in Senotherapy

[0002] The present invention relates to compounds of formula I: in which: each of A, B, and D is independently N or CH; each of X and Y is independently N, CH or CR9in which R9is F, Br, Cl, I or -OH, with the proviso that when one of X or Y is N or CR9, the other is CH; and R1, R2, R7and R8are CH3, and each of R3, R4, R5and R6is H; or one of R1or R2is CH3 and the other, together with R3or R4represents a bond and one of R7and R8is CH3 and the other, together with R5or R6, represents a bond; and stereoisomers or geometric isomers thereof; in free or salt form; for use as an anti-senescence medicament. Retinoids are a family of natural or synthetic compounds that are analogues of Vitamin A and its derivatives. Retinoids are essential for numerous cellular activities and as signalling molecules are involved in controlling important biological pathways from embryogenesis, through to adult homeostasis, as well as in aspects of stem cell development such as proliferation, differentiation and apoptosis. All-trans-retinoic acid (ATRA) is the most abundant endogenous retinoid and has been used as a model compound for the study of retinoids.

[0003] Retinoids act on a group of nuclear receptors known as retinoic acid receptors (RARs); inducible ligand-activated transcription factors which regulate multiple physiological mechanisms at a genomic level. Consequently, synthetic retinoids have been investigated as potential therapeutics for use in a range of diseases and conditions mediated or potentially mediated by RARs.

[0004] Senescence is the irreversible arrest of the cell cycle and loss of proliferative capacity of the cell. Senescent cells undergo changes in their morphology, gene expression and metabolism and are metabolically active. Senescent cells secrete a variety of factors which together are known as the Senescence-Associated Secretory Phenotype (SASP). The SASP includes pro- inflammatory cytokines and growth factors which induce chronic inflammation. As a result, senescent cells can have a significant pathological impact on the tissues and organs in which they accumulate.

[0005] Cellular senescence arises as a response to multiple stressors and as a result of telomere shortening. Telomeres are DNA-protein structures located at the end of the chromosomes; they function to protect the ends of the chromosomes from damage.

[0006] Each time a cell divides, a short portion of the telomere is lost. This is known as telomere shortening. When the telomere reaches a critical length, the DNA damage response (DDR) is induced which results in cellular senescence.

[0007] Telomere shortening is a normal consequence of ageing and results in the accumulation of senescent cells. This accumulation of senescent cells can lead to a chronic inflammatory state which can be causative of age-related chronic inflammatory diseases such as diabetes, osteoarthritis and osteoporosis.

[0008] As the development of modern medicine has improved human health and prolonged lifespan, age-related diseases are becoming an increasing occurrence. The World Health Organisation (WHO) estimates that 1 in 6 people, or 2.1 billion people worldwide, are expected to be aged 60 or over by 2030 ("Ageing and Health." World Health Organisation (WHO), 1 October 2022, https: / / www.who.int / news-room / fact-sheets / detail / ageing-and-health). Age-related diseases, such as age-related chronic inflammatory disease often have poor outcomes for the patient and result in disability and death. These diseases are proving to be an increasing burden on the social economy and public health and there is an unmet need for treatments targeting the pathophysiological mechanisms underlying the ageing process. There is a need for treatments which will slow down or even reverse the onset and / or progression of diseases or conditions associated with ageing. This is of great importance in an ageing global population to extend healthy lifespan and reduce the socioeconomic impacts of ageing.

[0009] Senotherapy is a term used to describe the therapeutic strategy of targeting cellular senescence. One group of compounds used in senotherapy are senolytics. Senolytics are compounds that target senescence by eliminating senolytic cells. These compounds include Dasatanib and Quercetin which have been clinically evaluated as therapies for the treatment of a range of diseases and conditions, including chronic kidney disease and frailty. Whilst these compounds are effective at selectively removing senescent cells, it would be advantageous if future senotherapies could prevent, or even reverse, cellular senescence thereby maintaining cells in a healthy state (rejuvenation), rather than eliminating cells in a senescent state.

[0010] Summary of the Invention

[0011] According to a first aspect of the present invention, there is provided a compound of formula in which: each of A, B, and D is independently N or CH; each of X and Y is independently N, CH or CR9in which R9is F, Br, Cl, I or -OH, with the proviso that when one of X or Y is N or CR9, the other is CH; and

[0012] R1, R2, R7and R8are CH3, and each of R3, R4, R5and R6is H; or one of R1or R2is CH3 and the other, together with R3or R4represents a bond and one of R7and R8is CH3 and the other, together with R5or R6, represents a bond; and stereoisomers or geometric isomers thereof; in free or salt form; for use as an anti-senescence medicament.

[0013] The term "anti-senescence" describes agents which reduce the number of senescent cells. Anti-senescent agents may delay or prevent cells from entering senescence, or reverse senescence in cells which have already entered a senescent state. The term "medicament" refers to agents used in the treatment and / or prevention of a medical disease or condition, and / or to agents used in the treatment or prevention of symptoms associated with a medical disease or condition. Medicaments may be diseasemodifying treatments. Disease-modifying treatments delay, slow or reverse the progression of a disease or condition. Disease-reversing treatments reverse the progression of a disease or condition.

[0014] Senescent cells can be identified by determining the presence of well-known specific biological markers such as senescence-associated beta galactosidase (SA- -gal), pl6INK4A(pl6), p21, Lamin Bl and IL6 (Huang, W., Hickson, L.J., Eirin, A. et al. Cellular senescence: the good, the bad and the unknown. Nat Rev Nephrol 18, 611-627 (2022); Freund A, Laberge RM, Demaria M, Campisi J. Lamin Bl loss is a senescence-associated biomarker. Mol Biol Cell. 2012 Jun;23(ll):2066-75. Epub 2012 Apr 11. PMID: 22496421; PMCID: PMC3364172.). Senescent cells can also be identified by specific cellular morphologies, including the presence of bean-shaped nuclei and heterochromatin aggregation.

[0015] The inventors have advantageously discovered that the compounds of the invention are surprisingly beneficial for use as an anti-senescence medicament. Even more surprisingly, the inventors have elucidated that the compounds of the invention act by reversing cellular senescence. The present inventors have termed compounds which act by halting or reversing senescence as "senoreversants". To date, it has been largely accepted that cellular senescence is an irreversible and permanent arrest of the cell cycle. For the first time, the present inventors have demonstrated reversal of senescence in cells by treatment with the compounds of the invention. This has been termed "senoreversal therapy"(rejuvenation).

[0016] Retinoids are known to have therapeutic benefit in the treatment of a range of diseases and conditions, such as neurodegenerative diseases. Retinoids are known to act genomically via the activation retinoic acid receptors (RARs) which regulate physiological mechanisms at a genomic level. Retinoids are also known to act non-genomically, either mediated by RARs or by independently of them, for example via activation of kinases such as extracellular signal- regulated kinase 1 / 2 (ERK1 / 2). However, retinoids have not previously been described as having anti-senescence activity. Surprisingly, the present inventors have for the first time, demonstrated anti-senescence activity by a group of synthetic retinoids (the compounds of formula I). Even more surprisingly, the inventors have for the first time demonstrated senoreversant activity (rejuvenation) for these compounds. This broadens the clinical pool of patients who may benefit from these compounds as anti-senescence medicaments, to include those who are not yet diagnosed, those at risk of developing, and those with minor or early symptoms of diseases or conditions associated with senescence, such as those diseases or conditions associated with the accumulation of senescent cells.

[0017] Accumulation of senescent cells is a well-described pathophysiological mechanism in a range of diseases and conditions, such as chronic inflammatory diseases (e.g. inflammatory bowel disease (IBD)), age-associated chronic inflammatory diseases (e.g. rheumatoid arthritis) and neurodegenerative diseases (e.g. Alzheimer's disease (AD)). Treatment options for many of these diseases and conditions are extremely limited and as such, there is currently a significant unmet need for new and improved therapies.

[0018] In addition to the above-described diseases and conditions, the accumulation of senescent cells is a major factor in ageing. There is a great need to ensure the ageing population remains as healthy as possible by preventing and / or slowing down the onset and / or progression of age-associated diseases and conditions.

[0019] The inventors have surprisingly observed that compounds of formula I can prevent cellular senescence and even reverse senescence where it has already occurred. As a result, these compounds may be surprisingly beneficial in the treatment of diseases and conditions whereby onset and / or symptoms are caused and / or exacerbated by the accumulation of senescent cells, as well as in anti-ageing treatments. These compounds are also envisioned to be beneficial in extending the healthy lifespan of ageing individuals by preventing and / or slowing down the onset and / or progression of age-associated diseases and conditions.

[0020] The compounds of the invention are compounds of formula I: in which each of A, B, and D is independently N or CH; each of X and Y is independently N, CH or CR9in which R9is F, Br, Cl, I or -OH, with the proviso that when one of X or Y is N or CR9, the other is CH; and

[0021] R1, R2, R7and R8are CH3, and each of R3, R4, R5and R6is H; or one of R1or R2is CH3 and the other, together with R3or R4represents a bond and one of R7and R8is CH3 and the other, together with R5or R6, represents a bond.

[0022] The compounds of formula I includes stereoisomers and geometric isomers thereof.

[0023] The compounds may be in free or salt form.

[0024] The compounds are for use as an anti-senescence medicament. In an embodiment, the compound is not: In an embodiment, the compound is of formula 1(a) or 1(b): ; in which A, B, D, X and Y are as previously defined.

[0025] In an embodiment, in formula 1(a) and / or 1(b) X and Y are both CH.

[0026] In an embodiment, in formula 1(a) and / or 1(b), D is CH. In an embodiment, the compound is of formula l(a)(i) or 1(b)(1): . In an embodiment, in formula l(a)(i) and / or l(b)(i), either one or both of A and B is N. In some embodiments, when either one or both of A and B is N, X and Y may both be CH.

[0027] In an embodiment, in formula I (a)(i) and / or I (b)(i), A and B are both CH.

[0028] In an embodiment, in formula I (a)(i) and / or I (b)(i), X is CR9or Y is N In an embodiment, in formula 1(a) and / or 1(b), X is CR9in which R9is F.

[0029] In an embodiment, the = compound of formula 1(a) or 1(b) is selected from: In an embodiment, the compound of formula l(a)(i) or l(b)(i) is selected from:

[0030] In an embodiment, the compound is of formula 1(a).

[0031] The term "senescent cell" means a cell in which cell senescence has been induced, which is a state in which a viable state is maintained and metabolic activity is exhibited but a proliferative ability is lost.

[0032] In an embodiment, there is provided the compound of formula I as described herein, wherein the anti-senescence medicament is for use in the treatment of a disease or condition selected from the following: chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, pancreatitis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age-related liver stenosis, age-related liver fibrosis, fibrotic pulmonary disease, chronic neuropathic pain, inflammatory pain, rheumatoid arthritis, macular degeneration, shingles, chronic obstructive pulmonary disease (COPD), coronary heart disease, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, Alzheimer's disease, amyotrophic lateral sclerosis, Lewy Body dementia, motor neurone disease, frontotemporal dementia, spinal muscular atrophy, primary Sjogren's disease, Progressive Supranuclear Palsy, Parkinson's disease, multiple sclerosis, Huntington's disease, schizophrenia, neuromuscular disease, senile dementia, vascular dementia and mixed dementia.

[0033] In an embodiment, there is provided the compound of formula I for use in the treatment of a disease or condition selected from the following: chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, pancreatitis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age-related liver stenosis, age- related liver fibrosis, fibrotic pulmonary disease, inflammatory pain, rheumatoid arthritis, macular degeneration, shingles, chronic obstructive pulmonary disease (COPD), coronary heart disease, inflammatory bowel disease, atopic dermatitis, psoriasis, and asthma.

[0034] The term "inflammatory bowel disease" includes Crohn's disease and ulcerative colitis.

[0035] In an embodiment, there is provided the compound of formula I as described herein, wherein the anti-senescence medicament is for use in the treatment of chronic inflammatory disease or condition, or in the treatment of a neurodegenerative disease or condition.

[0036] The term "chronic inflammatory disease or condition" is a disease or condition which is associated with an increase in levels of pro-inflammatory markers.

[0037] Chronic inflammatory diseases and conditions include, but are not limited to: chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, pancreatitis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age- related liver stenosis, age-related liver fibrosis, fibrotic pulmonary disease, inflammatory pain, rheumatoid arthritis, macular degeneration, chronic obstructive pulmonary disease (COPD), coronary heart disease, inflammatory bowel disease, atopic dermatitis, psoriasis, and asthma. The term "neurodegenerative disease or condition" refers to a group of diseases and conditions and diseases which result from the loss of function and / or death of nerve cells.

[0038] Neurodegenerative diseases and conditions include, but are not limited to: Alzheimer's disease, amyotrophic lateral sclerosis, Lewy Body dementia, motor neurone disease, frontotemporal dementia, spinal muscular atrophy, primary Sjogren's disease, Progressive Supranuclear Palsy, chronic neuropathic pain, Parkinson's disease, multiple sclerosis, Huntington's disease, schizophrenia, neuromuscular disease, senile dementia, vascular dementia, and mixed dementia.

[0039] In an embodiment, the chronic inflammatory disease or condition is an age-associated chronic inflammatory disease or condition.

[0040] The term "age-associated chronic inflammatory disease or condition" is a disease or condition which is associated with an age-related increase in levels of pro-inflammatory markers such as Rheumatoid arthritis.

[0041] Age-associated chronic inflammatory diseases and conditions include, but are not limited to: chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age- related liver stenosis, age-related liver fibrosis, fibrotic pulmonary disease, neuropathic pain, inflammatory pain rheumatoid arthritis, macular degeneration, rheumatoid arthritis, chronic obstructive pulmonary disease, and coronary heart disease.

[0042] In an embodiment, there is provided the compound of formula I as described herein, wherein the anti-senescence medicament is for use in the treatment of chronic inflammatory disease or condition wherein the disease is not a neurodegenerative disease or condition.

[0043] Aspects of the invention relate to compounds of formula I as described herein, wherein the anti-senescence medicament is for use in senotherapy.

[0044] Senotherapy is a therapy which specifically targets cellular senescence. The term senotherapy includes those which selectively target senescent cells to induce cell death (senolytics) and therapies which inhibit the pro-inflammatory senescent secretome (senomorphics), e.g. reduce IL-6 secretion. The term senotherapy also encompasses therapies which prevent cells from entering senescence or reverse senescence (cellular rejuvenation).

[0045] The compounds of formula I are senotherapeutics but do not act as senolytics. The compounds of formula I act by preventing cells from entering senescence or reversing senescence in cells which have already entered a senescent state. The inventors have termed compounds which reverse cellular senescence as "senoreversants".

[0046] In certain embodiments, there is provided the compound of formula I as described herein, wherein the anti-senescence medicament is for use in the treatment of a disease or condition associated with senescent cell accumulation.

[0047] According to the present invention, the term "disease or condition associated with senescent cell accumulation" is any disease or condition where there is a connection between the accumulation of senescent cells and said disease or condition. Accumulation of senescent cells may be the cause, or partial cause of the symptoms or, may exacerbate the symptoms, or may cause or contribute to the onset of the disease or condition ("tipping-point").

[0048] Diseases and conditions associated with senescent cell accumulation include: chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, pancreatitis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age- related liver stenosis, age-related liver fibrosis, fibrotic pulmonary disease, chronic neuropathic pain, inflammatory pain, rheumatoid arthritis, macular degeneration, , shingles, chronic obstructive pulmonary disease (COPD), coronary heart disease, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, Alzheimer's disease, amyotrophic lateral sclerosis, Lewy Body dementia, motor neurone disease, fronto-temporal dementia, spinal muscular atrophy, primary Sjogren's disease, Progressive Supranuclear Palsy, Parkinson's disease, multiple sclerosis, Huntington's disease, schizophrenia, neuromuscular disease, senile dementia, vascular dementia, and mixed dementia.

[0049] In embodiments, disease or condition associated with senescent cell accumulation does not include neurodegenerative diseases or conditions. In an aspect of the present invention there is provided the use of a compound of formula I, as defined herein, in the manufacture of a medicament for use as an anti-senescence medicament. In an embodiment, the medicament comprises a compound of formula I.

[0050] In embodiments, the anti-senescence medicament acts as a senescence reversant.

[0051] The term "senescence reversant" is used interchangeably with the term "senoreversant" and refers to agents which can induce the reversal of cells from a state of senescence to a healthy functional state. The reversal of cellular senescence can be identified in cells which are no longer positive for senescence markers, and the nuclear shape is normalised (circular).

[0052] In another aspect of the present invention there is provided a pharmaceutical composition comprising a compound of formula I as defined herein, optionally in conjunction with one or more pharmaceutically acceptable excipients, diluents or carriers, for use as an antisenescence medicament. The composition may optionally comprise one or more additional therapeutic agents.

[0053] In an aspect of the present invention, there is provided a method of treatment of a patient with a chronic inflammatory disease or condition, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I, wherein formula I is as defined herein.

[0054] In embodiments, the chronic inflammatory disease or condition is an age-associated chronic inflammatory disease or condition.

[0055] In an aspect of the present invention, there is provided a method of treatment of a patient with a neurodegenerative disease or condition, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I, wherein formula I is as defined herein.

[0056] In an aspect of the present invention, there is provided a method of treatment of a patient with a disease or condition associated with senescent cell accumulation, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I, wherein formula I is as defined herein. The term "therapeutically effective" amount, or "effective amount" refers to a quantity of the compound or composition of the present invention which is effective in producing the desired therapeutic, ameliorative, inhibitory or preventative effect.

[0057] The term "pharmaceutical composition" refers to a composition suitable for administration to a patient. Thus, the term "pharmaceutical composition" refers to compositions which comprise the compound of the invention or mixtures thereof, or salts, solvates, prodrugs, isomers or tautomers thereof, optionally in combination with one or more pharmaceutically acceptable excipients, carriers or diluents. The term "pharmaceutical composition" is also intended to encompass both the bulk composition (i.e. in a form that has not yet been formed into individual dosage units) and individual dosage units. Such individual dosage units include tablets, pills, caplets, ampoules and the like.

[0058] Those skilled in the art will recognize those instances in which the compounds of the invention may be converted to prodrugs and / or solvates. The term "prodrug" refers to a compound (e.g., a drug precursor) that is transformed in vivo to yield a compound of the invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood.

[0059] The compounds of the invention may be unsolvated or may be solvated with pharmaceutically acceptable solvents such as water, ethanol, and the like. For instance, it will be understood that a solvate may be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Suitable solvates include, but are not limited to, ethanolates, methanolates, hydrates, and the like.

[0060] Compounds for use in the invention include salts thereof, and reference to a compound of the invention is intended to include reference to salts thereof, unless otherwise stated. Suitable salts include for instance, acidic salts formed with inorganic and / or organic acids, basic salts formed with inorganic and / or organic bases, as well as zwitterions ("inner salts") which may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts may be useful in certain circumstances. Exemplary acid addition salts which may be useful include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like. Exemplary basic salts which may be useful include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen- containing groups may be quarternerized with agents such as lower alkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g. decyl, lauryl, and stearyl chlorides, bromides and iodides), arylalkyl halides (e.g. benzyl and phenethyl bromides), and others.

[0061] Compounds for use in the invention include pharmaceutically acceptable esters thereof, and may include carboxylic acid esters, obtained by esterification of the hydroxy groups, in which the non- carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, Ci-4 alkyl, or C1-4 alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4)phosphonate esters; and (5) mono-, di- or triphosphate esters.

[0062] Polymorphic forms of the compounds of the invention, and of the salts, solvates, esters and prodrugs of the compounds of the invention, are intended to be included in the present invention.

[0063] Suitable dosages for administering compounds of the invention to patients may be determined by those skilled in the art, e.g. by an attending physician, pharmacist, or other skilled worker and may vary according to factors such as patient weight, health, age, frequency of administration, mode of administration, the presence of any other active ingredients, and the condition for which the compounds are being administered.

[0064] Examples of excipients, diluents and carriers include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol and silicic derivatives. Binding agents may also be included. Adjuvants may also be included.

[0065] Optionally the compound of formula I may be administered in combination with one or more additional therapeutic agents. When used in combination with one or more additional therapeutic agents, the compounds of the invention may be administered together or sequentially.

[0066] The compositions may be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary, mucosal, intraocular and intranasal routes.

[0067] Suitable dosage forms will be recognised by one skilled in the art and include, among others, tablets, capsules, solutions, suspensions, powders, aerosols, ampules, pre-filled syringes, small volume infusion containers or multi-dose containers, creams, milks, gels, dispersions, microemulsions, lotions, impregnated pads, ointments, eye drops, nose drops, lozenges etc.

[0068] Various further features and aspects of the invention are defined in the claims.

[0069] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0070] Examples:

[0071] The invention will now be described by way of example only with reference to the accompanying figures, in which:

[0072] Figure 1 illustrates the synthesis of coupling partners;

[0073] Figure 2 illustrates the synthesis of exemplary compounds DC645 and RC8; Figure 3 illustrates cellular senescence in treated C6 glia cells under stress conditions;

[0074] Figure 4 illustrates p21 expression in treated C6 glia cells under stress conditions;

[0075] Figure 5 illustrates lamin Bl expression in treated C6 glia cells under stress conditions;

[0076] Figure 6 illustrates cell number in treated C6 glia cells under stress conditions;

[0077] Figure 7 illustrates LC3B expression in treated C6 glia cells under stress conditions;

[0078] Figure 8 illustrates IL-6 release in treated C6 glia cells under stress conditions;

[0079] Figure 9 illustrates the percentage of cells treated with control or DC645 which are p21- positive.

[0080] Example 1: Synthesis of exemplary compounds of formula I:

[0081] General experimental

[0082] Reagents were purchased from Sigma-Aldrich, Acros Organics, Alfa-Aesar, and Fluorochem. Reagents were purified, if required, by recrystallization or distillation / sublimation under vacuum. Solvents were used as supplied by Fisher Scientific or Sigma-Aldrich, and dried before use if required with appropriate drying agents. Thin-layer chromatography (TLC) was conducted using Merck Millipore silica gel 60G F254 25 glass plates and / or TLC-PET foils of aluminum oxide with fluorescent indicator 254 nm (40 mm x 80 mm) with visualization with a UV lamp or appropriate staining agents. Flash column chromatography was performed using SiOz from Sigma-Aldrich (230-400 mesh, 40-63 pM, 60 A), and monitored using TLC. Su bli mation / distil lation was performed using a Buchi Glass Oven B-585 Kugelrohr operating at a pressure between 4 and 10 Torr. NMR spectra were recorded using Varian VNMRS-700, Varian VNMRS-600, Bruker Avance-400, or Varian Mercury-400 spectrometers operating at ambient probe temperature. NMR peaks are reported as singlet (s), doublet (d), triplet (t), quartet (q), broad (br), septet (sept), combinations thereof, or as a multiplet (m), with reference to the following deuterated solvent signals: CDCh (1H = 7.26 ppm,13C = 77.0 ppm), (CDshSO (1H = 2.50 ppm,13C = 39.5 ppm). ESMS was performed using a TQD (Waters Ltd., U.K.) mass spectrometer with an Acquity UPLC (Waters Ltd., U.K.) system, and accurate mass measurements were obtained using a QtoF Premier mass spectrometer with an Acquity UPLC (Waters Ltd., U.K.). ASAP measurements were performed using an LCT Premier XE mass spectrometer and an Acquity UPLC (Waters Ltd., U.K.). IR spectra were recorded using a PerkinElmer FTIR spectrometer. Unless otherwise noted, all tested compounds were found to be >95% pure according to HPLC analysis.

[0083] 1.1 Synthesis of coupling partners

[0084] 1.1.1. Synthesis of 6-lodo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene, 1

[0085] The synthesis of 6-iodo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (1) is illustrated in Figure 1(a). l,l,4,4-Tetramethyl-l,2,3,4-tetrahydronaphthalene (11.46 g, 60.9 mmol), I2 (7.77 g, 30.6 mmol) and H5IO6 (3.49 g, 15.3 mmol) were added to a mixture of gl. ethanoic acid / acetic acid (AcOH) (250 mL), H2O (25 mL), and H2SO4 (13 mL), and the resultant solution was stirred at 70 °C for 6 hours. The solution was cooled, and extracted with ethyl ethanoate / ethyl acetate (EtOAc). The organics were washed with sat. Na2S2O3, H2O and brine, dried (MgSC ) and evaporated to give a crude orange oil (17 g). This was purified by dry column vacuum chromatography (eluting with heptane) to give compound 1 as a white solid (16.3 g, 85%): 1H NMR (700 MHz, CDCI3) 6 1.25 (s, 6H), 1.26 (s, 6H), 1.66 (s, 4H), 7.04 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 1.9 Hz, 1H), 7.60 (d, J = 1.9 Hz, 1H); 13C NMR (176 MHz, CDCI3) 6 31.9, 32.0, 34.4, 34.6, 35.0, 35.1, 91.3, 128.9, 134.8, 135.8, 144.8, 147.9; all other data matched the literature (V. B. Christie et al, Org. Biomol. Chem., 2008, 6, 3497-3507).

[0086] 1.1.2 Synthesis of 6-Ethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene, 3

[0087] The synthesis of 6-ethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (3) is illustrated in Figure 1(b). Triethylamine (Et3N) (150 mL) was degassed by sparging with N2 for 1 h. Compound 1 (10.0 g, 31.8 mmol), Pd(PPh3)2CI2(0.223 g, 0.32 mmol), Cui (0.061 g, 0.32 mmol) and trimethylsilylacetylene (5.29 mL, 38.2 mmol) were then added under N2 and the resultant slurry was stirred at RT for 20 h. The mixture was diluted with heptane and passed through Celite / SiC>2 (eluting with heptane), and the resultant solution was evaporated to give a crude brown oil (10.36 g). This was purified by SiCh chromatography (100% heptane) to give compound 2 as a yellow oil (9.99 g, >100%). Compound 2 (9.99 g, 35.1 mmol) was dissolved in methanol (MeOH) / methyl tert-butyl ether (MTBE) (1:1, 160 mL). A solution of sodium hydroxide (NaOH) (0.96 g, 24.0 mmol) in H2O (15 mL) was then added and the resultant solution was stirred at RT for 16 h. The solution was diluted with MTBE, washed with H2O and brine, dried (MgSC ) and evaporated to give a crude yellow oil (6.6 g). This was purified by SiO2 chromatography (100% heptane) to give compound 3 as a colourless oil that slowly solidified (6.06 g, 90% over two steps): all data matched the literature (V. B. Christie et al, Org. Biomol. Chem., 2008, 6, 3497-3507).

[0088] 1.1.3 Synthesis of 4-bromo-3-fluorobenzoate, 4

[0089] The synthesis of 4-bromo-3-fluorobenzoate (4) is illustrated in Figure 1(c). 4-Bromo-2- fluorobenzoic acid (25.0 g, 114.2 mmol) was suspended in MeOH (250 mL), whereupon cone. H2SO4 (4 mL) was added and the resultant solution was stirred at reflux overnight. The clear solution was then cooled, and H2O (100 mL) was added, whereupon a white precipitate formed. This was filtered, washed with H2O and dried to give a crude white solid. This was recrystallised from heptane to give compound 4 as a colourless crystalline solid (21.34 g, 80%):XH NMR (600 MHz, CDCI3) 6 3.91 (s, 3H), 7.31 - 7.36 (m, 2H), 7.80 (t, J = 8.0 Hz, 1H);13C NMR (151 MHz, CDCI3) 6 52.4, 117.6 (d, J = 9.9 Hz), 120.6 (d, J = 25.6 Hz), 127.5 (d, J = 3.9 Hz), 127.9 (d, J = 9.6 Hz), 133.1, 161.56 (d, J = 264.9 Hz), 164.1 (d, J = 3.9 Hz);19F NMR (376 MHz, CDCh) 6 -106.6; IR (ATR) Vmax / cm-13104w, 3086w, 2961w, 1712s, 1599s, 1571m, 1403m, 1215s, 882s; MS (ASAP): m / z = 233.0 [M+H]+; HRMS (ASAP) ealed. for C8H7O2BrF [M+H]+: 232.9613, found: 232.9621 (Zimmerman et al., J. Med. Chem. 2014, 57:2334-2356).

[0090] 1.1.4 Synthesis of methyl 4-ethynyl-3-fluorobenzoate, 6

[0091] The synthesis of methyl 4-ethynyl-3-fluorobenzoate (6) is illustrated in Figure 1(d). EtsN (120 mL) was degassed by sparging with N2 for 1 hour. Compound 4 (5.0 g, 21.45 mmol), trimethylsilylacetylene (3.56 mL, 25.74 mmol), Pd(PPh3)2Cl2 (301 mg, 0.429 mmol) and Cui (82 mg, 0.429 mmol) were then added under N2 and the resultant suspension was stirred at room temperature for 16 hours. The suspension was diluted with heptane and passed through Celite® / SiO2 and the extracts were evaporated to a give a crude oil (6.44 g). This was purified by dry column vacuum chromatography (100% heptane, to 9:1, heptane / EtOAc), and the isolated product further purified by Kugelrohr distillation (150 °C, 7.4 Torr) to give compound 5 as a yellow oil (5.58 g, >100%) which was carried directly to the next step:1H NMR (400 MHz, CDCI3) 6 0.27 (s, 9H), 3.92 (s, 3H), 7.50 (dd, J = 8.0, 6.8 Hz, 1H), 7.71 (dd, J = 9.6, 1.5 Hz, 1H), 7.75 (dd, J = 8.0, 1.6 Hz, 1H). To a MeOH / MTBE solution (5:50, 55 mL) was added compound 5 (5.58 g, 22.3 mmol) and K2CO3 (6.16 g, 44.6 mmol), and the resultant mixture was stirred under N2 for 6 hours at room temperature. The solution was then diluted with EtOAc, washed with sat. NH4CI, H2O and brine, dried (MgSC ) and evaporated to give a crude solid (3.6 g). This was purified by dry column vacuum chromatography (100% heptane, to 8:2, heptane / EtOAc) to give compound 6 as a white solid (3.07 g, 80% over two steps):TH NMR (600 MHz, CDCI3) 6 3.45 (s, 1H), 3.92 (s, 3H), 7.53 (dd, J = 8.0, 6.8 Hz, 1H), 7.72 (dd, J = 9.6, 1.6 Hz, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H);13C NMR (151 MHz, CDCI3) 6 52.5, 76.3, 85.1 (d, J = 3.3 Hz), 115.3 (d, J = 16.0 Hz), 116.5 (d, J = 22.9 Hz), 124.9 (d, J = 3.7 Hz), 132.2 (d, J = 7.4 Hz), 133.9 (d, J = 1.3 Hz), 162.9 (d, J = 253.5 Hz), 165.3 (d, J = 2.7 Hz);19F NMR (376 MHz, CDCI3) 6 -109.3; IR (ATR) vmax / cm13238m, 3090w, 2967w, 2111w, 1710s, 1564m, 1501m, 1440m, 1308s, 1212s, 766s; MS (ASAP) m / z = 179.0 [M+H]+; HRMS (ASAP) calcd. for CI0H8O2F [M+H]+: 179.0508, found 179.0495.

[0092] 1.1.5 Synthesis of methyl 5-bromopyridine-2-carboxylate, 8

[0093] The synthesis of methyl 5-bromopyridine-2-carboxylate (8) is illustrated in Figure 1(e). 5-Bromopyridine-2-carboxylic acid (20.0 g, 99.0 mmol) was suspended in MeOH (150 ml), whereupon cone. H2SO4 (5 mL) was carefully added and the resultant solution was stirred at reflux for 6 h. The solution was cooled, diluted with EtOAc, washed with H2O and brine (50 mL), dried (MgSO4) and evaporated to give a crude colourless solid. This was distilled in vacuo using a Kugelrohr (200 °C, 7.4 Torr), and the resultant white solid was further recrystallised from heptane / MeOH (10:1) to give compound 8 as a white solid (17.21 g, 80%):TH NMR (700 MHz, CDCI3) 6 3.90 (s, 4H), 7.86 - 7.93 (m, 3H), 8.68 (d, J = 2.0 Hz, 1H);13C NMR (176 MHz, CDCI3) 6 52.8, 124.8, 126.0, 139.5, 146.0, 150.7, 164.7; max / cm’13059w, 3008w, 2957w, 1710s, 1571w, 1558w, 1436m, 1305s, 1131s, 696s; MS (ASAP): m / z = 216.0 [M + H]+; HRMS (ASAP) calcd. for C10H9NOI [M + H]+: 215.9660, found: 215.9664 (Tung et al., Eur. J. Med. Chem. 2017, 126, 1011-1020).

[0094] 1.1.6 Synthesis of 2,2,5,5-tetramethylhexanedioic acid, 9 The synthesis of 2,2,5,5-tetramethylhexanedioic acid (9) is illustrated in Figure 1(f). To a 2L three-neck flask equipped with mechanical stirrer was added H2O (600 mL), cone. H2SO4 (7.5 mL), then pivalic acid (51.0 g, 500 mmol), and the resultant slurry was cooled to 0 °C. Over 15 mins, H2O2 (30%, 43 mL) and a solution of FeSO4.7H2O (139.0 g, 500 mmol) in H2O (288 mL) and cone. H2SO4 (27.5 mL) were added dropwise, with vigorous stirring. After completion of the addition, the suspension was stirred for a further 15 mins, before the solution was concentrated to ca. 250 mL. The precipitated solids were filtered, then dried further under vacuum using a rotary evaporator to give a crude residue. This was recrystallised from AcOH to give 9 as a colourless crystalline solid (3.39 g, 3%):1H NMR (400 MHz, DMSO-de) 6 1.06 (s, 12H), 1.37 (s, 4H), 12.05 (s, 2H).

[0095] 1.1.7 Synthesis of 1,6-diethyl 2,2,5,5-tetramethylhexanedioate, 10

[0096] The synthesis of 1,6-diethyl 2,2,5,5-tetramethylhexanedioate (10) is illustrated in Figure 1(g). To a solution of 9 (3.39 g, 16.76 mmol) in EtOH (40 mL) was added cone. H2SO4 (2 mL), and the resultant suspension was stirred at reflux for 16 h. The solution was cooled, and the solvent evaporated to a give crude residue which was dissolved in EtOAc. The organics were washed with sat. NaHCCh, H2O and brine, dried (MgSC ) and evaporated to give a crude oil (3.8 g). This was purified by SiCh chromatography (95:5, heptane / EtOAc) to give 10 as a colourless oil (3.41 g, 79%) :TH NMR (700 MHz, CDCI3) 6 1.14 (s, 11H), 1.24 (t, J = 7.1 Hz, 6H), 4.11 (q, J = 7.1 Hz, 4H);13C NMR (176 MHz, CDCI3) 6 14.2, 25.0, 35.5, 41.8, 60.2, 177.7; IR (ATR) Vmax / cm-12978m, 2934w, 2880w, 1726s, 1475m, 1308m, 1176s, 1110m,

[0097] 771w; MS(ES): m / z = 259.5 [M+H]+.

[0098] 1.1.8 Synthesis of trimethyl({3,3,6,6-tetramethyl-2-[(trimethylsilyl)oxy]cyclohex-l-en-l- yl}oxy)silane, 11

[0099] The synthesis of trimethyl({3,3,6,6-tetramethyl-2-[(trimethylsilyl)oxy]cyclohex-l-en-l- yl}oxy)silane, 11 is illustrated in Figure 1(h). To anhydrous toluene (50 mL) was added sodium (1.20 g, 52.1 mmol) under N2, and the resultant mixture was heated to reflux until the sodium melted. The flask was then removed from the heat, and then 10 (2.69 g, 10.41 mmol) and chlorotrimethylsilane (6.72 mL, 53.0 mmol) were added and the resultant suspension was then stirred at reflux overnight. The purple suspension was then cooled, and filtered under a flow of N2, washing with toluene, then tetra hydrofuran (THF). The filtrate was then evaporated to give a crude light yellow oil (3.2 g), which was purified by Kugelrohr distillation (120 °C, 3.6 Torr) to give 11 as a clear oil (2.64 g, Sl%):XH NMR (400 MHz, CDCI3) 6 0.19 (s, 18H), 1.03 (s, 12H), 1.44 (s, 4H). All other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp.409-419).

[0100] 1.1.9 Synthesis of 3,3,6,6-tetramethylcyclohexane-l,2-dione, 12

[0101] The synthesis of 3,3,6,6-tetramethylcyclohexane-l,2-dione is illustrated in Figure l(i). To a solution of 11 (2.6 g, 8.2 mmol) in dichloromethane (DCM) was added bromine (0.42 mL, 8.2 mmol) dropwise over 5 mins. The resultant yellow solution was stirred at RT for 1 h, before being diluted with DCM, and treated with sat. Na2S20s, then washed with H2O, dried (MgSC ) and evaporated to give a crude yellow solid (1.5 g). This was purified by recrystallisation from heptane to give 12 as a yellow crystalline solid (1.07 g, 78%) :1H NMR (700 MHz, CDCI3) 6 1.14 (s, 4H), 1.85 (s, 12H);13C NMR (176 MHz, CDCI3) 6 22.9, 34.7, 48.6, 207.3; IR (ATR) Vmax / crrT12973m, 2940w, 2870w, 1706s, 1599w, 1459m, 1372m, 1102m, 931m; MS(ES): m / z = 169.3 [M+H]+. All other data matched the literature. (Kikuchi et al. J. Med. Chem. 2000; 43: pp.409- 419).

[0102] 1.1.10 Synthesis of methyl 5,5,8,8-tetramethyl-5,6,7,8-tetrahvdroquinoxaline-2-carboxylate, 13

[0103] The synthesis of methyl 5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carboxylate, 13 is illustrated in Figure l(j). 12 (0.80 g, 4.76 mmol) and DL-2,3-diaminopropionic acid hydrochloride (0.67 g, 4.76 mmol) were combined in MeOH (30 mL). NaOH (0.76 g, 19.04 mmol) was added and the resultant mixture was stirred at reflux for 24 h. The solution was then cooled to 0°C, H2SO4 carefully added, and the solution was stirred at reflux for a further 6 h. The solution was cooled, and the solvent evaporated to give a crude residue which was dissolved with EtOAc, washed with sat. NaHCCh, H2O and brine, dried (MgSC ) and evaporated to give a crude yellow oil (0.9 g). This was purified by SiCh chromatography (95:5, heptane / EtOAc) to give 13 as a colourless oil (0.633 g, 54%):TH NMR (400 MHz, CDCI3) 6 1.33 & 1.36 (s, 12H), 1.81 (s, 4H), 3.98 (s, 3H), 9.00 (s, 1H). All other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp.409-419). 1.1.11 Synthesis of (5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)methanol, 14 The synthesis of (5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)methanol, 14 is illustrated in Figure l(k). To a solution of 13 (5.09 g, 20.5 mmol) in THF (80 mL) was added NaBF (2.33 g, 61.5 mmol). The solution was then heated to reflux, whereupon MeOH (16 mL) was slowly added over 1 h. The resultant solution was then stirred at reflux overnight. The solution was cooled, quenched with 1 M HCI, and the solvent then evaporated. The residue was dissolved in DCM, washed with water, dried (MgSC ) and evaporated to give a crude yellow oil (4 g). This was purified by SiCh chromatography (8:2, heptane / EtOAc, as eluent) to give 14 as a colourless oil (3.96 g, 88%):XH NMR (400 MHz, CDCI3) 6 1.33 & 1.36 (s, 12H), 1.80 (s, 4H), 3.50 (br, 1H), 4.75 (s, 2H), 8.32 (s, 1H); all other data matched the literature (Kikuchi et al. J. Med. Chem. 2000; 43: pp.409-419).

[0104] 1.1.12 Synthesis of 5,5,8,8-tetramethyl-5,6,7,8-tetrahvdroquinoxaline-2-carbaldehyde, 15 The synthesis of 5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline-2-carbaldehyde, 15 is illustrated in Figure 1(1). Oxalyl chloride (2.28 mL, 26.96 mmol) was added to anhydrous DCM (100 mL) under N2. The resultant solution was cooled to -78 °C, whereupon DMSO (3.83 mL, 53.92 mmol) was added dropwise so as to maintain the temperature below -60 °C. The solution was stirred for 15 mins before 14, as a solution in anhydrous DCM (3.96 g, 17.97 mmol, in 20 mL) was added dropwise so as to main the temperature below -60 °C. The solution was stirred for a further 15 mins before EtsN (18.03 mL, 129.38 mmol) was added. The solution was then stirred for 10 mins, before being allowed to reach room temperature (RT) over 30 mins. H2O was added, and the resultant mixture was diluted with DCM, washed with H2O, dried (MgSC ) and evaporated to give a crude oil (4 g). This was purified by dry column vacuum chromatography (DCVC) (heptane to 9:1, heptane / EtOAc) to give 15 as a colourless oil that slowly crystallises (3.39 g, 86%):1H NMR (700 MHz, CDCI3) 6 1.35 & 1.37 (s, 12H), 1.83 (s, 4H), 8.90 (s, 1H), 10.08 (s, 1H);13C NMR (176 MHz, CDCI3) 6 29.7, 29.7, 33.8, 33.8, 37.4, 37.9, 139.8, 144.2, 159.0, 163.7, 193.4; IR (ATR) Vmax / crn12979m, 2964m, 2928m, 2862m, 2823w, 1707s, 1553m, 1457m, 1126s, 1078s, 737s; MS(ES): m / z = 219.3 [M+H]+; HRMS (ES) calcd. for C13H19ON2 [M+H]+: 216.1497, found 216.1503.

[0105] 1.1.13 Synthesis of dimethyl l-diazo-2-oxopropylphosphonate, 16 The synthesis of Dimethyl l-diazo-2-oxopropylphosphonate, 16 is illustrated in Figure l(m). To a solution of dimethyl 2-oxopropylphosphonate (4.49 mL, 32.5 mmol) in anhydrous toluene (30 mL) at 0 °C was added NaH (60% dispersion in mineral oil, 1.20 g, 30.00 mmol) portionwise with vigorous stirring. After gas evolution had ceased 4- acetamidobenzenesulfonyl azide (7.21 g, 30.0 mmol), as a solution in anhydrous THF (lOmL), was added dropwise. The resultant suspension was stirred at RT for 16 h, whereupon heptane was added, and the suspension was filtered through Celite®, and rinsed with MTBE. The organic extracts were then evaporated to give a crude oil (5 g), which was purified by SiO2 chromatography (1:1, heptane / EtOAc) to give dimethyl l-diazo-2- oxopropylphosphonate as a light yellow oil (3.14 g, 54%) :1H NMR (400 MHz, CDCI3) 6 2.24 (s, 3H), 3.82 (d, J = 11.9 Hz, 6H), all other data matched the literature (Pietruszka et al. Synthesis, 2006, pp. 4266-4268).

[0106] 1.1.14 2-Ethvnyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline, 17

[0107] The synthesis of 2-ethynyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxaline, 17 is illustrated in Figure l(n). To a solution of 15 (1.18 g, 5.40 mmol) in anhydrous MeOH (50 mL) under ISh was added K2CO3 (1.49 g, 10.80 mmol) and dimethyl-l-diazo-2- oxopropylphosphonate (0.98 mL, 6.48 mmol), and the resultant suspension was stirred at RT for 16 h. The solution was diluted with EtOAc, washed with 5% NaHCCh, H2O and brine, dried (MgSC ) and evaporated to give a crude orange oil (0.4 g). This was purified by SiO2 chromatography (95:5, heptane / EtOAc, as eluent) to give 17 as a colourless oil that slowly crystallised (0.84 g, 73%) :TH NMR (700 MHz, CDCI3) 6 1.30 & 1.31 (s, 12H), 1.77 (s, 4H), 3.22 (s, 1H), 8.45 (s, 1H);13C NMR (176 MHz, CDCI3) 6 29.6, 29.7, 33.8, 34.0, 37.2, 37.3, 79.0, 81.0, 135.4, 144.5, 158.1, 158.6; IR (ATR) Vmax / crn’13279s, 2986w, 2945m, 2917m, 2863w, 2110w, 1519w, 1470m, 1459m, 1274m, 1078s, 674s; MS(ES): m / z = 215.3 [M+H]+; HRMS (ES) calcd. for C14H19N2 [M+H]+: 215.1548, found 215.1548.

[0108] 1.1.15 Ethyl (Z)-2-amino-2-hydrazineylideneacetate, 18

[0109] Ethyl thiooxamate (2.0 g, 15.0 mmol) was added to ethanol (30 mL) under N2- Hydrazine (IM in tetrahydrofuran, 15 mL, 15.0 mmol) was then added dropwise at RT, and the resultant solution was then stirred at RT for 1 h. The solvent was then evaporated to give compound 1

[0110] 18 as a red / orange solid (1.55 g, 79%) which was used without further purification: H NMR (400 MHz, DMSO-d6) 6 1.21 (t, J = 7.1 Hz, 3H), 4.12 (q, J = 7.1 Hz, 2H), 5.35 (s, 1H), 5.66 (br, 1H).

[0111] 1.1.16 Ethyl 5,5,8,8-tetramethyl-5,6,7,8-tetrahvdrobenzo[e][l,2,4]triazine-3-carboxylate, 19

[0112] Compound 12 (3.00 g, 17.8 mmol) was dissolved in anhydrous ethanol (50 mL) under N2, whereupon compound 18 (2.34 g, 17.8 mmol) was added in portions and the resultant solution was stirred at RT for 16 h. The solution was then heated at reflux for 1 h before being cooled, and the solvent was then evaporated. The crude material was then purified by dry column vacuum chromatography (100% heptane, to heptane / EtOAc, 9:1) to give compound

[0113] 19 as a yellow oil (4.2 g, 89%) :TH NMR (400 MHz, CDCI3) 64.53 (q, J = 7.1 Hz, 2H), 1.84 (s, 4H), 1.47 (s, 6H), 1.46 (t, J = 7.1 Hz, 3H), 1.38 (s, 6H).

[0114] 1.1.17 5,5,8,8-Tetramethyl-5,6,7,8-tetrahvdrobenzo[e][l,2,4]triazine-3-carbaldehyde, 20

[0115] Compound 19 (3.1 g, 11.77o mmol) was dissolved in anhydrous toluene under N2, and the solution was cooled to - 78 °C. DIBAL (1.0 M in toluene, 11.8 mL, 11.8 mmol) was carefully added dropwise down the side of the flask, and the resultant solution was stirred at -78 °C for 0.5 h before being allowed to slowly reach 0 °C over 3 h. 20% aq. NaOH was added and the mixture diluted with EtOAc. The organics were washed with H2O and brine, dried (MgSC ) and evaporated to give a crude yellow solid (2.7 g). This was purified by dry column vacuum chromatography (100% heptane to heptane / EtOAc, 9:1) to give compound 20 as a yellow solid (1.93 g, 75%):TH NMR (400 MHz, CDCI3) 6 1.40 (s, 6H), 1.50 (s, 6H), 1.86 (s, 4H), 10.30 (s, 1H).

[0116] 1.1.18 3-Ethynyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydrobenzo[e][l,2,4]triazine, 21

[0117] To a solution of compound 20 (500 mg, 2.28 mmol) in anhydrous tetrahydrofuran (14.0 mL) and anhydrous methanol (14.0 mL) under Ar, was added K2CO3 (630 mg, 4.56 mmol) and the Bestman-Ohira reagent (0.66 mL, 2.73 mmol). The yellow reaction mixture was then stirred for 18 h, after which the solution was passed through a short Celite plug, washed with dichloromethane. The solvent was removed under reduced pressure, before the solid was redissolved in dichloromethane (100 mL). The crude was washed with H2O (15 mL) and brine (15 mL) and the organic layers were dried (MgSC ). The crude material was concentrated under reduced pressure to give a brown solid that was purified by SiCh chromatography (hexane / EtOAc, 9:1) to afford the compound 21 as a yellow solid (0.18 g, 37 %);1H NMR (400 MHz, CDCI3) 6 1.35 (s, 6H), 1.46 (s, 6H), 1.81 (s, 4H), 3.26 (s, 1H);13C NMR (101 MHz, CDCI3) 6 29.2, 29.7, 33.3, 33.7, 36.8, 37.2, 78.3, 80.3, 151.4, 163.0, 164.4; IR (ATR) v^x / cm’13203s, 2961m, 2934m, 2867m, 2114s, 1512s, 1458m, 1362s, 1267m, 1097m, 743s; MS (ESI): m / z [M + H]+216.4 (100 %); HRMS (ESI): m / z calculated C13H17N3 [M + H]+216.1498, found 216.1501.

[0118] 1.2 Synthesis of methyl 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2- yl)ethvnyl]benzoate, 22

[0119] The synthesis of compound 22 is illustrated in Figure 2. EtsN (20 mL) was degassed by sparging with Ar for 1 h. Methyl 4-iodobenzoate (0.31 g, 1.20 mmol) compound 17 (0.30 g, 1.40 mmol), Pd(PPh3)2Cl2 (83 mg, 0.12 mmol) and Cui (22 mg, 0.12 mmol) were then added under Ar and the resultant suspension was stirred at RT for 16 h. The suspension was diluted with MTBE and passed through Celite’ / SiCh and the extracts were evaporated to a give a crude solid (0.5 g). This was purified by dry column vacuum chromatography (100% heptane to 85:15, heptane / EtOAc) to give an off-white solid which was subsequently recrystallised from MeOH to give DC641 as a colourless crystalline solid (0.29 g, 71%), which was carried directly to the next step:TH NMR (400 MHz, CDCI3) 6 1.33 & 1.35 (s, 12H), 1.81 (s, 4H), 3.94 (s, 3H), 7.64 - 7.71 (m, 2H), 8.01 - 8.08 (m, 2H), 8.52 (s, 1H).

[0120] 1.3 Synthesis of 4-[2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahvdroquinoxalin-2-yl)ethvnyl]benzoic acid, DC645 (compound of the invention)

[0121] The synthesis of exemplary compound DC645 is illustrated in Figure 2. Compound 22 (0.28 g, 0.8 mmol) was dissolved in THF (20 mL), 20% NaOH (2mL) added, and the resultant solution was stirred at reflux for 16 h. The mixture was cooled, acidified to pH 1 with 5% HCI, extracted with EtOAc, washed with H2O and brine, dried (MgSC ) and evaporated to give a crude white solid which was recrystallised from MeCN to give DC645 as a colourless crystalline solid (0.24 g, 89%):TH NMR (700 MHz, DMSO-d6) 6 1.29 (s, 12H), 1.78 (s, 4H), 7.73 - 7.79 (m, 2H), 7.98 - 8.02 (m, 2H), 8.68 (s, 1H), 13.24 (br, 1H);13C NMR (176 MHz, DMSO-d6) 6 29.4, 29.4, 33.1, 33.2, 37.0, 37.1, 88.9, 89.8, 125.2, 129.6, 131.3, 131.9, 135.2, 144.4, 157.6, 158.0, 166.5; IR (ATR) Vmax / cm-12961w, 2925w, 2958w, 2223w, 1683s, 1606m, 1558w, 1428m, 1282s, 862s, 769m; MS(ASAP): m / z = 334.2 [M]+; HRMS (ASAP) calcd. for C21H22N2O2 [M]+: 334.1681, found 334.1686.

[0122] 1.4 Synthesis of EC23 (4-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahydronaphthalen-2- ylethynyDbenzoic acid (compound of the invention)

[0123] 1.4.1 6-Bromo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene

[0124] To a solution of l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (10.0g, 53.0 mmol) in DCM (60 ml) at 0 °C under N2was added Br2(15.58 g, 97.5 mmol). BF3.Et2O (8.27 g, 58.3 mmol) in DCM (10 ml) was added dropwise over 2 h. The reaction mixture was diluted with 40 / 60 EtOAc / hexane (150 ml) and washed with saturated Na2SO3solution (100 ml), saturated NaHCO3solution (100 ml), and H2O (100 ml). The organic layer was dried (MgSC ), filtered and evaporated to give a dark brown oil. Kugelrohr distillation (120 °C, 8 x 10-3mbar) gave 6- bromo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene as pale yellow crystals (11.02 g, 78%); M.p. 43-45 °C. All13C and1H NMR data was identical to the literature [(a). G. Garipova, A. Gautier, S.R. Piettre; Tetrahedron Lett.; 2005; 61; 4755-4759 (b). H. kagechika, E. Kawachi, Y. Hashimoto, T. Himi, K. Shudo; J. Med. Chem., 1988, 31, 11, 2182-2192)], MS (El) 267 (m+). Anal. Calcd for Ci4Hi9Br: C, 62.93; H, 7.17; Found: C, 62.81; H, 7.16.

[0125] 1.4.2 Trimethyl-(5,5,8,8-tetramethyl-5,6,7,8-tetrahvdronaphthalen-2-ylethvnyl)silane

[0126] PdCl2 (75 mg, 0.43 mmol), Cu(OAc)2 (77 mg, 0.43 mmol), 6-bromo-l,l,4,4-tetramethyl- 1,2,3,4-tetrahydronaphthalene (1.14 g, 4.30 mmol), and PPh3(0.56 g, 2.14 mmol) were placed in a 250 ml Schlenk flask under N2. Dry, degassed triethylamine (100 ml) was added via cannula and TMSA (0.7 mL 5.14 mmol) added via syringe. After 18 h at 70 °C, triethylamine was evaporated and the residue was passed through a short silica gel column (hexane as eluent) to give the crude product as a viscous, pale yellow oil after evaporation which slowly solidified to give an off-white solid which was recrystallised from ethanol to give the TMSacetylene adduct trimethyl-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2- ylethynyl)silane (1.0 g, 81%); Mp 51-52 °C. 6H(400 MHz; CDCI3) 0.24 (9H, s, CH3), 1.25 (6H, s, CH3), 1.26 (6H, s, CH3), 1.66 (4H, s, CH2), 7.21 (2H, m, Ar), 7.40 (1H, m, Ar); 6C(400 MHz, CDCI3) 0.0 , 31.6, 31.7, 34.1, 34.2, 34.8, 34.9, 92.6, 105.8, 120.0, 126.4, 129.0, 130.2, 144.8, 145.7 (Ar-C); m / z MS (El): 284 (m+). Anal. Calcd for Ci9H28Si: C, 80.21; H, 9.92. Found: C, 80.04: H, 9.90.

[0127] 1.4.3 6-Ethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene

[0128] To a solution of 6-trimethylsilylethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (1.42 g, 5 mmol) in methanol (50 ml) and diethyl ether (50 ml), was added NaOH (0.14 g, 3.5 mmol) in water (2 ml). After 4 h, the mixture was extracted with diethyl ether, washed with water (3 x), dried (MgSC ) and evaporated to give 6-ethynyl-l, 1,4, 4-tetramethyl-l, 2,3,4- tetrahydronaphthalene as a pale yellow oil which slowly solidified to give a white solid (0.78 g, 74%); Mp 48-49 °C; IR (KBr disc, cm1) 3289, 2956, 2927, 2864, 2105 (CC), 1486, 1454, 1362, 1262, 1179, 1106;XH NMR (200 MHz, CDCI3) d 7.47 (s, 1H, Ar), 7.27 (s, 2H, Ar), 3.03 (s, 1H, CH), 1.70 (s, 4H, CH2), 1.29 (s, 6H, 2 x Me), 1.26 (s, 6H, 2 x Me); ^C H} NMR (100 MHz, CDCh) d 146.1, 145.1, 130.5, 129.2, 126.6, 119.1, 84.3, 75.9, 34.9, 34.8, 34.3, 34.2, 31.8, 31.7. MS (El): 212 (m+); Anal, calcd for CI6H20: C, 90.51; H, 9.49; found: C, 90.27; H, 9.57.

[0129] 1.4.4 Methyl 3-iodobenzoate

[0130] A solution of 3-iodobenzoic acid (24.80 g, 100 mmol) in methanol (300 ml) was treated with sulphuric acid (98%, 5 g). After heating at 70 °C for 5 h, the mixture was extracted with diethyl ether, washed with water (3 x), dried (MgSC ) and evaporated. The residue was passed through a short silica gel column (hexane as eluent), to give the product as a white crystalline solid (21.1 g, 81%); Mp 53-54 °C. All spectroscopic and analytical properties were identical to those reported in the literature [Wuest, Hans Heiner; Frickel, Fritz Frieder; Nuerrenbach, Axel. (BASF A.-G., Fed. Rep. Ger.). Ger. Offen. (1986), 23 pp. CODEN:

[0131] GWXXBX DE 3434946 Al 19860403 Patent. Application: DE 84-

[0132] 3434946 19840922. Priority: CAN 105:172069],

[0133] 1.4.5 4-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahvdronaphthalen-2-ylethvnyl)benzoic acid methyl ester

[0134] Methyl 4-iodobenzoate (3.59 g, 13.7 mmol), Pd(PPhs)2CI2(0.09 g, 0.14 mmol) and Cui (0.03 g, 0.14 mmol) were added to a stirred 500 ml Schlenk flask which was evacuated and purged with N2 (3 x). Triethylamine (200 ml) was added via cannula under N2, followed by 6-ethynyl- l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (3.50 g, 16.5 mmol). After 18 h at RT, triethylamine was evaporated and the residue was passed through a short silica gel column (hexane, followed by hexane:DCM, 4:1 as eluent) to give the crude product, which was recrystallised from ethanol to give the product 7a as a white crystalline solid (4.13 g, 87%); Mp 128-129 °C; IR (KBr disc, cm1) 2957, 2921, 2857, 2207 (CC), 1712 (C=O), 1605, 1433, 1276, 1177, 1106;XH NMR (400 MHz, CDCI3) d 8.03 (d, J = 9 Hz, 2H, Ar), 7.59 (d, J = 9 Hz, 2H, Ar), 7.51 (s, 1H, Ar), 7.31 (s, 2H, Ar), 3.94 (s, 3H, MeO), 1.71 (s, 4H, 2 x CH2), 1.28 (s, 12H, Me);13C{XH} NMR (100 MHz, CDCI3) d 166.8, 146.3, 145.4, 131.7, 130.3, 129.7, 129.4, 129.0, 128.6, 127.0, 119.8, 93.3, 87.2, 52.4, 35.2, 35.1, 34.6, 34.5, 32.0, 31.9; UV-vis (CHCI3) umax 310 nm (e 26400 M^cm1); Anal, calcd for C24H26O2: C, 83.20; H, 7.56; found: C, 83.03; H, 7.59.

[0135] 1.4.6 4-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahvdronaphthalen-2-ylethvnyl)benzoic acid (EC23); (compound of formula I)

[0136] A solution of 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid methyl ester (0.35 g, 1 mmol) in THF (20 ml) was treated with aqueous 20% NaOH (20 ml). After heating at 70 °C for 20 h, the reaction mixture was diluted with diethyl ether (150 ml) and water (150 ml), then IM HCI solution was added until mixture reached pH 1. The ether layer was separated, dried (MgSC ) and evaporated to give an off-white powder, which was re-crystallised from acetonitrile to give the product 4-(5, 5,8, 8-Tetramethyl-5, 6,7,8- tetrahydronaphthalen-2-ylethynyl)benzoic acid as a white crystalline solid (0.24 g, 72%); Mp 254-256 °C; IR (KBr disc, cm1) 2961, 2928, 2858, 2205 (CC), 1681 (C=O), 1606, 1423, 1314, 1292, 1177;TH NMR (400 MHz, CDCI3) d 8.09 (d, J = 8.5 Hz, 2H, Ar), 7.62 (d, J = 8.5 Hz, 2H, Ar), 7.51 (s, 1H, Ar), 7.31 (s, 2H, Ar), 1.68 (s, 4H, CH2), 1.31 (s, 12H, Me). ^C H} NMR (125 MHz, CDCh) d 171.5, 146.5, 145.4, 131.7, 130.4, 130.3, 129.6, 129.1, 128.4, 127.0, 119.7, 93.9, 87.8, 35.1, 35.0, 34.6, 34.5, 32.0, 31.9. MS (ESI): 377 (m+Na2), 331 (m-H). UV-vis (CHCI3) umax 310 nm (e 26900 M^cm1); HRMS calcd for C23H23O2: 331.16926 (m-H). Found: 331.16949.

[0137] 1.5 Synthesis of 3-Fluoro-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2- yl)ethvnyl]benzoic acid, DC526 (compound of formula I) 5-[2-(5, 5,8, 8-Tetramethyl-5, 6,7,8- tetrahvdronaphthalen-2-yl)ethvnyl]pyridine-2-carboxylic acid DC528 (compound of formula

[0138] 11

[0139] 1.5.1 6-lodo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene l,l,4,4-Tetramethyl-l,2,3,4-tetrahydronaphthalene (11.46 g, 60.9 mmol), l2(7.77 g, 30.6 mmol) and H5IO6 (3.49 g, 15.3 mmol) were added to a mixture of AcOH (250 mL), H2O (25 mL), and H2SO4 (13 mL), and the resultant solution was stirred at 70 °C for 16 h. The solution was cooled, and extracted with EtOAc. The organics were washed with sat. Na2S2O3, H2O and brine, dried (MgSC ) and evaporated to give a crude orange oil (17 g). This was purified by dry column vacuum chromatography (eluting with heptane) to give 6-iodo-l, 1,4,4- tetramethyl-l,2,3,4-tetrahydronaphthalene as a light yellow oil which slowly crystallizes on standing (16.25 g, 85%): all data matched the literature (Christie, V. B.; Barnard, J. H.; Batsanov, A. S.; Bridgens, C. E.; Cartmell, E. B.; Collings, J. C.; Maltman, D. J.; Redfern, C. P. F.; Marder, T. B.; Przyborski, S.; Whiting, A. Org. Biomol. Chem. 2008, 6, 3497-3507)

[0140] 1.5.2 6-Ethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene

[0141] EtsN (150 mL) was added to a 3-neck, 250 mL RBF under N2. The solution was degassed by sparging with N2 for 1 h. 6-lodo-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (10.0 g, 31.8 mmol), Pd(PPh3)2Cl2 (0.22 g, 0.32 mmol), Cui (0.061 g, 0.32 mmol) and trimethylsilylacetylene (5.3 mL, 38.2 mmol) were then added under N2 and the suspension was stirred at RT for 20 h. The mixture was diluted with heptane and passed through Celite / SiC>2 (eluting with heptane), and the resultant solution was evaporated to give a crude brown oil (10.36 g). This was purified by SiCh chromatography (100% heptane) to give trimethyl[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethynyl]silane as a yellow oil (9.99 g, >100%). This was dissolved in dissolved in MeOH / MTBE (1:1, 160 mL). A solution of NaOH (0.96 g, 24.0 mmol) in H2O (15 mL) was then added and the resultant solution stirred at RT for 72 h. The solution was diluted with MTBE, washed with H2O and brine, dried (MgSC ) and evaporated to give a crude yellow oil (6.6 g). This was purified by SiO2 chromatography (100% heptane) to give 6-ethynyl-l, 1,4, 4-tetramethyl-l, 2,3,4- tetrahydronaphthalene as a colourless oil that slowly solidified (6.06 g, 90% over two steps): all data matched the literature (Christie, V. B.; Barnard, J. H.; Batsanov, A. S.; Bridgens, C. E.; Cartmell, E. B.; Collings, J. C.; Maltman, D. J.; Redfern, C. P. F.; Marder, T. B.; Przyborski, S.; Whiting, A. Org. Biomol. Chem. 2008, 6, 3497-3507).

[0142] 1.5.3 3-Fluoro-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2- yl)ethvnyl]benzoic acid, DC526 (compound of formula I)

[0143] EtsN (80 mL) was degassed by sparging with N2 for 1 h. 6-Ethynyl-l, 1,4, 4-tetramethyl-l, 2, 3, 4- tetrahydronaphthalene (0.80 g, 2.55 mmol), DC550 (0.54 g, 3.05 mmol), Pd( PPhgJzCh (179 mg, 0.26 mmol) and Cui (49 mg, 0.26 mmol) were then added under isl and the resultant suspension was stirred at RT for 72 h. The suspension was diluted with MTBE and passed through Celite’ / SiCh and the extracts were evaporated to give a crude solid (1.1 g). This was purified by dry column vacuum chromatography (100% heptane to heptane / EtOAc, 95:5) to give DC553 as a colourless oil which slowly crystallized (0.82 g, 88%). This was dissolved in THF (40 mL), 20% NaOH (3 mL) added, and the resultant solution was stirred at reflux for 16 h. The mixture was cooled, acidified to pH 1 with 5% HCI, extracted with EtOAc, washed with H2O and brine, dried (MgSC ) and evaporated to give a crude white solid which was recrystallized from MeCN to give DC526 as a colourless crystalline solid (0.60 g, 77%):TH NMR (600 MHz, DMSO-ck) 1.24 & 1.26 (s, 12H), 1.64 (s, 4H), 7.32 (dd, J = 8.2, 1.8 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.53 (d, J = 1.8 Hz, 1H), 7.70 - 7.78 (m, 2H), 7.80 (dd, J = 7.9, 1.6 Hz, 1H), 13.44 (br, 1H);13C NMR (151 MHz, DMSO-d6) 6 31.3, 31.4, 33.9, 34.1, 34.2, 34.3, 81.0, 97.4 (d, J = 3.1 Hz), 115.2, 115.3 (d, J = 15.8 Hz), 116.0 (d, J = 22.3 Hz), 118.5, 125.4 (d, J = 3.4 Hz), 127.1, 128.7, 129.6, 132.7 (d, J = 7.1 Hz), 133.6, 145.2, 146.5, 161.4 (d, J = 250.3 Hz), 165.7 (d, J = 2.5 Hz);19F NMR (376 MHz, DMSO-d6) 6 -110.0; IR (ATR) Vmax / crn12967m, 2928m, 2857m, 2210w, 1686s, 1617m, 1566m, 1421m, 1307m, 1218m, 834s, 764m; MS(ASAP): m / z = 351.2 [M+H]+; HRMS (ASAP) calcd. for C23H24O2F [M+H]+: 351.1760, found 351.1766.

[0144] 1.5.4 Ethyl 5-bromopyridine-2-carboxylate

[0145] 5-Bromopyridine-2-carboxylic acid (20.0 g, 99.0 mmol) was suspended in MeOH (150 ml), whereupon cone. H2SO4 (5 mL) was carefully added and the resultant solution was stirred at reflux for 6 h. The solution was cooled, diluted with EtOAc, washed with H2O and brine (50 mL), dried (MgSO4) and evaporated to give a crude colourless solid. This was purified by Kugelrohr distillation (200 °C, 7.4 Torr), and the resultant white solid was further recrystallized from heptane / MeOH (10:1) to give compound Ethyl 5-bromopyridine-2- carboxylate as a white solid (17.21 g, 80%):TH NMR (700 MHz, CDCI3) 6 3.90 (s, 4H), 7.86 - 7.93 (m, 3H), 8.68 (d, J = 2.0 Hz, 1H);13C NMR (176 MHz, CDCI3) 6 52.8, 124.8, 126.0, 139.5, 146.0, 150.7, 164.7; Vmax / crn’13059w, 3008w, 2957w, 1710s, 1571w, 1558w, 1436m, 1305s, 1131s, 696s; MS (ASAP): m / z = 216.0, 218.0 [M+H]+; HRMS (ASAP) calcd. for C7H7NO2Br [M+H]+: 215.9660, found: 215.9664.

[0146] 1.5.5 5-[2-(5,5,8,8-Tetramethyl-5,6,7,8-tetrahvdronaphthalen-2-yl)ethvnyl]pyridine-2- carboxylic acid, DC528 (compound of formula I)

[0147] EtsN / THF (1:1, 120 mL) was added to an oven-dried 100 ml RBF under N2, and the solution was degassed by sparging with N7for l h. Pd(PPh3hCl2 (0.265 g, 0.38 mmol), Cui (0.072 g, 0.38 mmol), 6-ethynyl-l,l,4,4-tetramethyl-l,2,3,4-tetrahydronaphthalene (0.8 g, 4.80 mmol) and ethyl 5-bromopyridine-2-carboxylate (0.98 g, 4.52 mmol) were added under isl and the resultant solution was stirred at 50 °C for 40 h. The solution was diluted with heptane, eluted through a Celite® / SiO2 plug and the extracts were evaporated to give a crude brown solid (1.9 g). This was purified by dry column vacuum chromatography (100% heptane to heptane / EtOAc, 8:2), to give DC510 as a white solid (0.36 g, 27%), which was carried directly to the next step:XH NMR (400 MHz, CDCI3) 6 1.29 (d, J = 7.9 Hz, 12H), 1.69 (s, 4H), 4.02 (s, 3H), 7.31 (d, J = 1.1 Hz, 2H), 7.51 (s, 1H), 7.94 (dd, J = 8.1, 2.1 Hz, 1H), 8.12 (dd, J = 8.1, 0.9 Hz, 1H), 8.85 (dd, J = 2.1, 0.9 Hz, 1H). DC510 (0.33 g, 0.95 mmol) was dissolved in THF (30 mL), 20% NaOH (3 mL) added, and the resultant solution was stirred at reflux for 16 h. The mixture was cooled, acidified to pH 1 with 5% HCI, extracted with EtOAc, washed with H2O and brine, dried (MgSC ) and evaporated to give a crude white solid. This was recrystallized from MeCN to give DC528 as a colourless crystalline solid (0.30 g, 96%) :TH NMR (700 MHz, DMSO-d6) 6 1.25 & 1.27 (s, 12H), 1.65 (s, 4H), 7.35 (dd, J = 8.1, 1.8 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 8.06 (dd, J = 8.1, 0.9 Hz, 1H), 8.12 (dd, J = 8.1, 2.1 Hz, 1H), 8.85 (dd, J = 2.1, 0.9 Hz, 1H), 13.35 (s, 1H);13C NMR (176 MHz, DMSO-d6) 6 31.3, 31.4, 33.9, 34.1, 34.2, 34.3, 39.5, 84.8, 95.4, 118.4, 122.7, 124.3, 127.1, 128.7, 129.8, 139.5, 145.2, 146.4, 146.9, 151.3, 165.6; IR (ATR) Vmax / cm-13283br, 2955m, 2920m, 2856m, 2208m, 1752s, 1586m, 1336s,

[0148] 1247m, 1017m, 833m; MS(ES): m / z = 334.2 [M+H]+; HRMS (ES) calcd. for C22H24NO2 [M+H]+: 334.1807, found 334.1808.

[0149] Example 1.6 Synthesis of 4-(3-ethvnyl-5,8-dimethylquinoline)benzoic acid (MH21) (Compound of formula I)

[0150] 1.6.1 Synthesis of 5,8-dimethyl-3-hvdroxymethylquinoline

[0151] 5,8-Dimethylquinoline-3-carboxylic acid (494 mg, 2.46 mmol) was dissolved in dry THF (30 mL) at —78 °C under an atmosphere of argon. After 5 minutes of stirring, DIBAL-H in toluene (9.82 mL, 9.82 mmol, 1.0 M) was carefully added and the mixture was left stirring at room temperature for 4 days. 5% NaOH (25 mL) was added, upon which a yellow solution was formed. The resulting aqueous and organic phases were separated, and the aqueous phase was extracted with EtOAc (3 x 25 mL). The combined organic phases were washed with brine (3 x 25 mL) before being dried over MgSO4. The solvent was removed under reduced pressure, leaving a yellow oil. The product was then purified by SiO2 column chromatography eluting in hexanes followed by a slowly increasing ratio of hexane : EtOAc (up to 1:9), with the product eluting in 4:6 hexane / EtOAc. 5,8-Dimethyl-3-hydroxymethylquinoline (6) was acquired as a pale-yellow solid (278 mg, 1.50 mmol, 61%). M.p. 58-59 °C. IR spectrum Vmax / crn’13161br (O-H), 2917w (C-H), 1604w (C- Haromatic), 1376s (C Naromatic).TH NMR (400 MHz; CDCh) 6H: 8.90 (1H, s, HF) 8.26 (1H, s, HA) 7.45 (1H, d, J = 7.3 Hz, HD) 7.27 (1H, d, J = 7.4 Hz, He) 4.93 (2H, s, HG) 2.79 (3H, s, HE) 2.64 (3H, s, HB).13C NMR (101 MHz, CDCI3) 6C: 148.5 (C8), 147.0 (C7), 134.7 (Cl), 132.6 (C5), 132.3 (C2), 130.7 (C6), 129.2 (C3), 127.2 (C4), 127.0 (C9), 63.3 (C12), 18.6 (Cll), 18.2 (CIO). MS (ESI): m / z [M+H]+188.205. HRMS (ESI) for C12H13NO for 188.107 found [M+H]+188.1075.

[0152] 1.6.2 Synthesis of 5,8-dimethylquinoline-3-carbaldehyde

[0153] To a solution of 5,8-dimethyl-3-hydroxymethylquinoline (430 mg, 2.30 mmol) in dry THF (43 mL), was added activated MnCh (2.00 g, 23.0 mmol) under an atmosphere of argon. The solution was left stirring at room temperature for 4 days, after which the reaction mixture was passed through a Celite / silica plug to remove any solid MnCh. The plug was washed with EtOAc (30 mL), and the filtrate was concentrated under reduced pressure. The resulting orange-yellow solid was purified by SiO2 column chromatography, eluting in 9:1 hexane / EtOAc, yielding 5,8-dimethylquinoline-3-carbaldehyde as a pale-yellow solid (279 mg, 1.51 mmol, 66%). M.p. 89-91 °C. IR spectrum Vmax / cm’12980w (C-H), 1677vs (C=O), 1584s (C- Haromatic), 1194s (C-Naromatic).TH NMR (400 MHz; CDCh) 6H: 10.32 (1H, s, HG), 9.40 (1H, d, J = 2.0 Hz, HF), 8.83 (1H, d, J = 2.1 Hz, HA), 7.64 (1H, d, J = 7.2 Hz, HD), 7.40 (1H, d, J = 7.2 Hz, Hc), 2.83 (3H, s, HE), 2.77 (3H, s, HB).13C NMR (101 MHz, CDCI3) 6C: 191.3 (CIO), 150.1 (C8), 147.8 (C7), 136.5 (C9), 135.5 (Cl), 134.3 (C2), 132.6 (C6), 128.0 (C3), 127.7 (C5), 126.6 (C4), 18.5 (C12), 18.2 (Cll). MS (ESI): m / z [M+H]+186.191. HRMS (ESI) for C12H11NO for 186.0911 found [M+H]+186.0919.

[0154] 1.6.3 Synthesis of 3-ethynyl-5,8-dimethylquinoline

[0155] K2CO3 (417 mg, 3.02 mmol) and the Bestmann-Ohira reagent (348 mg, 4.1 ml, 1.81 mmol) were added to a solution of 5,8-dimethylmethylquinoline-3-carbaldehyde (279 mg, 1.51 mmol) in dry THF (9 mL) and dry MeOH (9 mL) under an argon atmosphere. The reaction mixture was stirred for 21 h, after which the solution was passed through a short celite plug, with DCM (200 mL) as the eluent. The solvent was removed under reduced pressure, and the solid redissolved in DCM (50 mL). Saturated NaCI (40 mL) was added, ensuring the solution pH was slightly basic, and the resulting aqueous and organic phases were separated. The aqueous phase was extracted with DCM (3 x 50 mL) and the organic phases were combined, before being dried over MgSC . The solvent was then removed under reduced pressure, yielding 3-ethynyl-5,8-dimethylquinoline as a light-brown solid (245 mg, 1.35 mmol, 90%). TLC and NMR spectral analysis of the product indicated sufficient purity had been obtained from the workup procedure, therefore no purification was necessary. M.p. 54-55 °C. IR spectrum Vmax / crn"13179w (C— H), 2105w (C=C), 1490w (C— H aromatic), 1355s (C Naromatic).

[0156] NMR (400 MHz; CDCI3) 6H: 8.99 (1H, d, J = 2.0 Hz, HF), 8.46 (1H, d, J = 2.0 Hz, HA), 7.48 (1H, d, J = 7.1 Hz, HD), 7.30 (1H, d, J = 7.2 Hz, He), 3.31 (1H, s, HG), 2.77 (3H, s, HE), 2.65 (3H, s, HB).13C NMR (101 MHz, CDCh) 6C: 150.5 (C8), 146.5 (C7), 136.3 (Cl), 135.1 (C5), 132.2 (C2), 130.4 (C6), 127.6 (C3), 126.5 (C4), 115.4 (C9), 81.5 (CIO), 80.2 (Cll), 18.4 (C13), 18.0 (C12). MS (ESI): m / z [M+H]+182.314. HRMS (ESI) for C13H11N for 182.0959 found [M+H]+182.0970.

[0157] 1.6.4 Synthesis of methyl 4-(3-ethvnyl-5,8-dimethylquinoline)benzoate

[0158] Cui (14.2 mg, 0.0747 mmol), PdCI2(PPh3)2 (26.2 mg, 0.0373 mmol), DIPEA (195 pL, 1.12 mmol) and 3-ethynyl-5,8-dimethylquinoline (203 mg, 1.12 mmol) were successively added to a solution of methyl 4-iodobenzoate (196 mg, 0.747 mmol) in dry THF (8.9 mL) under an argon atmosphere. The mixture was stirred at room temperature for 18 h before being treated with water (18 mL) and passed through a short celite plug, with saturated NH4CI (18 mL) as the eluent. The resulting aqueous and organic phases were separated, and the aqueous phase was extracted with EtOAc (3 x 45 mL). The organic phases were combined and dried over MgSC , before removing the solvent under reduced pressure. The resulting orange solid was purified by SiCh column chromatography, eluting in hexanes followed by a slowly increasing ratio of hexane : EtOAc (up to 7:3) once the first product had been isolated from the column, yielding methyl 4-(3-ethynyl-5,8-dimethylquinoline)benzoate as a pale-orange solid (221 mg, 0.699 mmol, 94%). However, NMR spectral analysis revealed 14% contamination of the product by the homo-coupled acetylene. The diyne was recrystallised from DCM / Et2O, followed by a second slow recrystallisation from DCM / hexane, yielding methyl 4-(3-ethynyl- 5,8-dimethylquinoline)benzoate (9) as a white solid (133 mg, 0.422 mmol, 57%). M.p. 124- 125 °C. IR spectrum Vmax / crn’12949w (C-H), 2206w (C=C), 1720vs (C=O), 1432s (C-Haromatic), 1272vs (C-0).XH NMR (400 MHz; CDCI3) 6H: 9.05 (1H, d, J = 2.1 Hz, HF), 8.51 (1H, d, J = 2.1 Hz, HA), 8.10-8.07 (2H, m, HH), 7.71-7.68 (2H, m, HG), 7.50 (1H, d, J = 7.2 Hz, HD), 7.33 (1H, d, J = 7.2 Hz, Hc), 3.97 (3H, s, Hi), 2.79 (3H, s, HE), 2.69 (3H, s, HB).13C NMR (101 MHz, CDCI3) 6C:

[0159] 166.5 (C16), 150.2 (C8), 146.4 (C7), 135.7 (Cl), 135.1 (C5), 132.3 (C2), 131.7 (C13), 130.4 (C6), 129.9 (C3), 129.6 (C14) 127.6 (C15), 127.4 (C12), 126.6 (C4), 116.0 (C9), 91.5 (CIO), 90.1 (Cll), 52.3 (C17), 18.5 (C19), 18.1 (C18). MS (ESI): m / z [M+H]+316.315. HRMS (ESI) for C21H17NO2 for 316.1344 found [M+H]+316.1338.

[0160] 1.6.5 Synthesis of 4-(3-ethvnyl-5,8-dimethylquinoline)benzoic acid (MH21, Compound of Formula I)

[0161] 20% NaOH (1.2 mL) was added to a solution of methyl 4-(3-ethynyl-5,8- dimethylquinoline)benzoate (170 mg, 0.209 mmol) in THF, and the mixture was refluxed for 17 h. The reaction mixture was then allowed to cool before being acidified to pH 4 with 5% HCI. The resulting aqueous and organic phases were separated, and the aqueous phase was extracted with EtOAc (3 x 30 mL). The combined organic phases were washed with water (3 x 30 mL) and brine (3 x 30 mL) before being dried over MgSC . The solvent was then removed under reduced pressure, yielding a light-pink solid which was recrystallised from MeCN to afford 4-(3-ethynyl-5,8-dimethylquinoline)benzoic acid as a pale-pink solid (111 mg, 0.368 mmol, 68%). M.p. 236-238 °C. IR spectrum Vmax / crn’12917br (O-H), 1673vs (C=O), 1424s (C- Haromatic), 1276s (C-O).TH NMR (400 MHz; DMSO-d6) 6H: 9.06 (1H, d, J = 2.1 Hz, HF), 8.68 (1H, d, J = 2.2 Hz, HA), 8.04-8.00 (2H, m, HH), 7.78-7.76 (2H, m, HG), 7.57 (1H, d, J = 7.1 Hz, HD), 7.40 (1H, d, J = 7.0 Hz, Hc), 2.69 (3H, s, HE), 2.66 (3H, s, HB).13C NMR (101 MHz, DMSO-d6) 6C: 167.2 (C16), 150.5 (C8), 146.3 (C7), 136.5 (Cl), 134.8 (C5), 133.3 (C2), 132.2 (C13), 131.4 (C6), 131.0 (C3), 130.1 (C14), 128.1 (C15), 126.6 (C12), 126.6 (C4), 115.8 (C9), 91.9 (CIO), 90.3 (Cll), 18.6 (C18), 18.1 (C17). MS (ESI): m / z 302.292. HRMS (ESI) for C20H15NO2 for 302.1192 found [M+H]+302.1181.

[0162] Example 1.7 Synthesis of 4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydrobenzo[e][l,2,4] triazin-3-yl)ethynyl)benzoic acid, 24 (RC8, compound of formula I)

[0163] 1.7.1 _ Methyl 4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydrobenzo[e][l,2,4]triazin-3- yl)ethynyl)benzoate, 23

[0164] Under Ar, Cui (6.7 mg, 0.0354 mmol), Pd(PPh3)2Cl2 (12.5 mg, 0.0177 mmol), N,N- diisopropylethylamine (91.5 pL, 0.531 mmol) and compound 21 (114 mg, 0.531 mmol) were successively added to a solution of methyl 4-iodobenzoate (92.8 mg, 0.354 mmol) in anhydrous tetrahydrofuran (5.00 mL). The mixture was stirred at room temperature for 72 h and then heated to reflux for 24 h. The solution was passed through a short Celite plug, washed with tetra hydrofuran and the solvent was removed under reduced pressure. The solid was redissolved in EtOAc (30.0 mL) and was washed with water (9.00 mL), sat. NH4CI (9.00 mL) and brine (9.00 mL) and the organic layer was dried (MgSC ). The crude material was concentrated under reduced pressure to afford a black solid that was purified by SiC>2 chromatography (hexane / EtOAc, 9:1) to yield compound 24as a yellow solid (0.0641 g, 34 %);XH NMR (400 MHz, CDCI3) 6 1.37 & 1.47 (s, 12H), 1.83 (s, 4H), 3.94 (s, 3H), 7.74 - 7.76 (m, 2H), 8.05 - 8.08 (m, 2H);13C NMR (101 MHz, CDCI3) 6 29.2, 29.8, 33.3, 33.8, 36.8, 37.3, 52.5, 88.8, 88.9, 126.1, 129.7, 131.0, 132.6, 152.3, 162.4, 164.3, 166.5; IR (ATR) v^x / crrT12959w, 2925s, 2863s, 2232m, 1719s, 1606m, 1454m, 1393s, 1279s, 1096s, 861s, 768s, 695s; MS (ESI): m / z [M + H]+350.6 (100 %); HRMS (ESI): m / z calculated C21H23N3O2 [M + H]+350.1875 found 350.1875.

[0165] 1.7.2 4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahvdrobenzo[e][l,2,4]triazin-3-yl)ethvnyl)benzoic acid, 24 (RC8, compound of formula I)

[0166] The synthesis of exemplary compound RC8 is illustrated in Figure 2. Compound 23 (20.0 mg, 0.0572 mmol) was dissolved in tetra hydrofuran (2.00 mL), 20% aq. NaOH (0.143 mL, 0.0286 mmol) added, and the resultant solution was stirred at reflux for 7 h. The mixture was cooled, acidified to pH 1 using 1.0 M HCI, and then extracted with EtOAc (5 x 8.00 mL). The organics were washed with H2O (13.0 mL) and brine (13.0 mL), dried (MgSC ) and the solvent was removed under reduced pressure to give the compound 24 (21.1 mg, 110 %);TH NMR (400 MHz, CDCI3) 6 1.38 & 1.47 (s, 12H), 1.83 (s, 4H), 7.80 - 7.82 (m, 2H), 8.12 - 8.14 (m, 2H);13C NMR (101 MHz, CDCI3) 6 29.3, 29.8, 33.4, 33.8, 36.8, 37.4, 76.9, 90.0, 126.3, 126.9, 130.3, 132.7, 152.2, 162.5, 164.7, 174.7; IR (ATR) v^ / crrr12928s, 2904w, 2222m, 1687s, 1604m, 1458m, 1401s, 1284s, 873s, 771s, 693s, MS (ESI): m / z [M + H]+336.6 (100 %); HRMS (ESI): m / z calculated C20H21N3O2 [M + H]+336.1723, found 336.1722.

[0167] Example 2: Biological Evaluation

[0168] 2.1 Materials and Methods

[0169] 2.1.1 Cell lines and culture

[0170] C6 (rat glioma) were obtained from Durham University and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin Streptomycin Solution (Pen-Strep, Lonza) at 37 °C in a humidified 5% incubator. The growth medium was changed every 2 days. When the culture reached 80% confluence, trypsin-EDTA was added and incubated for 3 to 5 minutes to detach adherent cells. Triturated cells were seeded 1:2 into 24-well plates or T75 flasks for further growth.

[0171] 2.1.2 Treatment of cells

[0172] After trypsinization, cells were plated at a density of 40000 cells / mL into chamber of a 24-well plate. The cells were cultured for 24 hours at 37°C and 5% CO2. Stress treatment was applied to cells by the addition of 20mM H2O2 or lOpg / ml lipopolysaccharide (LPS) for 24 hours. H2O2 is used to induce oxidative stress in cells. LPS is used to induce inflammatory stress in cells. Cells under serum starvation conditions were cultured in serum starvation media (DMEM / F12 supplemented with 1% FBS and 1% Pen-Strep) for 24 hours. Cells were then treated with lOnM DC645 or corresponding concentration of DMSO (control) for 24 hours.

[0173] 2.1.3 Lactate Dehydrogenase (LDH) release assay LDH release was measured using CytoTox 96® kit (ADG1781, Promega). 100 p.1 of the supernatant was taken out of each cell culture well and transferred to a 96-well tissue culture plate. 100 pl of the cytotoxicity detection kit LDH solution was added to each well and incubated for 30 minutes in the dark at room temperature. After 30 minutes, the reaction was stopped by adding 50 pl of stop solution. The optical density was then measured at 490 nm. This assay was normalised by freezing the remaining plate containing media only and later thawing it, then pipetting the contents of each well into Eppendorf tubes, centrifuging those for 10 minutes for the cells to settle down, and then taking out 100 pl of the supernatant from each Eppendorf tube and following the same procedure as described above. This gave an indication of total LDH and allowed normalisation.

[0174] 2.1.4 Enzyme-Linked Immunosorbent Assay (ELISA)

[0175] 24 hours after treatment, 100 pl of the cellular supernatant was collected from each well and ELISA was carried out using the Human IL-6 ELISA kit (abl78013, Abeam) in accordance with the manufacturer's protocol. The standard curve generated was used to calculate concentrations from the absorbance measurements.

[0176] 2.1.5 Senescence-Associated |3-Galactosidase (SA-|3-Gal) staining

[0177] Cells were plated at a density of 8000 / mL in 6-well (35mm) chambers onto 15mmxl5mm coverslips. Stress treatment was applied as described in section 2.1.2 and after 24 hours, Senescence-Associated [3-Galactosidase (SA-P-Gal) staining was carried out using the Senescence Cells Histostaining Kit (Sigma-Aldrich, CS0030-1KT) according to the manufacturer's protocol. Cell nuclei were then stained using DAPL The stained cells were counted and compared to the total number of cells, evaluated by counting the DAPI-stained nuclei.

[0178] 2.1.6 Immunocytochemistry staining

[0179] Cells were plated 8000 / mL with DMEM / F12, 10 %FBS, 1% penstrep, and MEM, 1% penstrep in 6-well (35mm) chambers onto 15mmxl5mm coverslips. 24 hours after plating, treatment was applied to cells as described in section 2.1.2. 24 hours after treatment, immunocytochemistry staining was carried out using the VECTASTAIN® Elite® ABC Universal Kit (PK-6200) and ImmPACT® DAB Substrate Kit, Peroxidase (SK-4105) according to the manufacturer's protocol. Primary antibody was diluted as follow: LC3B (1:200, PAI-46286, invitrogen). Images were captured by using a Leica microscope (Leica DM2500).

[0180] 2.1.7 Immunofluorescence staining

[0181] Cells were plated 8000 / mL in 6-well (35mm) chambers onto 15 mm x 15 mm coverslips. 24 hours after treatment, the cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature. Cells were washed three times for 5 minutes with phosphate-buffered saline (PBS) and then blocked using blocking buffer (1% bovine serum albumin (BSA), 1% fish skin gelatin and 0.3% Triton X-100 in PBS) at room temperature for 1 hour. Then the cells were incubated with primary antibody for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer as follows: P21 (1:200, G1814), LaminBl (1:500, Abeam, abl6048). Cells were then washed three times for 5 minutes in PBS and incubated with secondary antibodies (Goat Anti-Mouse IgG H&L Alexa Fluor® 488, 1:1000, Abeam, abl50113) for 1.5 hours at room temperature. Cells were then washed three times for 5 minutes with PBS, and incubated with DAPI (lpg / mL) for 5 minutes at room temperature. DAPI is used to stain the DNA for nuclear localization. Fluorescent images were captured by using a Zeiss fluorescent microscope (Zeiss ApoTome) and a Zeiss confocal microscope (Zeiss LSM 800).

[0182] 2.1.8 Senescence-reversing assay

[0183] C6 glial cells were plated at a density of 8000 / mL in 6-well (35 mm) chambers onto 15 mm x 15 mm coverslips and grow for 24h before being stressed with 20pM H2O2. After being stressed for 24 h, cells were treated with 10 nM of DC645 for another 24h. The cells were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. Cells were washed three times for 5 min with PBS and then blocked in PBS containing 1% bovine serum albumin, 1% fish skin gelatin and 0.3% Triton X-100 at room temperature for 1 h. Then, the cells were incubated with the primary antibodies (p-21) for 1 h at room temperature. The p21 primary antibodies were diluted in a ratio of 1:200. Cells were then washed three times for 5 min in PBS and incubated with secondary antibodies for 1.5 h at room temperature. Cells were then washed three times for 5 min with PBS and incubated with DAPI (1 pg / mL) for 5 min at room temperature to stain the DNA for nuclear localization. Fluorescent images were captured by using a Zeiss fluorescent microscope (Zeiss ApoTome, Cambridge, UK). Results are shown in Figure 9.

[0184] 2.1.9 Quantification and statistical analysis

[0185] The semi-quantitative detection of immunofluorescence images and immunocytochemistry images were applied using Fiji.

[0186] The average fluorescence intensity was measured with the mean gray value limited with the same threshold and the same exposure time.

[0187] Cell counting was conducted manually and blind to treatment condition.

[0188] The data were obtained from at least three independent experiments for each experimental condition. Data are expressed as means ± SD. Two-tailed t-tests were used to analyze differences between two groups, p values <0.05 are considered significant. All these analyses were performed using Graphpad Prism 8.

[0189] 2.2 Results

[0190] 2.2.1 Cellular Senescence in Compound of Formula I Treated C6 Glia Cells

[0191] Figure 3A shows C6 glia cells stained with SA-P-Gal, a biomarker of cellular senescence. Cells which show a positive SA-P-Gal stain are considered to be senescent cells.

[0192] Figure 3A panels (a) and (c) show C6 glia cells under control conditions (+ PBS), these cells displayed minimal SA-P-Gal staining indicative of low cellular senescence.

[0193] Figure 3A panels (b) and (d) show C6 glia cells under stress conditions (+ H2O2). Panel (b) shows control treated cells (+ DMSO) under stress conditions have increased levels of SA- - Gal staining, indicative of stress-induced cellular senescence. Panel (d) shows cells treated with DC645 under stress conditions. No increase in SA- -Gal staining is observed in the DC645 treated stress condition cells.

[0194] Figure 3B shows quantification of senescence in DC645 treated cells. Stressed cells treated with DC645 showed a statistically significant decrease in the percentage of senescent cells when compared to control (+ DMSO) stressed cells (p < 0.01). These data indicate that DC645 is protective against cellular senescence under stress conditions.

[0195] 2.2.2 p21 expression in treated C6 glia cells p21, also known as cyclin-dependent kinase inhibitor 1, is a protein which plays a role in the arrest of the cell cycle. Increased expression of p21 is widely used as a marker of cellular senescence.

[0196] Figure 4A shows immunofluorescence images showing p21 (shown in (a), (b), (e), and (f) as grey highlight) and DAPI (shown in (c), (d), (g), and (h) as grey highlight) expression in C6 glia cells under control (+ PBS) and stress (+ H2O2) conditions. DAPI is a DNA stain and is used to show nuclear localization.

[0197] Panels (a) to (d) show cells with control treatment (+ DMSO) and panels (e) to (h) show cells after treatment with DC645. The data demonstrates that p21 expression is reduced after treatment with DC645 both under stress (panel (f)) and control conditions (panel (e)) when compared to control treated cells (panels (b) and (a), respectively). This data indicates that treatment with DC645 reduces cellular senescence.

[0198] Figure 4B shows the percentage of p21 positive cells under control (+ PBS) and (+ H2O2) conditions. A statistically significant reduction in p21 positive cells can be observed after treatment with DC645 under both control (p < 0.01) and stress conditions (p < 0.05). This data demonstrates a reduction in senescence after treatment with DC645.

[0199] 2.2.3 Lamin Bl expression in treated C6 glia cells

[0200] Lamin Bl is a component of the nuclear lamina. Downregulation of lamin Bl is a biomarker indicating cellular senescence. In addition, lamin Bl can be used to visualize the nuclear structure and observe any structural changes which may also be indicative of cellular senescence.

[0201] Figure 5A shows immunofluorescence images of lamin Bl (shown in (a), (b), (e), and (f) as grey highlight) and DAPI (shown in (c), (d), (g), and (h) as grey highlight) expression in C6 glia cells under control (+ PBS) and stress (+ H2O2) conditions. DAPI is a DNA stain and is used to show nuclear localization and shape.

[0202] Panels (a) to (d) show cells with control treatment (+ DMSO) and panels (e) to (h) show cells after treatment with DC645. Figure 5A demonstrates that lamin Bl expression is increased under stress conditions after treatment with DC645 (panel (f)) in comparison with control cells under stress conditions (panel (b)). Also, DC645 reversed the nuclear shape within the cell from a stress-induced "bean-shape" back to a healthy circular shape. This finding indicates that treatment with DC645 reduces cellular senescence, particularly under stress conditions.

[0203] Figure 5B shows quantification of fluorescence intensity of lamin Bl expression in DC645 treated cells. A significant increase in lamin Bl expression is observed in treated cells under stress conditions (p <0.05) after treatment with DC645. These data provide further evidence that DC645 reduces senescence under stress conditions.

[0204] 2.2.4 Cell Number (optical density)

[0205] Figure 6 shows cell number after treatment with DC645 under control (+ PBS) and stress conditions (+ H2O2). The data shows an increase in cell number after treatment with DC645 under both stress conditions back to control levels. This data suggests that treatment with DC645 results in a higher number of cells, this may be a result of senescence being prevented or reversed in treated cells.

[0206] 2.2.5 Autophagy in treated C6 glia cells

[0207] Figure 7 shows LC3B staining in treated C6 glia cells under stress (serum starvation) conditions. LC3B is a marker of autophagy in cells. Autophagy suppresses senescence by removing damaged organelles and proteins to maintain homeostasis under stressful conditions. Chronic inhibition of autophagy induces cellular senescence mainly due to accumulation of defective mitochondria and increased levels of reactive oxygen species (ROS).

[0208] Figure 7A shows the C6 glia cells stained with LC3B. Panels (a) and (b) show control cells. Panels (c) and (d) show cells treated with DC645. Treated cells under stress conditions show an increased expression of LC3B indicating increased levels of autophagy. Figure 7B shows the average optical density of treated cells under stress conditions. Optical density is used to quantify LC3B expression. As can be seen in figure 7B, the average optical density was significantly higher in DC645 treated cells compared to control cells under stress conditions.

[0209] 2.2.6 IL-6 release in treated C6 glia cells

[0210] Figure 8 shows IL-6 release in DC645 treated C6 glia cells. Interleukin 6 (IL-6) is a pro- inflammatory cytokine which is present in the senescence-associated secretory phenotype (SASP), An increase in IL-6 levels can therefore be used as a marker of senescence.

[0211] Figure 8 shows a significant decrease in IL-6 release under stress conditions (+ LPS) in DC645 treated cells compared to control treated cells. This significant decrease in IL-6 release indicates that DC645 is preventing and / or reversing senescence under stress conditions.

[0212] 2.2.7 Senescence-reversing assay

[0213] Figure 9 shows the percentage of cells treated with control or DC645 which were positive for p21 expression. Treatment with DC645 reduced the expression of p21 significantly* (*p<0.05, n=6), indicating that DC645 reverses senescence.

[0214] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0215] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as "open" terms (e.g., the term "including" or "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations).

[0216] It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.

Claims

CLAIMSin which: each of A, B, and D is independently N or CH; each of X and Y is independently N, CH or CR9in which R9is F, Br, Cl, I or -OH, with the proviso that when one of X or Y is N or CR9, the other is CH; andR1, R2, R7and R8are CH3, and each of R3, R4, R5and R6is H; or one of R1or R2is CH3 and the other, together with R3or R4represents a bond and one of R7and R8is CH3 and the other, together with R5or R6, represents a bond; and stereoisomers or geometric isomers thereof; in free or salt form; for use as an anti-senescence medicament.

2. A compound of formula I as claimed in claim 1, wherein the compound is of formula 1(a) or 1(b):

3. A compound of formula I as claimed in claim 1 or claim 2, wherein the compound is of formula l(a)(i) or l(b)(i):Formula l(b)(i).

4. A compound as claimed in any preceding claim, wherein X and Y are both CH.

5. A compound as claimed in any preceding claim, wherein at least one of A or B is N.

6. A compound as claimed in any preceding claim, wherein A and B are both CH.

7. A compound as claimed in claim 6, wherein X is CR9or Y is N.

8. A compound as claimed in claim 7, wherein X is CR9in which R9is F.

9. A compound of formula I as claimed in claim 1, wherein the compound is selected from:

10. A compound of formula I as claimed in any preceding claim, wherein the antisenescent medicament is for use in the treatment of a disease or condition selected from chronic kidney disease, idiopathic pulmonary fibrosis, diabetes, diabetic bone disease, atherosclerosis, pancreatitis, sarcopenia, osteoarthritis, osteoporosis, obesity, cardiovascular disease, age-related liver stenosis, age-related liver fibrosis, fibrotic pulmonary disease, chronic neuropathic pain, inflammatory pain, rheumatoid arthritis, macular degeneration, shingles, chronic obstructive pulmonary disease (COPD), coronary heart disease, inflammatory bowel disease, atopic dermatitis, psoriasis, asthma, Alzheimer's disease, amyotrophic lateral sclerosis, Lewy Body dementia, motor neurone disease, fronto-temporal dementia, spinal muscular atrophy, primary Sjogren's disease, Progressive Supranuclear Palsy, Parkinson's disease, multiple sclerosis, Huntington's disease, schizophrenia, neuromuscular disease, senile dementia, vascular dementia, and mixed dementia.

11. A compound of formula I as claimed in claim 10 , wherein inflammatory bowel disease includes Crohn's disease and ulcerative colitis.

12. A compound of formula I as claimed in any preceding claim, wherein the antisenescent medicament is for use in the treatment of chronic inflammatory disease or condition or in the treatment of a neurodegenerative disease or condition.

13. A compound of formula I as claimed in claim 12, wherein the chronic inflammatory disease or condition is an age-associated chronic inflammatory disease or condition.

14. A compound of formula I as claimed in any preceding claim, wherein the antisenescent medicament is for use in senotherapy.

15. A compound of formula I as claimed in any preceding claim, wherein the antisenescent medicament is for use in the treatment of a disease or condition associated with senescent cell accumulation.

16. A compound of formula I as defined in any of claims 1 to 15, in the manufacture of a medicament for use as an anti-senescence medicament.

17. A compound of formula I as defined in any of claims 1 to 16, wherein the antisenescence medicament is a senescence reversant.

18. A pharmaceutical composition comprising a compound of formula I as claimed in any of claims 1 to 17, optionally in conjunction with one or more pharmaceutically acceptable excipients, diluents, or carriers, for use as an anti-senescence medicament.

19. A method of treatment of a patient with a chronic inflammatory disease or condition, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I as defined in any of claims 1 to 17.

20. A method of treatment as claimed in claim 19, wherein the chronic inflammatory disease or condition is an age-associated chronic inflammatory disease or condition.

21. A method of treatment of a patient with a neurodegenerative disease or condition, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I as defined in any of claims 1 to 17.

22. A method of treatment of a patient with a disease or condition associated with senescent cell accumulation, the method comprising administering to a patient a therapeutically effective amount of a compound of formula I as defined in any of claims 1 to 17.

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