WO2008014602A1 - Quinoline derivatives - Google Patents

Quinoline derivatives Download PDF

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Publication number
WO2008014602A1
WO2008014602A1 PCT/CA2007/001324 CA2007001324W WO2008014602A1 WO 2008014602 A1 WO2008014602 A1 WO 2008014602A1 CA 2007001324 W CA2007001324 W CA 2007001324W WO 2008014602 A1 WO2008014602 A1 WO 2008014602A1
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WIPO (PCT)
Prior art keywords
methyl
hydroxyquinolin
chloro
pyridin
acetamide
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PCT/CA2007/001324
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French (fr)
Inventor
Siegfried Hekimi
Kevin Mcbride
Abdelmadjid K. Hihi
Irenej Kianicka
Ying Wang
Steven Leonard Hayes
Marie-Pierre Guimond
Guy SÉVIGNY
Daniel Dumas
Julian Smith
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Envivo Pharmaceuticals, Inc.
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Publication date
Application filed by Envivo Pharmaceuticals, Inc. filed Critical Envivo Pharmaceuticals, Inc.
Priority to EP07815779A priority Critical patent/EP2079699A1/en
Priority to AU2007280984A priority patent/AU2007280984A1/en
Priority to CA002658793A priority patent/CA2658793A1/en
Priority to JP2009521076A priority patent/JP2009544631A/en
Publication of WO2008014602A1 publication Critical patent/WO2008014602A1/en

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Abstract

The invention relates to new quinoline derivatives which are active CLK-1 inhibitors. More specifically, the CLK-1 inhibitors of the invention are compounds of formula (A). The invention also relates to pharmaceutical compositions comprising such compounds and to methods for the prophylaxis and/or treatment of disorders or their associated symptoms for which the inhibition of CLK-1 is beneficial.

Description

QUINOLINE DERIVATIVES
FIELD OF THE INVENTION
The present invention relates to new quinoline derivatives as CLK-1 inhibitors. More specifically, the present invention relates to new quinoline derivatives or their pharmaceutically acceptable salts or pro-drugs as CLK-1 inhibitors, and to pharmaceutical compositions comprising the same and to methods for the prophylaxis and/or treatment of disorders or its associated symptoms for which the inhibition of CLK-1 is beneficial.
BACKGROUND OF THE INVENTION
Role of clk-1 and CLK-1
The clk-1 gene encodes a 21 kDa mitochondrial protein which is conserved amongst eukaryotes with sequence identity of 85% between mouse and human sequence and 50% with the homologous sequence of the nematode worm Caenorhabditis elegans. CLK-1 participates in one of the last steps of ubiquinone biosynthesis and is responsible for demethoxyubiquinone (DMQ) hydroxylation. Mutants in which CLK-1 is inactive do not synthesize detectable amounts of ubiquinone (Q), and rather accumulate DMQ (Miyadera et al. 2001).
Q has numerous functions in the cell. It is involved in electron transport in the inner mitochondrial membrane, the plasma membrane, and the membrane of the lysosome. It is also a cofactor for several cellular enzymes (e.g. dihydroorotate dehydrogenase, which is necessary for nucleic acid biosynthesis) and other proteins and complexes such as the mitochondrial uncoupling proteins (UCP) which regulate metabolic heat production and the mitochondrial permeability transition pore (MPTP) which regulates programmed cell death.
DMQ is a quinone that can transport electrons, although less efficiently than ubiquinone. Mouse embryos in which the clk-1 homologue (mclk-1) has been knocked out also accumulate DMQ, and are not viable (Levavasseur et al. 2001) but mice heterozygous for mclk-1 display an increased lifespan phenotype. Transgenic expression of CLK-1 in the mouse knock-out rescues the lethality phenotype, and allows ubiquinone synthesis (Nakai et al. 2004). Interestingly, heterologous expression of C. elegans CLK-1 in slow-growing coq7 (yeast clk-1) yeast mutants is functional, and rescues yeast slow-growth phenotype (Ewbank et al. 1997). Altogether, these observations indicate that CLK-1 is the only DMQ hydroxylase in the studied organisms, and that there are no functional compensatory mechanisms for CLK-1 activities.
In C. elegans, reduction of CLK-1 activity is also translated in an increase of lifespan. Lifespan is a cumulative phenotype that integrates variations in worm physiology, and that is sensitive to environmental cues, and to genetic mutations. As such, conditions that extend lifespan are often considered as beneficial. For example, a favourable repair vs. damage balance would manifest itself by increasing lifespan (Hekimi et al. 2001).
Age-related disorders
Age-related diseases refer to diseases for which the incidence and/or prevalence increase with age. These diseases include by example, cancers, cerebrovascular disease (stroke), neurodegenerative diseases, pneumonia, respiratory diseases, arthritis, heart diseases, diabetes, hearing impairment, vision impairment and kidney disease.
CLK-1 activity and age-related disorders
Inhibition of CLK-1 activity has similar lifespan-lengthening effects in mice and in nematodes. This strongly shows a causal link between CLK-1 biological activity and the physiological aging process. It is generally accepted that physiological aging is a risk factor for numerous diseases, the so-called age-dependent diseases. Many of these diseases are lethal and limit lifespan. It has been shown that when the level of mouse CLK1 (mCLK1) protein is reduced by half in mclk1 +/- heterozygotes (Levavasseur et al., 2001), it increases the mean, median and the maximum lifespan of these animals in three different experiments. Each experiment was carried out in a different genetic background (129SV/J, C57BL/6 and 129SV/J x Balb/c) (Liu et al., 2005). Animals in these backgrounds are known to die from a variety of age-dependent diseases, whose pattern is different in each background (Blackwell et al., 1995; Cosgrove et al., 1978; Frith et al., 1983; Smith et al., 1973). In all three experiments most of the wild type animals and the mutant animals die within a relatively short period of time, typical of the genotype (mclk1 +/+ or mclk1 +/-) (Liu et al., 2005). The fact that mclki reduction acts in different genetic background, and that maximum and median lifespans are increased indicates that all or most causes of lethality must have been partially suppressed by this reduction. Indeed, when a single cause of lifespan is affected by a genetic manipulation, such as cancer, lifespan lengthens only minimally or not at all (Matheu et al., 2004; Miller, 2005). In contrast, manipulations, such as caloric restriction and reduction of growth hormone signaling, which are known to slow down physiological aging, reduce the incidence of age-dependent diseases (Bartke, 2005; Berrigan et al., 2002; Hursting et al., 2004). Moreover, animals in which the aging process has been accelerated demonstrate increase prevalence and early onset of disease (Fenton et al., 2004; Takeda et al., 1997). These observations, together with the evolutionary conservation of the effect of c//c- 1/mclk1 on lifespan (Liu et al., 2005), indicate that reducing mCLK1 activity slows down physiological aging, and therefore that inhibition of CLK-1 can delay the onset and severity of age-dependent diseases. It is well recognized that slowing down physiological aging is the best way to reduce the incidence and severity of a variety of diseases (Finkel, 2005; Miller, 2005).
Thus, inhibiting CLK-1 by using chemicals that are useful as CLK-1 inhibitors leads to the prevention or reduction of age-related diseases. Quinoline derivatives
Compounds of the type of quinoline derivatives are disclosed in CA 2.493.536 A1 , US 2006/0074104 A1 and WO 2004/087160 A1 for use in the treatment, amelioration and/or prophylaxis of neurological conditions in particular those associated with or facilitated by oxidative stress. The neurological disorders contemplated include any condition leading to cognitive impairment such as pre- or mild cognitive impairment or memory loss.
Although the above mentioned quinoline derivatives have been shown to be useful oxidative stress regulators, they are described as exerting their therapeutic effects principally through metal chelation. There is no prior art reference known to date showing that similar compounds could be used to prevent and/or treat age-related disorders for which the inhibition of CLK- is beneficial.
In this context, the present inventors have developed a new family of compounds which are active to inhibit CLK-1. Such compounds are active on age-related disorders as well as on ischemia-reperfusion injury and inflammation disorders.
SUMMARY
Therefore, an object of the present invention is to provide new quinoline derivatives of formula A, which have CLK-1 inhibitory activity.
More specifically, the present invention relates to the compounds of formula A as defined below, or their pharmaceutically acceptable salts or their pro-drugs:
Figure imgf000006_0001
wherein X = H, methyl or halogen and the substituents Ri and R2 are defined as follows:
a) when R2 is hydrogen, then Ri is H or halogen, or R1 is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or R1 is -CH(R3)NR4R5 with ■ R4 = H or C1-C4 alkyl;
Figure imgf000006_0002
and Re is selected from the group consiting of: C1-Ce alkyl, Cs-Cβ aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and ■ R3 is selected from the group consisting of:
H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, 4- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, 4- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1 H-pyrazol-1-yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl,
3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, 4- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, 1 H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
or Ri is — c — N — R7 and R7 is -(ChMn-Rs where Rs is a Cs-Cε aryl optionally
Il o substituted by one or two methoxyl groups, and n= 0 or 1 ; or
b) when X and Ri both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl;
with the proviso that each of the compounds of formula A is not one of the compounds identified in Annex 1.
The compounds of Formula A of the invention when comprising at least one asymmetric centre are either in the form of one of their optically active isomers like enantiomers or a mixture thereof including for example the racemate. The invention also relates to a method for inhibiting CLK-1 activity in a cell which comprises the following steps: a) providing a cell wherein CLK-1 activity needs to be inhibited, b) contacting the cell with a compound of formula (B) as defined below, or a pharmaceutically acceptable salts or a pro-drug thereof:
Figure imgf000008_0001
wherein X, Ri and R2 have the same definition than previously mentioned; the compound of formula (B), when comprising at least one asymmetric centre is in the form of one of its optically active isomer or a mixture thereof; and c) determining the CLK-1 activity in the cell.
The invention also relates to a method for the prophylaxis and/or treatment of a disorder or its associated symptoms for which inhibition of CLK-1 is beneficial, in animal, which comprises the following steps: a) identifying an animal having a disorder for which inhibition of CLK-1 is beneficial; and b) administering to the animal a compound of formula (B), a pharmaceutically acceptable salts or a pro-drug thereof.
The invention also relates to a pharmaceutical composition comprising a compound of formula A of the invention, a pharmaceutically acceptable salt or a pro-drug thereof, and at least one pharmaceutically acceptable carrier. The invention further relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula A or B as defined hereinabove, a pharmaceutically acceptable salt or a pro-drug thereof, and at least one pharmaceutically acceptable carrier, to effectively reduce and/or inhibit totally or partially CLK-1 activity.
BRIEF DESCRIPTION OF THE FIGURES Figure 1: HPLC profiles showing the induction of DMQ in quinone profiles after treatment with a CLK-1 inhibitor. Mouse macrophages (RAW264.7) were treated with a) an inactive compound or b) the active compound 135 (1μM) for 24hr, followed by cell lysis and hexane/ethanol extraction of quinones. Samples were run with methanol/ethanol gradients on HPLC with UV detection to determine levels of ubiquinone (Q) and its precursor, demethoxyubiquinone (DMQ).
Figure 2: HPLC profiles showing relative quinone peaks in RAW264.7 cells treated with a) DMSO or b) 10 μM of Compound 7 (Q: Ubiquinone, DMQ: Demethoxyubiquinone).
Figure 3: HPLC profiles showing relative quinone peaks in RAW264.7 cells treated with a) 1 μM or b) 3 μM of Compound 23 (Q: Ubiquinone, DMQ: Demethoxyubiquinone).
Figure 4: HPLC profiles showing relative quinone peaks in RAW264.7 cells treated with a) 5 μM or b) 10 μM of Compound 69 (Q: Ubiquinone, DMQ: Demethoxyubiquinone).
Figure 5: HPLC profiles showing relative quinone peaks in RAW264.7 cells treated with a) 5 μM or b) 10 μM of Compound 47 (Q: Ubiquinone, DMQ: Demethoxyubiquinone).
Figure 6: HPLC profiles showing relative quinone peaks in RAW264.7 cells treated with a) 5 μM or b) 10 μM of Compound 53 (Q: Ubiquinone, DMQ: Demethoxyubiquinone). Figure 7: Specificity of CLK-1 inhibitor. Effect of Compound 7 on a) mouse CLK-1 (JF496-A77c//c7 testing system), and b) the bacterial DMQ hydroxylase UbiF (JF496-ub/F testing system). JF496 bacteria expressing a) mouse CLK-1 and b) the bacterial DMQ-hydroxylase UbiF were treated with Compound 7 for 5h. Quinones were extracted and analyzed using HPLC. The HPLC trace shows that CLK-1 activity is inhibited by 7, as ubiquinone (Q) synthesis is lowered with a concomitant increase of demethoxyubiquinone (DMQ). In contrast to that, UbiF activity is not affected, and Q is the major quinone species detected. This shows that Compound 7 has a specific CLK-1 inhibition activity.
Figure 8: Specificity of CLK-1 inhibitor. Effect of Compound 52 on a) mouse CLK- 1 (JF496-/77C//C/ testing system), and b) the bacterial DMQ hydroxylase UbiF (JF496-t/b/F testing system). JF496 bacteria expressing a) mouse CLK-1 and b) the bacterial DMQ-hydroxylase UbiF were treated with Compound 52 for 5hrs. Quinones were extracted and analyzed using HPLC. The HPLC trace shows that CLK-1 activity is inhibited by 52, as ubiquinone (Q) synthesis is lowered with a concomitant increase of demethoxyubiquinone (DMQ). In contrast to that, UbiF activity is not affected, and Q is the major quinone species detected. This shows that Compound 52 has a CLK-1 -specific inhibition activity.
Figure 9: ROS lowering effect of CLK-1 inhibitors (7 and 135). (A) table showing quinone profiles for CLK-1 inhibitor in mouse macrophage cells; (B) cells pre- incubated with CLK-1 inhibitors were treated with the fluorescence marker DCF, which produces green fluorescence when oxidized by cellular ROS. H2O2 is used to induce ROS in cells, and the reduction observed with CLK-1 inhibitors is shown as percentage relative to control; and (C) COMET assay whereby fluorescent tails of DNA leaking from cell nuclei are used to measure DNA damage induced by ROS. Again H2O2 was used to induce ROS, and data are expressed as percentage of cells with tails compared to control. The reduction of cells showing comets (% of total cells) in the presence of CLK-1 inhibitors is indicated. Figure 10: Effects of Compounds 7 or 52 on serum creatinine levels in a rat model of ischemia-reperfusion.
Figure 11: Effects of Compounds 7 or 52 on Blood Urea Nitrogen (BUN) levels in a rat model of ischemia-reperfusion.
Figure 12: Effects of Compounds 7 or 52 on creatinine clearance in a rat model of ischemia-reperfusion.
Figure 13: Effects of Compounds 7 or 52 on urine protein concentration (g/L) in a rat model of ischemia reperfusion.
Figure 14: Effects of Compound 7 on lipoxygenase (LPO) levels in rats with lipopolysaccharide (LPS) induced lung injuriy.
Figure 15: Effects of Compound 7 on protein contents in rats with LPS induced lung injury.
Figure 16: Effects of Compound 7 on total cells and neutrophils levels in rats with LPS induced lung injury.
Figure 17: Effects of Compound 7 on TNF-α levels in rats with LPS induced lung injury.
Figure 18: Effects of a treatment with Compound 138 in various dosages on soluble Aβ1-40 determined by ELISA in hAPP751SL transgenic mice in the TBS fraction.
Figure 19: Effects of a treatment with Compound 138 in various dosages on soluble AB1-42 determined by ELISA in hAPP751SL transgenic mice in the TBS fraction. Figure 20: Effects of a treatment with Compound 138 in various dosages on soluble Aβi-40 determined by ELISA in hAPP751SL transgenic mice in the Triton X-100 fraction.
Figure 21: Effects of a treatment with Compound 138 in various dosages on soluble AB1-42 determined by ELISA in hAPP751SL transgenic mice in the Triton X- 100 fraction.
Figure 22: Effects of a treatment with Compound 138 in various dosages on bound AB1-40 determined by ELISA in hAPP751SL transgenic mice in the FA fraction.
Figure 23: Effects of a treatment with Compound 138 in various dosages on bound AB1-42 determined by ELISA in hAPP751SL transgenic mice in the FA fraction.
DETAILED DESCRIPTION OF THE INVENTION The present invention has yielded the unexpected discovery of a new class of compounds consisting of quinoline derivatives of formula A defined below which present CLK-1 inhibitory activity.
Definitions As used herein, a pharmaceutically acceptable salt is meant to include any salt of the compounds of the invention that are suitable for use in contact with the tissues of human and lower animals without undue toxicity, irritation, or allergic response and are commensurate with a reasonable risk/benefit ratio. Pharmaceutically acceptable salts are well known in the art (Pharmaceutical Salts Properties, Selection, and Use, Stahl, P. Heinrich / Wermuth, Camille G. (eds.), 2002. Monograph - ISBN 3-906390-26-8 - Verlag Helvetica Chimica Acta, Zurich). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately, by reacting a free basic function of the compounds of the present invention with a suitable acid or by reacting a free acidic function of the compounds of the invention with a suitable base such as, but not limited to, hydroxide, carbonate or bicarbonate of a pharmacologically acceptable metal cation. Pharmaceutically acceptable acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfonate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphtalenesulfonate, oxalate, pramoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, nitrate, sulfate, bisulfate, phosphate, acid phosphate salts, glutamate, bicarbonate, p-toluensulfonate and undecanoate. Also the basic nitrogen-containing groups can be quaternized with such agents as alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides and iodides; and arylalkyl halides such as benzyl and phenylethyl bromides. Pharmaceutically acceptable salts also include, but are not limited to, cations based on alkali metals or alkaline earth metals such as aluminum, calcium, lithium, magnesium, potassium, sodium salts and the like. As used herein, a pro-drug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the parent compound, for example, by hydrolysis in blood. To produce a pro-drug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The pro-drug may be designed to alter the metabolic stability or the transport characteristics of a drug (including improvement of bioavailability), to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, could design pro-drugs of the compound. The present invention also contemplates metabolites formed by in vivo transformation of compounds having formula A or B. The term metabolite refers to compounds formed by in vivo biotransformation of compounds having formula A or B by oxidation, reduction, hydrolysis, or conjugation.
As used herein, the term alkyl refers to the saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, and cycloalkyl (alicyclic) groups.
The term aryl as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms (S, N, O), for example, unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
As used herein, the term halogen designates F, Cl, Br or I.
As used herein, the term optically active isomer designates any isomeric form of a claimed compound such as but not limited to an enantiomer, a diastereoisomer, a racemate or a mixture thereof.
By pharmaceutically acceptable carrier, it is meant a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients of the composition, namely the compound of formula A, and which is not toxic to the host or patient. Furthermore, the carrier is advantageously a compound with minimum probability of being rejected by the immune system of the subject being treated. Suitable carriers are of common knowledge to one skilled in the art and will not be further detailed.
As used herein, the term treatment refers to a process by which a disorder or its associated symptoms, for which inhibition of CLK-1 is beneficial, are alleviated or completely eliminated. For instance, one may understand that such treatment of the disorder or its associated symptoms may be alleviated or completely eliminated by, for instance, giving a compound of the present invention in appropriate formulation at appropriate doses to patients with high risk of/or early stages of disease.
As used herein, the term prophylaxis refers to a process by which a disorder, or its associated symptoms, including but not limited to an age-related disorder, is obstructed, delayed or prevented.
As used herein, the expression a disorder for which inhibition of CLK-1 is beneficial refers to a disorder or its associated symptoms for which inhibition of CLK-1 is known or anticipated to be beneficial. More particularly, this expression refers to a disorder or its associated symptoms is alleviated or eliminated upon inhibition of CLK-1. Even more particularly, this expression refers to, but is not limited to, inflammation disorders, disorders caused by or exacerbated by oxidative stress and/or free radical-induced damage, including ROS mediated diseases, such as hypoxia / reoxygenation injury and ischemia / reperfusion injury. This expression also encompasses age-related disorders.
As used herein, the expression an age-related disorder refers to a disorder for which the incidence and/or prevalence increase with age, including, without being limited to, cardiovascular diseases, such as atherosclerosis, coronary artery disease and stroke; peripheral vascular disease; metabolic disorders such as Type Il diabetes, dyslipidemia, and hypertriglyceridemia; cancer, such as skin cancer, papillomas and age-dependent cancers; ischemia / reperfusion injury, such as renal, heart, cerebral ischemia and radiocontrast-induced nephropathy; inflammation; neurodegenerative disorders and dementia such as Alzheimer's disease, Parkinson's disease, Huntington's disease, memory disorders, and psychosis; Bladder and kidney disorders such as nephritis, nephropathy, end- stage renal disease (ESRD); Diabetes complications such as neuropathy, impaired wound healing, and retinopathy; eyes disorders such as age-related macular degeneration, dry-eye disease, glaucoma, retinitis pigmentosa, and cataracts; lung and respiratory disorders such as chronic obstructive pulmonary disease (COPD), and idiopathic pumunory fibrosis (IPF); musculoskeletal disorders such as inflammatory arthritis, gout, and osteoporosis; and skin conditions such as skin cancer, and skin ageing like photoaging.
As used herein the term inflammation is meant to intend a localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute or wall off both the injurious agent and the injured tissue, characterized in the acute form by the classical sequence of pain, heat, redness, swelling, and loss of function, and histologically involving a complex series of events, including dilatation of the arterioles, capillaries, and venules with increased permeability and blood flow, exudation of fluids including plasma proteins, and leukocyte migration into the inflammatory focus. It is also characterized by massive release of TNF-α.
As used herein the expression ischemia / reperfusion injury refers to the condition suffered by tissues and organs when deprived of blood flow, mostly due to the effects of inadequate nutrient and oxygen. Reperfusion injury refers to the tissue damage inflicted when blood flow is restored after an ischemic period of generally more than about ten minutes. Ischemia and reperfusion can cause serious or fatal damage to afflicted tissues.
The quinoline derivatives according to the present invention are the compounds having the following general formula (A), or their pharmaceutically acceptable salts or their pro-drugs:
Figure imgf000016_0001
wherein X = H, methyl or halogen, and the substituents Ri and R2 are defined as follows:
a) when R2 is hydrogen, then Ri is H or halogen, or R1 is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or Ri is -CH(R3)NR4R5 with ■ R4 = H or Ci-C4 alkyl;
Figure imgf000017_0001
and R6 is selected from the group consiting of: Ci-C6 alkyl, C5-C6 aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and
■ R3 is selected from the group consisting of: H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, A- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, A- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, A- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1 H-pyrazol-1-yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl, 3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, A- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, 1 H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
LJ or Ri is — c — N — R7 and R7 is -(CH2)n-Rs where R8 is a C5-C6 aryl
Il o optionally substituted by one or two methoxyl groups, and n= 0 or 1 ; or b) when X and Ri both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl;
with the proviso that each of the compounds of formula (A) is not one of the compounds identified in Annex 1.
The compounds of Formula (A) of the invention comprising at least one asymmetric centre are either in the form of one of their enantiomers or a mixture thereof.
More preferably, the compounds of formula (A) are the compounds wherein R4 is
H or methyl, R6 is selected from methyl, ethyl, n-propyl, /-propyl, n-butyl, /-butyl, ter-butyl, phenyl, benzyl, dimethylaminomethyl, phenylethyl, cyclohexyl, diphenylmethyl, pyridin-2-yl and pyridin-3-yl, the other substituents having the same meaning as previously defined.
According to another preferred embodiment, the compounds of formula (A) are chosen in the group consisting of:
N-((3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl)acetamide 2,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)benzamide 3,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-N- methylacetamide 8,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-2,2- diphenylacetamide 11, N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-2- phenylacetamide Λ2,
7-(amino(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-ol 13, N-(1-(5-chloro-8-hydroxyquinolin-7-yl)-2-phenylethyl)acetamide 14, N-((2-butyl-1H-imidazol-4-yl)(5-chloro-8-hydroxyquinolin-7-yl)methyl)acetamide
15,
N-((5-chloro-8-hydroxyquinolin-7-yl)(2-methoxypyridin-3-yl)methyl)acetamide 16,
N-((5-chloro-8-hydroxyquinolin-7-yl)(quinolin-4-yl)methyl)acetamide 17, N-((5-chloro-8-hydroxyquinolin-7-yl)(2-morpholin-4-ylpyridin-3-yl)methyl)- acetamide 18,
N-((5-chloro-8-hydroxyquinolin-7-yl)(quinolin-3-yl)methyl)acetamide 19, 4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl) methylbenzoate
21,
3-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl) methylbenzoate
22,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-cyanophenyl)methyl)acetamide 23, N-((5-chloro-8-hydroxyquinolin-7-yl)(4-pyridin-2-ylphenyl)methyl)acetamide
24, N-((5-chloro-8-hydroxyquinolin-7-yl)(4-(1H-pyrazol-1-yl)phenyl)methyl)acetamide
25,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-hydroxyphenyl)methyl)acetamide 26, N-((5-chloro-8-hydroxyquinolin-7-yl)(3-hydroxyphenyl)methyl)acetamide 27.
N-((5-chloro-8-hydroxyquinolin-7-yl)(6-methoxypyridin-3-yl)methyl)acetamide 28,
N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-2-yl)methyl)acetamide 29,
N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-4-yl)methyl)acetamide 30, N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-4-yl)methyl)acetamidehydrochloride
31,
N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-3-yl)methyl)acetamide 32,
N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-3-yl)methyl)acetamidehydrochloride
33, N-((5-chloro-8 hydroxyquinolin-7-yl)(8-hydroxyquinolin-2-yl)methyl)acetamide
34,
N-((5-chloro-8-hydroxyquinolin-7-yl)(6-morpholin-4-ylpyridin-3-yl)methyl)- acetamide 35,
N-((5-chloro-8-hydroxyquinolin-7-yl)(1 H-indol-3-yl)methyl)acetamicle 36, N-((5-chloro-8-hydroxyquinolin-7-yl)(1H-indol-4-yl)methyl)acetamide 37, N-((5-chloro-8-hydroxyquinolin-7-yl)(1 H-indol-7-yl)methyl)acetamide 38, 4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N,N-dimethyl- benzamide 39,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-(morpholin-4-ylcarbonyl)phenyl)methyl)- acetamide 40,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)pyridine-2- carboxamide 41.
4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N-(3-methoxypropyl)- benzamide 44, 4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N-methylbenzamide
45, N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-N,N-dimethyl- glycinamide 46,
N-(1-(5-chloro-8-hydroxyquinolin-7-yl)ethyl)acetamide 47,
N-((8-hydroxyquinolin-7-yl)methyl)acetamide 48, N-(2-furylmethyl)-8-hydroxyquinolin-7-yl carboxamide 53, N-(2-thienylmethyl)-8-hydroxyquinolin-7-yl carboxamide 54, N-(2-methoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 55, 5-chloro-7-(3-hydroxyphenyl)quinolin-8-ol 57, 5-chloro-7-(4-hydroxyphenyl)quinolin-8-ol 58, N-(2-(5-chloro-8-hydroxyquinolin-7-yl)phenyl)acetamide 59, 5-chloro-7-(2-methoxypyridin-3-yl)quinolin-8-ol 6_1,
5-chloro-7-pyridin-4-ylquinolin-8-ol hydrochloride 62, N-(3,4-dimethoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 63, N-(3-methoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 64, N-(3-methoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 65, N-(3,4-dimethoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 66, N-(2-methoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 67, N-(pyridin-3-ylmethyl)-8-hydroxyquinolin-7-yl carboxamide 68, N-(8-hydroxyquinolin-2-yl)pyridin-2-yl carboxamide 69, N-(8-hydroxyquinolin-2-yl)-2-pyridin-2-yl acetamide 70, 2-(3-hydroxyphenyl)-N-(8-hydroxyquinolin-2-yl)acetamide 71, 2-(4-hydroxyphenyl)-N-(8-hydroxyquinolin-2-yl)acetamide 72, N-(8-hydroxyquinolin-2-yl)-2-(2-thienyl)acetamide 73. 2-(((3-methoxybenzyl)amino)methyl)quinolin-8-ol 74, 2-(((3-methoxyphenyl)amino)methyl)quinolin-8-ol 75. 2-(((2-thienylmethyl)amino)methyl)quinolin-8-ol 76,
2-(((2-methoxyphenyl)amino)methyl)quinolin-8-ol 77, N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]acetamide 78, N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]acetamide 79, N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]acetamide 80, N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]butanamide 8J., N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]butanamide 82, N-[(8-hydroxyquinolin-7-yl)(4-methylphenyl)methyl]-3-phenylpropanamide
92, N-[(2-chlorophenyl)(8-hydroxyquinolin-7-yl)methyl]-3-phenylpropanamide
93, N-^^-dichlorophenyOCδ-hydroxyquinolin^-yOmethyll-S-methylbutanamide 94,
N-[(8-hydroxyquinolin-7-yl)(phenyl)methyl]-3-methylbutanamide 110, N-[(8-hydroxyquinolin-7-yl)(4-methoxyphenyl)methyl]-3-phenylpropanamide
111. N-[(3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl]-3-methylbutanamide 112,
N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]-3-methylbutanamide 113, N-[(4-chlorophenyl)(8-hydroxyquinolin-7-yl)methyl]cyclohexanecarboxamide
121. N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]propanamide 122, N-[(3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl]-3-phenylpropanamide
124, N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]pentanamide 125, N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]pentanamide 126,
N-[[4-(diethylamino)phenyl](8-hydroxyquinolin-7-yl)methyl]pentanamide 127, N-[(8-hydroxyquinolin-7-yl)(4-methoxyphenyl)methyl]cyclohexanecarboxamide
128, N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]butanamide 129.
N-[(8-hydroxyquinolin-7-yl)(4-methylphenyl)methyl]cyclohexanecarboxamide
130. and N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]cyclohexanecarboxamide 131.
According to a preferred embodiment, the pro-drugs of the compounds of formula (A) of the invention correspond to the compounds wherein the hydroxyl group in position 8 of the quinoline moiety is replaced by one of the following groups:
— (T O— F-OH or — O^^O— P-O-M+
OH 0-M+ wherein M is selected from Li, Na, Ca and K.
According to another preferred embodiment, the above defined dihydrogen phosphate pro-drugs are in the form of their hydrochloride or dihydrochloride salts.
The preferred pro-drugs according to the invention are the following:
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 5, ((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 6,
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 136, ((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 137.
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate dihydrochloride 138, and ((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate hydrochloride 139.
As previously mentioned, asymmetric or chiral centers may exist in the compounds of the present invention. The present invention contemplates the various optically active isomers of the compounds of formula (A) and mixtures thereof. As an example, an individual enantiomer of a compound of the present invention is prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of mixtures of enantiomeric compounds followed by resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by a) attachment of a racemic mixture of enantiomers to a chiral auxiliary, separation of the resulting diasteroisomers by recrystalization or chromatography and liberation of the optically pure product from auxiliary or b) direct separation of the mixture of optical enantiomers on the chiral chromatographic columns.
The invention also relates to a method for inhibiting CLK-1 activity in a cell which comprises the following steps: a) providing a cell wherein CLK-1 activity needs to be inhibited, b) contacting the cell with a compound of formula (B):
Figure imgf000023_0001
wherein X = H, methyl or halogen and the substituents Ri and R2 are defined as follows: i) when R2 is hydrogen, then Ri is H or halogen, or Ri is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or Ri is -CH(R3)NR4R5 with
■ R4 = H or CrC4 alkyl;
Figure imgf000024_0001
and R6 is selected from the group consiting of: Ci-C6 alkyl, C5-C6 aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and
■ R3 is selected from the group consisting of:
H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, A- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, A- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl,
4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, A- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1H-pyrazol-1-yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl,
3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, A- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1 H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, 1 H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
LJ or Ri is — c — N — R7 and R7 is -(CH2)n-R8 where Rs is a C5-C6 aryl optionally
Il o substituted by one or two methoxyl groups, and n= 0 or 1 ; or ii) when X and Ri both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl; the compound of formula (B), when comprising at least one asymmetric centre, being in the form of one of its enantiomers or a mixture thereof; and c) determining the CLK-1 activity in the cell.
Advantageously, the compound of formula (B) used for inhibiting CLK-1 activity in a cell is selected from the group of compounds identified in Table 1.
Even more advantageously, the compound of formula (B) used for inhibiting CLK-
1 activity in a cell is selected from the group of:
N-[(5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl]acetamide
Z, 5-chloro-7-iodoquinolin-8-ol 52,
5-chloro-7-(2-hydroxyphenyl)quinolin-8-ol 56,
5-chloro-7-(4-(dimethylamino)phenyl)quinolin-8-ol 60, and
N-[(5-chloro-8-hydroxyquinolin-7-yl)(2-furyl)methyl]acetamide 135.
The invention further relates to a method for the prophylaxis and/or treatment of a disorder or its associated symptoms for which inhibition of CLK-1 is beneficial, in animals, which comprises the following steps: a) identifying an animal having a disorder for which inhibition of CLK-1 is beneficial; and b) administering to the animal a compound of formula (B) as defined above, a pharmaceutically acceptable salt or a pro-drug thereof.
Advantageously, the compound of formula (B), its salt or its pro-drug or an optically active isomer, used for the prophylaxis and/or treatment in animal, of a disorder or its associated symptoms for which inhibition of CLK-1 is beneficial, is selected from the group of compounds identified in Table 1. Even more advantageously, the compound of formula (B), its salt or its pro-drug, used for the prophylaxis and/or treatment in animal, of a disorder or its associated symptoms for which inhibition of CLK-1 is beneficial, is selected from the group of: ((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 5,
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 6,
N-[(5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl]acetamide 7,
N-[(5-chloro-8-hydroxyquinolin-7-yl)(2-furyl)methyl]acetamide 135.
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyldihydrogen phosphate 136.
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyldisodium phosphate 137.
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate dihydrochloride 138, and
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate hydrochloride 139.
According to a preferred embodiment the disorder for which inhibition of CLK-1 is beneficial is an ischemia / reperfusion injury, an inflammation or a neurodegenerative disorder or dementia.
More preferably, but not limited to, the ischemia / reperfusion injury is a renal, heart, myocardium, lung, brain, or spinal cord ischemia / reperfusion injury.
According to a preferred embodiment, the inflammation is a lung inflammation, a liver inflammation or an inflammation of any other organ and any clinical indications in which such inflammation is promoted.
According to another embodiment, a neurodegenerative disorder is Alzheimer's disease. Since it has been previously shown that inhibiting CLK-1 activity in mice slows down physiological aging (Liu et al., 2005), this indicates that the inhibition of CLK-1 activity in animals can delay the onset and severity of age-dependent diseases.
In this context, the present inventors have now disclosed that the compounds of formula (B) of the present invention are also effective to treat or prevent an age- related disorder or its associated symptoms. Thus, more preferably, the invention relates to a method for the prophylaxis and/or treatment of an age-related disorder or its associated symptoms, in animals, which comprises the following steps: a) identifying an animal having an age-related disorder; and b) administering to the animal a compound of formula (B), a pharmaceutically acceptable salt or a pro-drug thereof; with the proviso that said compound of formula (B) is not one of the following compounds, a pharmaceutically acceptable salt or a pro-drug thereof: 5-chloro-7-iodoquinolin-8-ol 52, 5-chloro-7-(2-hydroxyphenyl)quinolin-8-ol 56, or 5-chloro-7-(4-(dimethylamino)phenyl)quinolin-8-ol 60.
According to a preferred embodiment the above method is used for the prophylaxis and/or treatment of an age-related disorder or its associated symptoms selected from the group consisting of: cardiovascular diseases, such as atherosclerosis, coronary artery disease and stroke; peripheral vascular disease; metabolic disorders such as Type Il diabetes, dyslipidemia, and hypertriglyceridemia; cancer, such as skin cancer, papillomas and age-dependent cancers; ischemia / reperfusion injury, such as renal, heart, cerebral ischemia and radiocontrast-induced nephropathy; inflammation; neurodegenerative disorders and dementia such as Alzheimer's disease, Parkinson's disease, Huntington's disease, memory disorders, and psychosis; Bladder and kidney disorders such as nephritis, nephropathy, end-stage renal disease (ESRD); Diabetes complications such as neuropathy, impaired wound healing, and retinopathy; eyes disorders such as age-related macular degeneration, dry-eye disease, glaucoma, retinitis pigmentosa, and cataracts;, lung and respiratory disorders such as chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF); musculoskeletal disorders such as inflammatory arthritis, gout, and osteoporosis; and skin conditions, both cosmetic and dermatologic, including skin ageing like photoaging and skin cancer.
The animal to be treated with the compound of formula (B) of the invention may be a human or any animal of commercial or domestic value including but not limited to cow, horse, pig, guinea pig, hamster goat, rabbit, chicken, dog, cat and birds. Even more preferably the animal treated is a human.
The invention also relates to a pharmaceutical composition comprising a compound of formula (A), a pharmaceutically acceptable salt or a pro-drug thereof as defined herein above, or any of the pro-drugs 5, 6, 136, 137, 138 and 139 defined above, formulated together with one or more non-toxic pharmaceutically acceptable carrier.
The present inventors have also shown that the compounds of formula (A) or (B) of the invention have a CLK-1 inhibitory activity.
Accordingly, the invention also relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula (A) or (B), a pharmaceutically acceptable salt or a pro-drug thereof, to effectively reduce and/or inhibit CLK-1 activity.
The invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable amount of any one of the pro-drug 5, 6, 136, 137, 138 and 139 to effectively reduce and/or inhibit CLK-1 activity. According to a preferred embodiment, the compounds of the present invention could be combined with another drug known to be active to treat an age-related disorder or its associated symptoms, such as for example combination with a statin for the treatment of dyslipidemia.
The pharmaceutical compositions according to the invention may be specially formulated for oral administration in solid or liquid form, for parenteral injection, for rectal administration or topical administration. The pharmaceutical compositions of this invention can be administrated to human and other animals orally, rectally, parenterally, intra cisternally, intravaginally, intraperitoneal^, topically (as by powder, cream, ointment, or drops), bucally, or as an oral or nasal spray. These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and gelatine.
In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug thus depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or fillers or extenders such as starches, lactose, sucrose, glucose, mannitol.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.
The compounds of the invention may also be administered in the form of liposomes deriving from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in aqueous medium. Any non-toxic, physiologically acceptable and metabolizable liquid capable of forming liposomes can be used. The preferred lipids are the phospholipids and the phosphatidyl cholines, both natural and synthetic.
Dosage forms for topical administration of a compound of the invention include powders, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.
Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
The present invention will be more readily understood by referring to the following examples, with reference to the accompanying figures. These examples are illustrative of the wide range of applicability of the present invention and are not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.
EXAMPLES EXAMPLE 1 : Specificity of CLK-1 inhibitors
Demethoxyubiquinone (DMQ) hydroxylation is carried by CLK-1 in eukaryotes and by UbiF in eubacteria. CLK-1 inhibitor molecules were tested in a modified JF496 E. coli strain, which carries a mutation in the ubiF gene, and lacks DMQ hydroxylation activity. This strain is complemented either with mouse clk-1 gene, or with E. coli ubiF gene. This complementation is functional, and restores DMQ hydroxylation. As a consequence, the rescued strains are capable of synthesizing ubiquinone (Q), instead of accumulating DMQ. Test molecules are evaluated for specificity regarding Q synthesis inhibition, using the two modified strains.
Materials and methods
Bacterial strains and media
Escherichia coli strain JF496 (ubiF-) was transformed with plasmids containing the mouse clk-1 gene, or the E. coli ubiF gene. For general growth, a clone of each strain is grown overnight at 37°C in 5 ml LB supplemented with ampicillin
(50 μg/ml). The cultures are diluted 1/10 and OD6oo is measured. The culture is diluted to an OD60O of 0.03 in M9-LB (0.5/0.5:v/v). This mix consists of M9 medium supplemented with (1 mM MgSO4, 20 μM CaCI2, 0.5 μg/mL thiamine, 0.12% casamino acids, 40 μg/mL D-L-methionine, 100 μg/mL L-asparagine, trace metals, 0,5% glucose), and of LB medium. Media contain ampicillin (50 μg/mL) for plasmid selection. For HPLC quinone analysis, the JF496-ubiF and JF496- mclk-1 strains are diluted from an overnight preculture into M9-LB (0.5/0.5:v/v) to an OD6Oo of 0.03. A volume of 750 μl of bacteria is transferred to 15 ml tubes. The bacterial cultures are incubated for 5 hours at 370C with agitation.
Quinones extraction
Cells are harvested by centrifugation, washed with distilled water and centrifuged again. Pellets are frozen at -8O0C overnight. Cells are then thawed and resuspended in 1 ml sodium phosphate buffer (0,1 M, pH 7.0), and 50 ng Q6 is added as a quinone extraction control. To extract quinones, 1 ml ethanol is added and cells are vortexed 30 seconds. 1 ml hexane is subsequently added. Cells are then vortexed 2 minutes and centrifuged. The upper phase (hexane) is collected. The lower phase is extracted a second time and centrifuged. The upper phase is then collected and added to the previous extract. Upper phases are freeze-dried for approximately 1 hour and the pellet is kept at -800C. Pellets are resuspended in 300 μl of mobile phase before HPLC analysis.
HPLC method
Samples are analyzed using a Beckman System Gold HPLC with a photo diode array detector and a Beckman Ultrasphere ODS (4.6mm x 25 cm) column. Data are analyzed using the 32 Karat software (Beckman). Quinones are separated using a mobile phase gradient of methanol-ethanol (beginning at 70% methanol-
30% ethanol, ending 6 minutes later at 30% methanol-70% ethanol), at a flow rate of 1 ml/min for 20 minutes. Demethoxyubiquinone (DMQ) and ubiquinone (Q) peaks are detected at 275 nm.
Results and short discussion on drugs specificity testing
JF496 bacteria expressing mouse CLK-1 and the bacterial DMQ-hydroxylase UbiF were treated with compound 7 (Figure 1), and with compound 52 (Figure 2). The quinone distributions resulting from these treatments were analyzed using HPLC. It appears that 7 and 52 can inhibit CLK-1 DMQ hydroxylase activity, as indicated by the accumulation of the DMQ precursor. In contrast to that, UbiF activity is not challenged by any of the compounds, since DMQ is not accumulating in treated cells. This differential effect is indicative of a specificity of action on CLK-1 compared to UbiF, even though these two enzymes carry an identical function which is DMQ hydroxylation. The effects seen on CLK-1 are not due to some non-specific activities of the compounds in E. coli, as both CLK-1 and UbiF are expressed in the same bacterial genetic background. EXAMPLE 2: Measurement of CLK-1 inhibition
To confirm that a positive compound identified in bacterial screening was a bona fide inhibitor of CLK-1 , we utilized an HPLC-based secondary assay to measure the quinone content of mammalian cells. CLK-1 is a demethoxyquinone (DMQ) hydroxylase that catalyzes the penultimate step in Q biosynthesis. Inhibition of CLK-1 leads to accumulation of the reaction precursor, DMQ. This assay measures the endogenous cellular levels of Q, and its precursor DMQ. In all cells tested the levels of DMQ present normally are negligible, so robust inhibition of CLK-1 leads to a profound change in the HPLC quinone profiles of treated cells.
Materials and Methods
Fresh dilutions of compounds that are identified from the bacterial screen were dissolved overnight in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. Dilutions of the compound (in DMSO) were added to wells of RAW264.7 cells, an Abelson's murine leukemia virus-transformed murine macrophage cell line. The cells were plated (1 x 105 cells per well) in Dulbecco's Modified Eagle's medium (supplemented with 10% Fetal Bovine serum, 1% Penicillin/Streptomycin, 100 μM Sodium Pyruvate) the day before compound treatment and left in a humidified CO2 incubator (5% CO2, 370C) overnight. Following addition of the compound, the plates were incubated for 24 hours, in humidified CO2 (5% CO2, 370C).
After 24 hours, the medium was aspirated from the wells; the cells were washed with PBS, before 500 μl RIPA buffer was added to each well (15 minutes, room temperature) with gentle rocking. After this time, samples were removed to a 1.5 ml microcentrifuge tube. 500 μl of HPLC grade ethanol, followed by 500 μl of HPLC grade hexane, was added to each tube, which was then vortexed vigorously for one minute, before being centrifuged (3000 x g, 15 minutes, room temperature). After centrifugation, 2 distinct layers could be seen in each tube. The top layer was carefully removed to a fresh microcentrifuge tube, capped with a needle perforated lid, and then subjected to freeze drying. Once the samples were dry, 320 μl of 70% HPLC grade methanol: 30% ethanol (v/v), was added to each tube. The tubes were then vortexed vigorously for 20 seconds, centrifuged for 10 seconds (14 000 x g), before the samples were transferred to a capped HPLC vial. Each sample was then subjected to HPLC analysis. The analysis was done by passing samples through a 250 mm C18 ODS column, using an ethanol: methanol gradient and quinones were detected at 275 nm, using a photodiode array detector. Visual inspection of chromatograms was used to determine the activity of various doses of each compound of interest.
Table 1 shows a list of compounds which have been determined to be active CLK-1 inhibitors.
Results and brief discussion of the inhibition of CLK-1 in mammalian cells
Shown in Figure 3 is a typical HPLC profile obtained from quinone extraction of a sample of RAW 264.7 mouse macrophage cells. The graph in the upper panel represents the quinone extracted from untreated macrophages, wherein the major species evident is ubiquinone-9, or Q. When we treat cells with a CLK-1 inhibitor we observe the appearance of a second quinine peak, which represents the accumulation of Q's precursor, DMQ. This profile represents cells treated with compound 135. Thus CLK-1 inhibitors are readily identified by a clear and simple visual analysis of HPLC profiles of cells treated with putative candidate inhibitors. A variety of human and mouse cell lines were examined, with no major difference between them other than variations in levels of endogenous ubiquinone.
This means that the only conceivable fashion by which we can detect a loss of Q in cells, with a corresponding accumulation of DMQ, is by inhibition of CLK-1. This secondary assay is thus extremely useful and very robust in its capacity to rapidly identify CLK-1 inhibitors. Figures 4-8 show the HPLC profiles of mouse macrophages treated with variable concentrations of compound 7, 23, 69, 47 or 53. Here we see the effects of increasing compound concentrations on the distribution of DMQ9 and Qg in treated cells. EXAMPLE 3: Measure the effect of CLK-1 inhibition
CLK-1 activity may have impacts on cellular ROS levels by regulating the cellular content of Q, which contributes to ROS production via the electron transport chain and ROS scavenging. Therefore, it is to be expected that inhibition of CLK- 1 will lead to alterations in cellular ROS levels. Additionally, one of the macromolecules that is targeted by ROS in cells is DNA, resulting in DNA damage. One might expect that DNA damage would also be reduced as a consequence of CLK-1 inhibition. We therefore used two assays to measure these factors in living cells, as outlined below.
Materials and Methods
ROS assay
DCF-DA (2,7-dichlorofluorescein diacetate) was used to assess cellular ROS levels. The lipophilic DCF-DA is transported across the cell membrane to the cytosol, and enzymatically converted to hydrophilic 2,7-dichlorofluorescein (DCFH) by cytosolic esterase(s). Cellular ROS oxidize DCFH to DCF, which is a fluorescent molecule. To evaluate the effect of CLK-1 inhibition on ROS production, firstly, mouse macrophage cells (RAW264.7) seeded (1 x 105 per well) in 96-well plates were treated with CLK-1 inhibitors for 24h. The cells were then incubated with 20 μM DCF-DA in Hanks' Balanced Salt Solution (HBSS) for 30 min at 37°C. The fluorescence from cellular DCF was then monitored using a fluorescence plate reader (excitation 488 nm and emission 520 nm). The protein amount in the tested wells was determined using a commercially available BCA protein assay kit. Cellular ROS levels were subsequently expressed as mean DCF fluorescence/mg protein from 3 replicates.
COMET assay
RAW264.7 cells were seeded (2.5 x 106 cells per well) in the presence/absence of a CLK-1 inhibitory compound in 12-well plates. After 24 hours, the cells were treated with 1 mM H2O2 for 1 hour, to induce ROS-mediated DNA damage. The cells were then washed twice in ice-cold PBS, before being scraped into 575 μl of ice cold PBS. 75μl of this mixture was removed and added to 750μl ice cold PBS. A 10μl aliquot was removed and mixed with 90μl of molten low-melting point agarose. 75 μl of this cell-agarose mix was pipetted onto a glass slide, which was placed flat at 40C, and shielded from the light for 15 minutes.
After this time, the slide was immersed in a pre-chilled lytic solution and left for 1 hour, at 40C, shielded from the light. Subsequently, the slide was removed and after removal of excess lytic solution, immersed in a freshly prepared alkaline solution (pH 13). The slide was then left for 1 hour, shielded from the light, at room temperature.
Excess alkaline solution was removed, before the slide was immersed for 5 minutes, at room temperature in 1x Tris-Borate/EDTA buffer (TBE). Again shielded from the light, the slide was placed in a 1x TBE-filled, electrophoretic apparatus and a voltage applied for 20 minutes. The slide was removed after 20 minutes and immersed, shielded from the light, in 70% ethanol, for 5 minutes, before being allowed to air dry at 370C until the agarose forms a thin layer on the slide.
Following this 370C incubation, 50 μl of diluted SYBR green is added to each dried agarose disk for 10 min, in the dark, before the dye is washed off by immersion in distilled water. Excess water is blotted off, before the slide was allowed to air dry, once again in the dark. Once completely dry, the slide is viewed on a fluorescence microscope and examined for the presence of DNA tails (comets) trailing the nuclei of cells. Data are usually represented as percentage of cells containing comets, having counted 10 fields.
Results and brief discussion of the effect of CLK-1 inhibitors on cellular ROS levels
Three panels showing the effect of archetype CLK-1 inhibitors, namely compounds 7 and 135, in quinone profiling, cellular ROS levels, and DNA damage are presented in Figure 9. These molecules were found to lower the induction of ROS by H2O2 in macrophages, with 7 being more potent (Panel B). These molecules have a quite striking effect in DNA damage induced by H2O2 in macrophages (Panel C), with 7 being extremely potent. These data underline that we are reducing the cellular levels of ROS and the subsequent damage incurred.
EXAMPLE 4: CLK-1 inhibtors are useful to treat kidney ischemia-reperfusion injury in the rat
The following study evaluates the utility of CLK-1 inhibitor administration for preventing and/or reducing reperfusion injuries in a model of renal ischemia.
Brief description of Experimental Methods
Adult (9 to 11 week-old) male Sprague-Dawley rats were anaesthetized and underwent right nephrectomy through a mid-line abdominal incision. Transient ischemia of the left renal artery was achieved by occlusion of the left renal artery and vein for 60 minutes. The treatment for all groups (Sham: no ischemia, received saline (0.9% NaCI), Naive: ischemia plus the vehicle, Treatment: ischemia plus compound 7 or 52) was performed once daily intraperitoneal^ (Lp.) for 15 consecutive days starting three days before the surgical intervention. Treatment group received compound 7 at 2.5 mg/kg/day Lp., (1 mg/ml), or compound 52 at 20 mg/kg/day (4 mg/ml). The renal functions were evaluated on Days 0, 3, 7, 11 and 14 through determination of Creatinine and Blood Urea Nitrogen (BUN) in serum and Creatinine and protein content in urine. Creatinine clearance (ml/min/100g) was calculated using the formula - CrCI = (uCr * uV) / (sCr x 1440 x weight). Urine protein concentration in 24 hr urine samples were determined using the method of Coomassi. Histopathologic changes in renal tissue (Hematoxylin and eosin; H&E stained slides) were analyzed by light microscopy for tubular epithelial cell necrosis, tubular dilation, proteinaceous casts, and medullar congestion as suggested by Racusen (2). The statistical analysis of the collected data was performed using One-way ANOVA followed by Turkey post-hoc test when indicated. The results with p < 0.05 were considered statistically significant. Brief description of ischemia-reperfusion injury Results
Renal function: Serum creatinine
The initial results demonstrated that the serum creatinine level in each group was not statistically different on day 0. The serum creatinine at day 3 to day 14, in groups treated with compound 7 or compound 52, was significantly lower than in group NAlVE. (Figure 10)
Renal function: Blood urea nitrogen (BUN) Initial results demonstrated that on day 0, BUN in each group was not statistically different. On day 3, 7, 11 and 14 BUN in groups treated with compounds 7 and 52 was significantly lower than in group NAΪVE (Figure 11).
Renal function: Creatinine clearance On day 0, creatinine clearance in each group was not statistically different. On days 3 and 7 creatinine clearance in group treated with compound 7 or compound 52 was significantly higher than in group NAΪVE. On days 11 and 14 creatinine clearance in group treated with compounds 7 or 52 was not statistically different compared with group NAΪVE, however was higher than in group NAΪVE on day 14 (Figure 12).
Renal function: Urine protein
On day 0, 24-hr urine protein in each group was not statistically different. On day 3, 24-hr urine protein in group treated with compounds 7 or 52 was significantly lower than in group NAΪVE. On day 7 and 14, 24-hr urine protein in group treated with compound 7 or 52 was not statistically different compared with group NAΪVE. On day 11 , 24-hr urine protein in group treated with compounds 7 or 52 was significantly lower than in group NAΪVE (P=0.033) (Figure 13). Brief discussion of the effect of CLK-1 inhibiotors on kidney ischemia- reprefusion injury
The renal function data (serum creatinine, blood urea nitrogen (BUN), creatinine clearance and urine protein) showed that once a day intraperitoneal treatment with compound 7 (2.5 mg/kg) and compound 52 (20 mg/kg) starting three days before the ischemic event and continued for 11 consecutive days effectively prevented, in a similar extent, the renal ischemia/reperfusion injury in one kidney rat model after 60-min ischemia when compared with the Naϊve control groups. In all cases, renal functions in the SHAM groups were not affected. The protective effect on several parameters of the renal function was particularly pronounced on days 3 and 7 after the ischemia/reperfusion.
EXAMPLE 5: Effects of CLK-1 inhibitors on lipopolysaccharide-induced acute lung injury
The following study evaluates the utility of CLK-1 inhibitors in an in vivo relevant rat model of acute lung inflammation elicited by systemic administration of lipopolysaccharide (LPS).
Brief description of Experimental Methods
Male Sprague-Dawley rats weighing 225 to 250 g (n=5 to 10 per group) were treated with lipopolysaccharide (LPS) in order to induce lung injury. Drug-treated rats received intraperitoneal^ (i.p.) Compound 7 or N-Acetyl-L-cysteine (NAC) as reference. Compound 7 was administered i.p. in vehicle containing DMA/PG/Tween 80 and water. Doses were given once a day, for three consecutive days, with the last dose administered 1 h before LPS exposure. Control rats received drug vehicles as indicated. Freshly prepared drugs were administered in a volume of 5 or 10 ml/kg or 5 ml/kg. Twenty-four hours after i.p. LPS or saline administration, the animals were sacrificed by an overdose of urethane and blood alveolar fluid (BALF) was collected for total and differential cell counts, LPO and TNF-α determination and protein contents. Brief description and discussion of LPS-induced lung injury results
The results showing the effects of Compound 7 (5 mg/kg or 10 mg/kg) given Lp., once a day for three days before LPS (6 mg/kg, Lp.) on total cell and neutrophil counts, protein and TNF-α content, LPO levels in BALF from rats exposed to saline (control) or LPS are shown in Figures 14 to 17. N-Acetylcysteine (NAC; 0.5 g/kg, Lp., once a day for three days before LPS) was given as reference compound. Briefly, Compound 7 effectively reduced the increase in BALF total cells and neutrophils to levels comparable to those observed in NAC-treated rats. This compound did not produce any effect on BALF total cell counts when given alone. Similarly, Compound 7 effectively reduced the increase in LPO in BALF to levels comparable to those observed in NAC-treated rats. Compound 7 failed to reduce the increase in BALF protein. Compound 7 (10 mg/kg) was effective to significantly reduce BALF TNF-α levels. Moreover, histological analysis (not shown) demonstrated that Compound 7 at 5 mg/kg, Lp. was effective to reduce the intensity of the interstitial pattern (alveolar wall is less thick in treated lungs) and the inflammatory response (lower number of inflammatory cells).
Altogether, these experiments demonstrated the utility of Compound 7 in reducing the BALF total and neutrophil counts, lipid hydroperoxide, and histological pulmonary lesions (not shown) in LPS-challenged rats. TNF-α level reduction was also observed with Compound 7 at 10 mg/kg, i.p. dose regimen.
As neutrophil influx and LPO are well known markers of inflammation for which
CLK-1 inhibitors are protective, the utility of the novel derivatives of quinoline for clinical indications in which such inflammation is promoted represents a particularly useful embodiment.
Example 6: Assessment of the effects of a treatment with Compound 138 on brain β-amyloid1-40 and β-amyloid1-42 levels of hAPP751SL transgenic mice. Animal: Male hAPPSL transgenic (tg) mice (C57BL/6 background) aged 5 months (± 2 weeks) at the starting date of the study. Transgenic hAPP751SL animals constitutively over-express human APP751 with the London (V717I) and the Swedish (K670M/N671 L) mutations under the regulatory control of the neuronal tissue specific murine-Thy-1 promoter. Due to the London mutation, high levels of β-amyloid1-42 are expressed all over the brain but mainly in cortex and hippocampus. High levels of β-amyloid1-42 are associated with amyloid plaque formation in the CNS at a much earlier age beginning at 3 months. Enhanced brain Aβi-42 levels as seen in this mouse model are associated with earlier plaque formation. Aβi-42 accelerates amyloid deposition and promotes formation of denser deposits; while Aβi-40 might have opposite effects. Therefore, the potential to influence the generation of this fatal amyloid peptide might be advantageous, in particular with regard to a possible treatment of Alzheimer's disease patients.
Treatment: Compound 138 was formulated in carboxymethyl cellulose (CMC) and administrated per os (p.o.) to animals once daily and twice daily (at the highest dose), respectively, for 60 consecutive days. Treatment doses were 5, 20, 50 and 2 x 50 mg/kg b.w./day, respectively or with vehicle (CMC).
Tissue Preparation: Transgenic mice were transcardially perfused with physiological (0.9%) saline until the whole blood was washed out; then brains were rapidly removed and the right hemisphere was immersion fixed in fresh 4% paraformaldehyde for one hour. After that the hemispheres were transferred to a 15% sucrose solution for cryprotection. On the next day, brains were frozen on dry ice and stored at -8O0C. The left brain hemisphere was immediately snap frozen on dry ice to determine β-amyloid1-40 and β-amyloid1-42 in four brain homogenate fractions.
Measurements: One brain hemisphere (including the bulbus olfactorius but without Cerebellum) of each animal was used for evaluation of brain Aβ1-40 and Aβi-42 in four (4) brain fractions which are TBS, Triton X-100 and FA. Aβ1-40 and AB1-42 was using high sensitivity ELISA kits (TK40HS™; TK42HS™) manufactured by The GENETICS Company, Switzerland.
Results:
Levels of Soluble Aβ1-40 and Aβ1-42
TBS extract fractions contain the water soluble AB1-40 and AB1-42 fraction of the brain tissue while in the Triton x-100 fraction smaller polymers like protofibrils are solubilized. The formic acid (FA) fractions contain the insoluble polymerised Aβ. Results are shown in Figures 18 to 23. Compound 138 significantly reduces Aβ1- 42 levels in the TBS (5 and 20mg/kg; Fig.19) and in the Triton x-100 fraction (5, 20 and 50mg/k; Fig.21). In the TBS fractions (Fig. 18 and 19) but not in the Triton fractions (Fig. 20 and 21) a similar but, however, not significant effect can be seen in terms of Aβ1-40 where all individual data points are very close together in the two Compound 138 groups.
In the FA fraction, mean Aβ1-42 values of all dosages are below that of the vehicle control whereby effects are significant in concentrations of 5 and 20mg/kg (Fig 23).
Thus, Compound 138 has a significant effect on APP processing and on generation of Aβ peptide, though the effects were more pronounced on Aβ1-42. Clear AB1 -42 lowering effects can be seen across various brain fractions, which represent different polymerization states of β-amyloid (monomers, oligomers, fibrils).
Conclusion:
In the brain, clear Aβ-amyloid 1-42 lowering effects of Compound 138 can be seen. The most striking and unique result is the capacity of Compound 138 to penetrate across brain fractions (monomers, oligomers, fibrils) to lower Aβ1-42 levels. The significance of the results is underlined by the fact that in this model, the AB1-42 level is extremely high, even compared with other transgenic models such as Tg2576 mouse. Thus, even in this aggressive model, Compound 138 reduces AB1-42 levels across brain fractions. Although a clearest effect was seen on Aβ1-42, Compound 138 also reduced Aβ1-40 to some extent in brain fractions. The more pronounced effect of Compound 138 on the toxic Aβ1-42 peptide leads to a reduction in the Aβ1-42/Aβ1-40 ratio, and this is beneficial therapeutically.
EXAMPLE 7: Synthesis of compounds from Series A
Compounds in series A can be prepared by employing the strategy depicted below. Groups other than the 5-Cl1 e.g. F or Me, would be equally well tolerated in this chemistry.
Figure imgf000045_0001
Example 7.1: N-[(5-chloro-8-hydroxyquinolin-7-yl)(3,4- dimethoxyphenyl)methyl]acetamide (Compound 7) {Method A_1}
Figure imgf000045_0002
intima rte mixture of 5-chloro-8-hydroxyquinoline (1.5g, 8.7mmol), 3,4- dimethoxybenzaldehyde (2.85g, 17.4mmol) and acetamide (1.03g, 17.4mmol) was sealed in a microwave reactor vessel. The vessel was placed in a microwave reactor (Biotage Initiator) and heated to 18O0C for 1 hour. The cooled reaction mixture was triturated with ethyl acetate and the resulting solid filtered off. This solid was further triturated with ethanol to yield the title compound as a white solid (2.2g, 65%). MS 387 (MH+); 1H NMR (DMSO-d6), 400 MHz δ: 10.28 (bs, 1H), 8.93-8.97 (m, 1H), 8.74 (d, 1H), 8.47 (d, 1H), 7.69-7.75 (m, 2H), 6.91- 6.94 (m, 1 H), 6.87 (d, 1H), 6.74 (d, 1H), 6.62 (d, 1 H), 3.70 (s,3H), 3.71(s, 3H), 1.94 (s, 3H).
Example 7_2_: N-((5-chloro-8-hydroxyquinolin-7-yl)(quinolin-3- yl)methyl)acetamide (Compound 19) {Method A_1}
Figure imgf000045_0003
An intimate mixture of 5-chloro-8-hydroxyquinoline (0.25g, 1.4mmol), quinoline-3- carbaldehyde (0.44g, 2.8mmol) and acetamide (0.165g, 2.8mmol) was sealed in a microwave reactor vessel. The vessel was placed in a microwave reactor (Biotage Initiator) and heated to 18O0C for 0.5 hour. The cooled reaction mixture was triturated with ethyl acetate and the resulting solid filtered off. This solid was further triturated with ethanol to yield the title compound as a white solid (0.236g, 44%). MS 377 (M+); 1H NMR (DMSO-d6), 400 MHz δ: 10.52 (bs, 1 H), 8.96-9.01 (m, 2H), 8.85 (s, 1 H), 8.51 (d, 1 H), 8.11 (s, 1 H), 7.95-8.01 (dd, 2H), 7.71-7.79 (m, 4H), 7.58 (t, 1 H), 6.87 (d, 1 H), 2.01 (s, 3H).
Example 7.3: N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-3-yl)methyl)acetamide
Figure imgf000046_0001
An intimate mixture of 5-chloro-8-hydroxyquinoline (0.25g, 1.4mmol), nicotinaldehyde (0.299g, 2.8mmol) and acetamide (0.165g, 2.8mmol) was sealed in a microwave reactor vessel. The vessel was placed in a microwave reactor (Biotage Initiator) and heated to 18O0C for 0.5 hour. The cooled reaction mixture was triturated with ethyl acetate and the resulting solid filtered off. This solid was further triturated with ethanol to yield the title compound as a white solid (0.123g, 27%). MS 327 (M+); 1H NMR (DMSO-d6), 400 MHz δ: 10.49 (bs, 1 H), 8.97 (s, 1H), 8.90 (d, 1 H), 8.47-8.51 (m, 3H), 7.72-7.77 (m, 2H), 7.64 (d, 1 H)1 7.33-7.36 (dd, 1 H), 6.70 (d, 1 H), 1.97 (s, 3H).
EXAMPLE 8: Synthesis of compounds from Series B1
Compounds in series B1 can be prepared by employing the strategy depicted below. This complements the synthesis employed for Series A compounds and is particularly appropriate for compounds where R1 is alkyl c.f. aryl. Groups other than the 5-CI, e.g. F or Me, would be equally well tolerated in this chemistry.
Figure imgf000047_0001
NEt3 B1 7
Figure imgf000047_0002
Figure imgf000047_0003
Figure imgf000047_0004
Method B1_1 Synthesis of intermediate: 2-((5-chloro-8-hydroxyquinolin-7- yl)methyl)-1 H-isoindole-1 ,3(2H)-dione
Figure imgf000047_0005
2-(Hydroxymethyl)-1 H-isoindole-1 , 3(2H)-dione (14.8g, 83mmol) was added portionwise to a well stirred solution of 5-chloro-8-hydroxyquinoline (15g, 83mmol) in concentrated sulphuric acid (150ml) cooled to O0C. After addition was complete the reaction was heated to 1000C overnight. The mixture was allowed to cool to RT then poured onto crushed ice (1000ml) and the resulting yellow precipitate filtered, washed with water and dried in vacuo overnight. The solid was triturated with ethanol and hexane to yield an off-white solid (16.7g, 59%). MS 338 (M+); 1H NMR (DMSO-d6), 400 MHz δ: 10.24 (bs, 1 H), 8.95 (d, 1 H), 8.48 (d, 1H), 7.41-7.92 (m, 4H), 7.72 (dd, 1 H), 7.57 (s, 1 H), 4.96 (s, 2H).
Method B1_2 Synthesis of intermediate: 2-((8-(benzyloxy)-5-chloroquinolin-7- yl)methyl)-1 H-isoindole-1 ,3(2H)-dione
Figure imgf000048_0001
2-((5-chloro-8-hydroxyquinolin-7-yl)methyl)-1 H-isoindole-1 ,3(2H)-dione (16.68g, 49mmol), benzyl bromide (12.63g, 73.5mmol) and potassium carbonate (13.61g,
98mmol) were dissolved/suspended in dimetnylformamide (250ml) and heated to
6O0C for 1.5 hours. The reaction was poured into cold water (1000ml) and the off-white precipitate filtered and washed well with water. The solid was dried in vacuo to yield the pure product (20.8g, 98%). MS 446 ([M+H2O]+); 1H NMR (DMSO-de), 400 MHz δ: 9.09 (d, 1H), 8.55 (d, 1H), 7.83-7.91 (m, 4H), 7.75 (dd,
1H), 7.64 (s, 1H), 7.55-7.61 (m, 2H), 7.347.44 (m, 3H), 5.53 (s, 2H), 4.96 (s, 2H).
Method B1_3 Synthesis of intermediate: 1-(8-(benzyloxy)-5-chloroquinolin-7- yl)methanamine
Figure imgf000048_0002
Hydrazine monohydrate (2.28ml, 46.6mmol) was added to a suspension of 2-((8- (benzyloxy)-5-chloroquinolin-7-yl)methyl)-1 H-isoindole-1 ,3(2H)-dione (2.Og, 4.7mmol) in methanol (50ml). The mixture was refluxed for 1hour and then solvent removed in vacuo. The residue was triturated with hot ethanol (~10ml) and filtered hot. The ethanol was concentrated to yield a light brown solid (1.1g, 79%) used in subsequent reactions without any further purification. MS 299 (MH+); 1H NMR (CDCI3), 400 MHz δ: 9.02 (d, 1H), 8.54 (d,1 H), 7.59 (s, 1H), 7.51 (dd, 1H), 7.41-7.44 (m, 2H), 7.31-7.39 (m, 3H), 5.50 (s, 2H), 3.87 (s, 2H). Method B1_4 Synthesis of intermediate: 1-(8-(benzyloxy)-5-chloroquinolin-7-yl)- N-(diphenylmethylene)methanamine
Figure imgf000049_0001
A mixture of 1-(8-(benzyloxy)-5-chloroquinolin-7-yl)methanamine (407mg, 1.36mmol) and benzophenone imine (247mg, 1.36mmol) were heated together at 6O0C. After 1hour the resultant solid was triturated with methanol and filtered to yield the title compound as a pale brown solid (480mg, 76%). MS 463 (M+); 1H NMR (DMSO-de), 400 MHz δ: 9.06 (d, 1H), 8.57 (d,1 H), 7.96 (s, 1 H), 7.73 (del, 1 H), 7.54-7.62 (m, 5H), 7.39-7.48 (m, 3H), 7.20-7.31 (m, 7H), 5.29 (s, 2H), 4.61 (s, 2H).
Method B1_5 Synthesis of intermediate: 1-(8-(benzyloxy)-5-chloroquinolin-7-yl)- N-(diphenylmethylene)ethanamine
Figure imgf000049_0002
Methyl iodide (147mg, 1.03mmol) and 1-(8-(benzyloxy)-5-chloroquinolin-7-yl)-N- (diphenylmethylene)methanamine (480mg, 1.03mmol) were dissolved in anhydrous tetrahydrofuran (0.5ml) and cooled to O0C. To this mixture was added dropwise a solution of potassium fe/f-butoxide (128mg, 1.16mmol) in tetrahydrofuran (0.13ml). The mixture was allowed to warm to RT and after 1 hour the reaction was concentrated. The residue was dissolved in ethyl acetate and washed with water and brine. The organic extract was dried over anhydrous magnesium sulphate and reduced in vacuo to yield the title compound as a white solid (480mg, 97%). MS 477 (M+); 1H NMR (CDCI3), 400 MHz δ: 8.89 (d, 1 H), 8.47 (d, 1 H), 8.15 (s, 1 H), 7.60 (d, 2H), 7.42 (dd, 1 H), 7.15-7.38 (m, 10H), 7.03- 7.09 (m, 3H), 5.20 (d, 1 H), 5.18 (q, 1 H), 4.81 (d, 1H), 1.23 (d, 3H)). Method B1 6 Synthesis of intermediate: 1-(8-(benzyloxy)-5-chloroquinolin-7- yl)ethanamine
Figure imgf000050_0001
To a solution of 1-(8-(benzyloxy)-5-chloroquinolin-7-yl)-N-
(diphenylmethylene)ethanamine (550mg, 1.15mmol) in methanol (5ml) was added hydroxylamine hydrochloride (144mg, 2.07mmol) and the mixture stirred at RT overnight. The reaction was concentrated and the residue treated with hydrochloric acid (5ml, 5% aqueous solution) and extracted with ethyl acetate to remove benzophenone. The aqueous layer was basified with sodium hydroxide solution (5M) and extracted with ethyl acetate (3x5ml). The combined organic extracts were washed with brine, dried over anhydrous magnesium sulphate, filtered and concentrated to yield the title compound (230mg, 64%). MS 313 (M+); 1H NMR (CDCI3), 400 MHz δ: 9.00 (d, 1 H)1 8.49 (d, 1 H), 7.68 (s, 1 H), 7.51 (dd, 1 H), 7.31-7.44 (m, 5H), 5.50 (q, 2H), 4.59 (q, 1 H), 1.46 (bs, 2H), 1.26 (d, 3H)).
Method B1 7 Synthesis of intermediate: N-(1-(8-(benzyloxy)-5-chloroquinolin-7- yl)ethyl)acetamide
Figure imgf000050_0002
To an ice-cold stirred solution of 1-(8-(benzyloxy)-5-chloroquinolin-7- yl)ethanamine (220mg, OJmrnol) and triethylamine (0.12ml, 0.84mmol) in dichloromethane (2.5ml) was added dropwise a solution of acetyl chloride (66mg, 0.84mmol) in dichloromethane (0.5ml). The reaction was stirred at RT overnight then diluted with dichloromethane (5ml). The mixture was washed with sequentially with saturated sodium carbonates solution, water and brine and dried over anhydrous magnesium sulphate. Removal of solvent in vacuo yielded the title compound (240mg, 96%). MS 354 (M+); 1H NMR (CDCI3), 400 MHz δ: 8.90 (d, 1H), 8.44 (d, 1 H), 7.54 (d, 2H), 7.40-7.45 (m, 2H), 7.26-7.37 (m, 3H), 6.22 (bd, 1 H), 5.61 (d, 1H), 5.46 (d, 1 H), 5.36 (q, 1H), 1.77 (s, 3H), 1.36 (d, 3H). Example 8.1 Synthesis of. N-(1-(5-chloro-8-hydroxyquinolin-7-yl)ethyl)acetamide (Compound 47)
Figure imgf000051_0001
To an ice-cold stirred solution of N-(1-(8-(benzyloxy)-5-chloroquinolin-7- yl)ethyl)acetamide (0.7g, 1.97mmol) in dichloromethane (20ml) was added a solution of boron trichloride (1M in dichloromethane, 4ml, 4mmol) in dichloromethane (5ml) under a nitrogen atmosphere. The mixture was then stirred at RT for 3 hours. The reaction was then cautiously poured into ice-cold water (100ml) and extracted with dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous magnesium sulphate, filtered and concentrated. The crude material was purified by chromatography on silica (0-10%MeOH: 100-90% EtOAc) to yield the title compound as a white solid (0.45g, 86%). MS 265 (M+); 1H NMR (DMSO-d6), 400 MHz δ: 10.17 (bs, 1H), 8.95 (d, 1 H), 8.47 (d, 1 H), 8.39 (d, 1H), 7.70 (dd, 1H), 7.64 (s, 1 H), 5.41 (q, 1H), 1.88 (s, 3H), 1.35 (d, 3H).
EXAMPLE 9: Synthesis of compounds from Series B2
Compounds in series B2 can be prepared by either of the methods depicted below:
Figure imgf000051_0002
Method B2 1
Figure imgf000051_0003
Method B2 2 Example 9.1: N-(8-hydroxyquinolin-2-yl)pyridine-2-carboxamide (Compound 69) {Method B2_2}
Figure imgf000052_0001
To a suspension of picolinoyl chloride hydrochloride (89mg, 0.5mmol) in tetrahydrofuran at O0C was added triethylamine ( 0.42ml, 3mmol) and 2-amino-8- hydroxyquinoline (160mg, LOmmol). The mixture was allowed to warm to RT and left to stir overnight. Solvent was removed in vacuo and the crude material purified by HPLC to yield the desired product (15mg, 11%). MS 265 (M+); 1H NMR (DMSO-de), 400 MHz δ: 10.79 (bs, 1H), 9.70 (bs, 1H), 8.81 (d, 1H), 8.53 (d, 1H), 8.43 (d, 1 H), 8.28 (d, 1 H), 8.16 (t, 1 H), 7.75-7.80 (m, 1 H), 7.36-7.41 (m, 2H), 7.08 (d, 1H).
Example 9.2: N-(8-hydroxyquinolin-2-yl)-2-pyridin-2-ylacetamide (Compound 70) {Method B2_1}
Figure imgf000052_0002
4-Pyridylacetic acid hydrochloride (87mg, O.δmmol) was dissolved in dimethylformamide (4ml). To this solution was added hydroxybenzotriazole (68mg, O.δmmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (96mg, O.δmmol) and triethylamine (0.28ml, 2.0mmol) and the mixture stirred at RT for 30 mins. 2-Amino-8-hydroxyquinoline (80mg, O.δmmol) was added to the reaction which was stirred at RT overnight. Solvent was removed in vacuo, the residue dissolved in dichloromethane (2ml) and washed with water. The organic phase was concentrated in vacuo and the crude product purified by HPLC to yield the desired product (11mg, 8%). MS 280 (MH+); 1H NMR (DMSO-d6), 400 MHz δ: 11.00 (bs, 1 H), 9.48 (bs, 1H), 8.52 (d, 2H), 8.27 (d, 1 H), 8.21 (d, 1 H), 7.39 (d, 2H), 7.28-7.34 (m, 2H), 7.07 (d, 1 H), 3.88 (s, 2H). (10mg, 7%). MS 280 (MH+); 1H NMR (DMSO-d6), 400 MHz δ 10.92 (bs, 1 H), 9.35-9.55 (bs, 1 H), 8.53 (d, 1H), 8.17-8.29 (m, 2H), 7.78 (t, 1 H), 7.44 (d, 1 H), 7.26-7.35 (m, 3H), 7.08 (d, 1H), 4.04 (bs, 2H). Example 9.3: 2-(3-hydroxyphenyl)-N-(8-hydroxyquinolin-2-yl)acetamide (Compound 71) {Method B2_1}
Figure imgf000053_0001
The title compound was similarly prepared according to Method B2 1 as described above in example 9.2. Yield = 12mg (9%). MS 295 (MH+); 1H NMR (DMSO-de), 400 MHz δ 10.84 (bs, 1 H), 9.43 (bs, 1 H), 9.34 (bs, 1 H), 8.21-8.28 (dd, 2H), 7.27-7.35 (m, 2H), 7.11 (t, 1 H), 7.07 (d, 1 H), 6.81 (s, 1 H), 6.79 (d, 1 H), 6.64 (d, 1H), 3.70 (s, 2H).
EXAMPLE 10: Synthesis of compounds from Series B3
Compounds in series B3 can be prepared by employing the scheme exemplified below:
Figure imgf000053_0002
Method, B3_1
Figure imgf000053_0003
Method B3 3 1 Synthesis of intermediate: 5-chloro-7-iodo-8-isopropoxyquinoline
Figure imgf000054_0001
To a solution of 5-chloro-7-iodo-8-hydroxyquinoline (18g, 58.9mmol) in dimethylsulfoxide was added potassium carbonate (32.57g, 235.6mmol) and 2- bromopropane (10.87g, 88.4mmol). The mixture was stirred at RT overnight and then poured into saturated ammonium chloride solution. This mixture was extracted with dichloromethane (3 x 300ml). The combined organic extracts were washed successively with sodium hydroxide (2M), water and brine and dried over anhydrous magnesium sulphate. Removal of solvent in vacuo and chromatography on silica eluting with ethyl acetate; hexane mixtures yielded the desired compound (11.0g, 55%). MS 347 (M+); 1 H NMR (CDCI3), 400 MHz δ: 8.92 (d,1 H), 8.50 (d, 1H), 7.97 (s, 1 H), 7.51 (dd, 1H), 5.38 (sept, 1 H), 1.42 (d, 6H). Method B3_2 Synthesis of intermediate: 3-(5-chloro-8-isopropoxyquinolin-7- yl)phenol
Figure imgf000054_0002
5-chloro-7-iodo-8-isopropoxyquinoline (500mg, 1.7mmol), (3- hydroxyphenyl)boronic acid (238mg, Ummol) and PdPCy3 (18mg, 0.028mmol) were dissolved/suspended in a mixture of acetonitrile/water (4ml: 1ml respectively) and sodium carbonate solution (2M, 5ml) added. The mixture was then heated to 80oC under N2 with stirring for 1 hour. TIc indicated complete consumption of starting iodo compound. The solvent was removed in vacuo, the residue dissolved/suspended in ethyl acetate and washed with water and brine. The organic phase was dried over anhydrous magnesium sulphate and concentrated in vacuo to yield essentially pure product (tic and nmr) (350mg, 77%). MS 314 (MH+); 1 H NMR (CDCI3), 400 MHz δ: 9.01 (bs, 1 H), 8.92 (d, 1H), 8.51 (d, 1H), 7.55 (s, 1 H), 7.47 (dd, 1H), 7.35 (t, 1 H), 7.32 (s, 1 H), 6.97-7.02 (m, 2H), 4.36 (sept, 1 H), 1.12 (d, 6H). Example 10.1 : 5-chloro-7-(3-hydroxyphenyl)quinolin-8-ol (Compound 57) {Method B3_3}
Figure imgf000055_0001
3-(5-chloro-8-isopropoxyquinolin-7-yl)phenol (475mg, 1.52mmol) was dissolved in dichloromethane (4ml) and cooled to -78oC under N2. A solution of boron trichloride dimethylsulfide complex in dichloromethane (2M, 3.04ml, 6.08mmol) was added dropwise and the mixture stirred at -78oC for 2 hours. Methanol (20ml) was added cautiously and the reaction allowed to warm to RT. Solvent was removed in vacuo and a further aliquot of methanol (20ml) added and again concentrated in vacuo. This was repeated a further two times to yield the crude product which was purified by HPLC to give the title compound (315mg, 76%). MS 272 (MH+); 1 H NMR (DMSO-d6), 400 MHz δ 9.5-10.5 (vbs, 1 H), 9.02 (d, 1H), 8.56 (d, 1 H), 7.78 (dd, 1 H), 7.72 (s, 1 H), 7.29 (t, 1 H), 7.17 (bs, 1 H), 7.11 (d, 1H), 6.80 (d, 1 H).
Method B3_2 Synthesis of intermediate: 5-chloro-8-isopropoxy-7-(2- methoxypyridin-3-yl)quinoline
Figure imgf000055_0002
The title compound was similarly prepared according to Method B3_2 as described above. MS 329 (MH+); 1 H NMR (CDCI3), 400 MHz δ 8.96 (d, 1H), 8.54 (d, 1H), 8.21 (d, 1H), 7.77 (d, 1 H), 7.63 (s, 1 H), 7.48-7.53 (dd, 1 H), 6.99- 7.02 (dd, 1 H), 4.57 (sept, 1 H), 3.97 (s, 3H), 1.06 (d, 6H).
Example 10.2: 5-chloro-7-(2-methoxypyridin-3-yl)quinolin-8-ol (Compound 61.) {Method B3_3}
Figure imgf000055_0003
The title compound was similarly prepared from 5-chloro-8-isopropoxy-7-(2- methoxypyridin-3-yl)quinoline according to Method B3_3 as described above in example 10.1. MS 288 (MH+); 1H NMR (DMSO-d6), 400 MHz δ 10.15 (bs, 1H), 9.00 (d, 1H), 8.54 (d, 1 H), 8.23 (d, 1H), 7.76-7.81 (m, 2H), 7.64 (s, 1 H), 7.12 (dd, 1H), 3.86 (s, 3H).
Method B3_2 Synthesis of intermediate: 4-(5-chloro-8-isopropoxyquinolin-7-yl)- N,N-dimethylaniline
Figure imgf000056_0001
The title compound was similarly prepared according to Method B3_2 as described above. MS 341 (MH+); 1 H NMR (CDCI3), 400 MHz δ 8.95 (d, 1H), 8.49 (d, 1 H), 7.69 (s, 1H), 7.63 (d, 2H), 7.43-7.46 (dd, 1 H), 6.80 (d, 2H), 4.51 (sept, 1H), 3.02 (s, 6H), 1.11 (d, 6H).
Example 10.3: 5-chloro-7-(4-(dimethylamino)phenyl)quinolin-8-ol (Compound 60) {Method B3_3}
Figure imgf000056_0002
The title compound was similarly prepared from 4-(5-chloro-8-isopropoxyquinolin- 7-yl)-N,N-dimethylaniline according to Method B3_3 as described above in example 10.1. MS 300 (MH+); 1 H NMR (DMSO-d6), 400 MHz δ 9.02 (d, 1H), 8.57 (d, 1 H), 7.77-7.86 (m, 4H), 7.55-7.68 (bs, 2H), 3.13 (s, 6H).
EXAMPLE 11 : Synthesis of compounds from Series B4
Compounds in series B4 can be prepared by employing the scheme exemplified below:
Figure imgf000056_0003
Method B4-1 Example 11.1 : 2-(((2-methoxyphenyl)amino)methyl)quinolin-8-ol (Compound 77) {Method B4_1}
Figure imgf000057_0001
To a solution of 8-hydroxyquinoline-2-carbaldehyde (87mg, O.δmmol) in 1 ,2- dichloroethane (2ml) was added 2-methoxyaniline (62mg, O.δmmol) and the mixture stirred for 30 minutes before the addition of sodium triacetoxyborohydride (95mg, 45mmol). The reaction was then stirred at RT overnight before being evaporated to dryness in vacuo. The residue was taken up in dichloromethane and washed with water. The aqueous phase was back extracted with dichloromethane and the combined organic extracts dried and reduce in vacuo to yield the crude product which was purified by HPLC (74mg, 53%). MS 281 (MH+); 1H NMR (DMSO-d6), 400 MHz δ 9.51 (bs, 1 H), 8.22 (d, 1 H), 7.52 (d, 1H), 7.32-7.43 (q, 2H), 7.09 (d, 1 H), 6.83 (d, 1 H), 6.67 (t, 1 H), 6.48-6.56 (dd, 2H), 5.96 (t, 1H), 4.59 (d, 2H), 3.84 (s, 3H).
Example 11.2: 2-(((3-methoxyphenyl)amino)methyl)quinolin-8-ol (Compound 75) {Method B4_1}
Figure imgf000057_0002
The title compound was similarly prepared according to Method B4_1 as described above in example 11.1. Yield = 71 mg (51%). MS 281 (MH+); 1H NMR (DMSO-de), 400 MHz δ 9.74 (bs, 1 H), 8.27 (d, 1 H), 7.53 (d, 1 H), 7.36-7.45 (q, 2H), 7.11 (d, 1H), 7.08 (t, 1 H), 6.70 (t, 1 H), 6.36 (d, 1 H), 6.32 (s, 1 H), 6.16 (d, 1H), 4.54 (d, 2H), 3.69 (s, 3H).
EXAMPLE 12: Synthesis of phosphate pro-drugs Phosphate Pro-drug (compound 5)
The scheme and protocols discussed in this section could equally well be applied to other compounds in the series. Synthetic scheme:
Figure imgf000058_0001
a) NaHCO3, n-Bu4NHSO4, H2O-CH2CI2, O 0C ~ RT.; b) K2CO3, DMF, 25 0C; c) 1 ,4-cyclohexandiene, 10% Pd/C, EtOH. Synthesis of benzyl chloromethyl phosphate A
Figure imgf000058_0002
Dibenzyl phosphate (2.64 g, 9.48 mmol), sodium bicarbonate (3.18 ml_, 11.97 mmol) and tetra-n-butylammonium hydrogen sulfate (3.2 g, 9.48 mmol) were dissolved in water (80 ml). Dichloromethane (DCM, 90 ml_) was added and the mixture was vigorously stirred at O °C for 10 min, followed by the addition of chloromethyl chlorosulfate (1.17 ml_, 11.37 mmol) in DCM (15 ml_) with continuous vigorous stirring overnight at room temperature. The organic layer was separated, washed with brine, dried (MgSO4) and evaporated. The residue was purified by flash silica gel column chromatography using ethyl acetate/hexane (1 :1) as eluent to give 1.39 g (44.8 %) of pure material. MS 327 (MH+); Η NMR (CDCI3), 400 MHz δ 5.09 (d, 2H, J = 8 Hz), 5.62 (d, 2H, J = 16 Hz), 7.41-7.28 (m, 10Hz).
Synthesis of the Pro-drug derivative: Synthesis of compound 5:
Figure imgf000059_0001
Protocol: Compound 7 (0.5 g, 1.29 mmol), potassium carbonate solid (0.36 g, 2.58 mmol) were suspended in dry N, N-Dimethylformamide (DMF, 5 ml_), and then dibenzyl chloromethyl phosphate (A, 0.42 g, 1.29 mmol) dissolved in DMF (2 mL) was added while stirring at 0 0C under a N2 atmosphere. The reaction mixture was stirred for 16 hrs at room temperature under a N2 atmosphere. The reaction mixture was poured into water (50 mL), and extracted with EtOAc (3 x 25 mL). The combined extracts were washed with brine, dried (MgSO4) and evaporated to dryness. An LC-MS of the crude residue showed that the oily residue was 97 % pure at 254 nm. So no purification was attempted at that stage and the oily residue was used in the next step. MS 677 (MH+) and isotope pattern consistent; 1H NMR (DMSO), 400 MHz δ 1.99 (s, 3H), 3.64 (s, 3H), 3.68 (s, 3H), 4.72 (d, 1 H, J = 4 Hz), 4.74 (d, 1 H, J = 3.5 Hz), 4.89 (d, 2H1 J = 7.8 Hz), 5.96-6.05 ( m, 2H), 6.47 (d, 1H, J = 8.08 Hz), 6.58 (d, 1H, J = 8.33 Hz), 6.64 (dd, 1H, J = 8.58 & 2.02 Hz), 6.80 (d, 1H, J = 2.02 Hz), 7.10-7.23 (m, 10H), 7.43 (dd, 1 H, J = 8.58 & 8.58 Hz), 7.54 (s, 1 H), 7.55 (d, 1 H, J = 8.33 Hz), 8.45 (dd, 1 H, J = 6.82 &1.76 Hz), 8.80 (dd, 1 H1 J= 4.04 & 1.76 Hz).
Synthesis of compound 5:
Figure imgf000060_0001
Protocol: To a suspension of 10 % Pd/C in EtOH was added drop-wise under a N2 atmosphere a solution of B in EtOH (3 ml_), followed by cyclohexadiene (0.25 ml_; 10 eq, 2.58 mmol). The reaction mixture was stirred for 12 hrs at room temperature. The catalyst was filtered on celite and the filtrate evaporated nearly to dryness (~10 % of EtOH being still present after the evaporation). The crude residue was then triturated with dry Et2O. The solid that precipitated was filtered and rinsed with Et2O to give 0.065 g (60%) of pure material. MS 497 (MH+) and isotope pattern consistent; 1 H NMR (DMSO), 400 MHz δ 1.52 (s, 3H), 3.3 (s, 3H), 3.49 (s, 3H), 5.63 (m, 2H), 6.40 (d, 1H, J = 9.8 Hz), 6.61 (m, 2H), 6.88 (s, 1 H), 7.46 (m, 2H), 8.29 (d, 1 H, J = 8.58 Hz), 8.74 (d, 1 H, J = 3.2 Hz), 8.83 (d, 1 H, 7.7 Hz).
Phosphate Pro-drug (compound 138)
Step 1: synthesis of P(OBut)2(=O)(OCH2CI) I I is synthesized according to the following scheme:
Figure imgf000060_0002
12.O g 12 O g CH2CI2 / H2O i (59 %) 8.8 g
Step 2: synthesis of 138
I (8.8 g) is reacted with compound 7 (as synthesized in Example 7.1) in the presence of K2CO3 in dimethylformamide (DMF) to form ((7-((acetylamino)(3,4- dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl di-ter-butyl phosphate
II (9.6 g, HPLC yield 96%). Il (5.0 g) is then reacted with HCI in a mixture of EtOAc/Et2O to form 138 in a 85% yield. Table 1
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
REFERENCES
■ Bartke, A. (2005). Minireview: role of the growth hormone/insulin-like growth factor system in mammalian aging. Endocrinology 146, 3718-3723.
■ Berrigan, D., Perkins, S. N., Haines, D. C, and Hursting, S. D. (2002). Adult-onset calorie restriction and fasting delay spontaneous tumorigenesis in p.
53-deficient mice. Carcinogenesis 23, 817-822.
■ Blackwell, B. N., Bucci, T. J., Hart, R. W., and Turturro, A. (1Θ95). Longevity, body weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet. Toxicol Pathol 23, 570-582. ■ Cosgrove, G. E., Satterfield, L. C, Bowles, N. D., and Klima, W. C. (1978). Diseases of aging untreated virgin female RFM and BALB/c mice. J Gerontol 33, 178-183.
■ Fenton, M., Huang, H. L, Hong, Y., Hawe, E., Kurz, D. J., and Erusalimsky, J. D. (2004). Early atherogenesis in senescence-accelerated mice. Exp Gerontol 39, 115-122.
■ Finkel, T. (2005). Opinion: Radical medicine: treating ageing to cure disease. Nat Rev MoI Cell Biol 6, 971-976.
■ Frith, C. H., Highman, B., Burger, G., and Sheldon, W. D. (1983). Spontaneous lesions in virgin and retired breeder BALB/c and C57BL/6 mice. Lab Anim Sci 33, 273-286.
■ Hursting, S. D., Lavigne, J. A., Berrigan, D., Donehower, L. A., Davis, B. J., Phang, J. M., Barrett, J. C, and Perkins, S. N. (2004). Diet-gene interactions in p53-deficient mice: insulin-like growth factor-1 as a mechanistic target. J Nutr 134, 2482S-2486S. ■ Levavasseur, F., Miyadera, H., Sirois, J., Tremblay, M. L., Kita, K., Shoubridge, E., and Hekimi, S. (2001). Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration. J Biol Chem 276, 46160-46164.
■ Liu, X., Jiang, N., Hughes, B., Bigras, E., Shoubridge, E., and Hekimi, S. (2005). Evolutionary conservation of the clk-1 -dependent mechanism of longevity: loss of mclk1 increases cellular fitness and lifespan in mice. Genes Dev 19, 2424- 2434. ■ Matheu, A., Pantoja, C, Efeyan, A., Criado, L. M., Martin-Caballero, J., Flores, J. M., Klatt, P., and Serrano, M. (2004). Increased gene dosage of Ink4a/Arf results in cancer resistance and normal aging. Genes Dev 18, 2736- 2746. ■ Miller, R. A. (2005). Genetic approaches to the study of aging. J Am Geriatr Soc 53, 284-286.
Smith, G. S., Walford, R. L., and Mickey, M. R. (1973). Lifespan and incidence of cancer and other diseases in selected long-lived inbred mice and their F 1 hybrids. J Natl Cancer Inst 50, 1195-1213. ■ Takeda, T., Matsushita, T., Kurozumi, M., Takemura, K., Higuchi, K., and Hosokawa, M. (1997). Pathobiology of the senescence-accelerated mouse (SAM). Exp Gerontol 32, 117-127.
■ Miyadera, H., Amino, H., Hiraishi, A., Taka, H., Murayama, K., Miyoshi, H., Sakamoto, K., Ishii, N., Hekimi, S. and Kita, K., Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans, J Biol Chem, 276(11), 7713, 2001.
■ Nakai, D., Shimizu, T., Nojiri, H., Uchiyama, S., Koike, H., Takahashi, M., Hirokawa, K. and Shirasawa, T., coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q, Aging Cell, 3(5), 273, 2004.
■ Ewbank, J.J., Barnes, T.M., Lakowski, B., Lussier, M., Bussey, H. and Hekimi, S., Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1 , Science, 275(5302), 980, 1997.
■ Hekimi, S., Benard, C, Branicky, R., Burgess, J., Hihi, A.K. and Rea, S., Why only time will tell, Mech Ageing Dev, 122(7), 571 , 2001.

Claims

1- \ compound having formula (A):
Figure imgf000071_0001
a pharmaceutically acceptable salt or a pro-drug thereof, wherein X = H, methyl or halogen, and the substituents R1 and R2 are defined as follows: a) when R2 is an hydrogen, then Ri is H or halogen, or Ri is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or Ri is -CH(R3)NR4R5 with ■ R4 = H or Ci-C4 alkyl;
Figure imgf000071_0002
and Re is selected from the group consiting of: Ci-Cβ alkyl, C5-C6 aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and
■ R3 is selected from the group consisting of: H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, 4- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, 4- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1 H-pyrazol-1 -yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl, 3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, 4- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, I H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
LJ or Ri is — c — N — R7 and R7 is -(ChMn-Rβ where Re is a C5-C6 aryl optionally
Il o substituted by one or two methoxyl groups, and n= 0 or 1 ; or
b) when X and Ri both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl;
with the proviso that said compound of formula (A) is not one of the compounds identified in Annex 1 ;
said compound of formula (A), when comprising at least one asymmetric centre, being in the form of one of its enantiomers, or a mixture thereof.
2- The compound of claim 1 wherein R4 is H or methyl, and R6 is selected from the group consisting of methyl, ethyl, n-propyl, /-propyl, π-butyl, /-butyl, ter-butyl, phenyl, benzyl, dimethylaminomethyl, phenylethyl, cyclohexyl, diphenylmethyl, pyridin-2-yl and pyridin-3-yl.
3- The compound of claim 2 selected from the group consisting of: N-((3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl)acetamide 2, N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)benzamide
3,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-N- methylacetamide 8, N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-2,2- diphenylacetamide H,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-2- phenylacetamide 12,
7-(amino(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-ol 13, N-(1-(5-chloro-8-hydroxyquinolin-7-yl)-2-phenylethyl)acetamide 14,
N-((2-butyl-1 H-imidazol-4-yl)(5-chloro-8-hydroxyquinolin-7-yl)methyl)acetamide
15,
N-((5-chloro-8-hydroxyquinolin-7-yl)(2-methoxypyridin-3-yl)methyl)acetamide
16, N-((5-chloro-8-hydroxyquinolin-7-yl)(quinolin-4-yl)methyl)acetamide 17, N-((5-chloro-8-hydroxyquinolin-7-yl)(2-morpholin-4-ylpyridin-3-yl)methyl)- acetamide 18,
N-((5-chloro-8-hydroxyquinolin-7-yl)(quinolin-3-yl)methyl)acetamide 19, 4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl) methylbenzoate 21,
3-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl) methylbenzoate
22,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-cyanophenyl)methyl)acetamide 23, N-((5-chloro-8-hydroxyquinolin-7-yl)(4-pyridin-2-ylphenyl)methyl)acetamide 24,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-(1H-pyrazol-1-yl)phenyl)methyl)acetamide 25,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-hydroxyphenyl)methy!)acetamide 26, N-((5-chloro-8-hydroxyquinolin-7-yl)(3-hydroxyphenyl)methyl)acetamide 27, N-((5-chloro-8-hydroxyquinolin-7-yl)(6-methoxypyridin-3-yl)methyl)acetamide
28, N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-2-yl)methyl)acetamide 29, N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-4-yl)methyl)acetamide 30, N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-4-yl)methyl)acetamidehydrochloride 31,
N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-3-yl)methyl)acetamide 32, N-((5-chloro-8-hydroxyquinolin-7-yl)(pyridin-3-yl)methyl)acetamidehydrochloride
33,
N-((5-chloro-8 hydroxyquinolin-7-yl)(8-hydroxyquinolin-2-yl)methyl)acetamide
34,
N-((5-chloro-8-hydroxyquinolin-7-yl)(6-morpholin-4-ylpyridin-3-yl)methyl)- acetamide 35,
N-((5-chloro-8-hydroxyquinolin-7-yl)(1 H-indol-3-yl)methyl)acetamide 36,
N-((5-chloro-8-hydroxyquinolin-7-yl)(1 H-indol-4-yl)methyl)acetamide 37,
N-((5-chloro-8-hydroxyquinolin-7-yl)(1 H-indol-7-yl)methyl)acetamide 38,
4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N,N-dimethyl- benzamide 39,
N-((5-chloro-8-hydroxyquinolin-7-yl)(4-(morpholin-4-ylcarbonyl)phenyl)methyl)- acetamide 40,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)pyridine-2- carboxamide 4I-, 4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N-(3-methoxypropyl)- benzamide 44,
4-((acetylamino)(5-chloro-8-hydroxyquinolin-7-yl)methyl)-N-methylbenzamide
45,
N-((5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl)-N,N-dimethyl- glycinamide 46,
N-(1 -(5-chloro-8-hydroxyquinolin-7-yl)ethyl)acetamide 47,
N-((8-hydroxyquinolin-7-yl)methyl)acetamide 48,
N-(2-furylmethyl)-8-hydroxyquinolin-7-yl carboxamide 53,
N-(2-thienylmethyl)-8-hydroxyquinolin-7-yl carboxamide 54, N-(2-methoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 55,
5-chloro-7-(3-hydroxyphenyl)quinolin-8-ol 57,
5-chloro-7-(4-hydroxyphenyl)quinolin-8-ol 58, N-(2-(5-chloro-8-hydroxyquinolin-7-yl)phenyl)acetamide 59, 5-chloro-7-(2-methoxypyridin-3-yl)quinolin-8-ol 61,
5-chloro-7-pyridin-4-ylquinolin-8-ol hydrochloride 62, N-(3,4-dimethoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 63, N-(3-methoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 64, N-(3-methoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 65, N-(3,4-dimethoxybenzyl)-8-hydroxyquinolin-7-yl carboxamide 66, N-(2-methoxyphenyl)-8-hydroxyquinolin-7-yl carboxamide 67, N-(pyridin-3-ylmethyl)-8-hydroxyquinolin-7-yl carboxamide 68, N-(8-hydroxyquinolin-2-yl)pyridin-2-yl carboxamide 69, N-(8-hydroxyquinolin-2-yl)-2-pyridin-2-yl acetamide 70, 2-(3-hydroxyphenyl)-N-(8-hydroxyquinolin-2-yl)acetamide 71, 2-(4-hydroxyphenyl)-N-(8-hydroxyquinolin-2-yl)acetamide 72, N-(8-hydroxyquinolin-2-yl)-2-(2-thienyl)acetamide 73, 2-(((3-methoxybenzyl)amino)methyl)quinolin-8-ol 74, 2-(((3-methoxyphenyl)amino)methyl)quinolin-8-ol 75, 2-(((2-thienylmethyl)amino)methyl)quinolin-8-ol 76,
2-(((2-methoxyphenyl)amino)methyl)quinolin-8-ol 77. N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]acetamide 78, N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]acetamide 79,
N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]acetamide 80, N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]butanamide 8_1, N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]butanamide 82, N-[(8-hydroxyquinolin-7-yl)(4-methylphenyl)methyl]-3-phenylpropanamide 92,
N-[(2-chlorophenyl)(8-hydroxyquinolin-7-yl)methyl]-3-phenylpropanamide
93,
N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]-3-methylbutanamide
94. N-[(8-hydroxyquinolin-7-yl)(phenyl)methyl]-3-methylbutanamide 110,
N-[(8-hydroxyquinolin-7-yl)(4-methoxyphenyl)methyl]-3-phenylpropanamide ill. N-[(3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl]-3-methylbutanamide
112,
N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]-3-methylbutanamide 113, N-[(4-chlorophenyl)(8-hydroxyquinolin-7-yl)methyl]cyclohexanecarboxamide 121,
N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]propanamide 122.
N-[(3,4-dimethoxyphenyl)(8-hydroxyquinolin-7-yl)methyl]-3-phenylpropanamide
124,
N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]pentanamide 125. N-[(8-hydroxyquinolin-7-yl)(4-isopropylphenyl)methyl]pentanamide 126.
N-[[4-(diethylamino)phenyl](8-hydroxyquinolin-7-yl)methyl]pentanamide 127.
N-[(8-hydroxyquinolin-7-yl)(4-methoxyphenyl)methyl]cyclohexanecarboxamide
128.
N-[(2,4-dichlorophenyl)(8-hydroxyquinolin-7-yl)methyl]butanamide 129. N-[(8-hydroxyquinolin-7-yl)(4-methylphenyl)methyl]cyclohexanecarboxamide
130. and N-[(8-hydroxyquinolin-7-yl)(2-thienyl)methyl]cyclohexanecarboxamide 131.
4- A pro-drug selected from the group consisting of: ((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 5,
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 6,
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 136.
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 137.
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate dihydrochloride 138. and ((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate hydrochloride 139. 5- A method for inhibiting CLK-1 activity in a cell, said method comprising the steps of: a) providing a cell wherein CLK-1 activity needs to be inhibited, b) contacting said cell with a compound of formula (B):
Figure imgf000077_0001
wherein X = H, methyl or halogen, and the substituents Ri,and R2 are defined as follows:
i) when R2 is an hydrogen, then Ri is H or halogen, or Ri is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or Ri is -CH(R3)NR4R5 with ■ R4 = H or Ci-C4 alkyl;
Figure imgf000077_0002
and R6 is selected from the group consiting of: Ci-Cβ alkyl, Cs-Cβ aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and ■ R3 is selected from the group consisting of:
H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, 4- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, 4- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1 H-pyrazol-1-yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl, 3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, 4- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1 H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, 1 H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
LJ or Ri is — c — N — R7 and R7 is -(CH2)n-R8 where R8 is a Cδ-Cβ aryl optionally
O substituted by one or two methoxyl groups, and n= 0 or 1 ; or
ii) when X and Ri both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl;
the compound of formula (B), when comprising at least one asymmetric centre, being in the form of one of its enantiomers or a mixture thereof; and c) determining the CLK-1 activity of the cell.
6- The method of claim 5 wherein said compound of formula (B) is selected from the group of compounds identified in Table 1. 7- The method of claim 6 wherein said compound of formula (B) is selected from the group consisting of: N-[(5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl]acetamide
L
5-chloro-7-iodoquinolin-8-ol 52, 5-chloro-7-(2-hydroxyphenyl)quinolin-8-ol 56, 5-chloro-7-(4-(dimethylamino)phenyl)quinolin-8-ol 60, and N-[(5-chloro-8-hydroxyquinolin-7-yl)(2-furyl)methyl]acetamide 135.
8- A method for the prophylaxis and/or treatment of a disorder or its associated symptoms for which inhibition of CLK-1 is beneficial, in an animal, said method comprising the steps of: a) identifying an animal having a disorder for which inhibition of CLK-1 is beneficial; and b) administering to said animal a compound of formula (B)
Figure imgf000079_0001
a pharmaceutically acceptable salt or a pro-drug thereof,
wherein X = H, methyl or halogen, and the substituents Ri and R are defined as follows:
i) when R2 is an hydrogen, then Ri is H or halogen, or Ri is selected from the group consisting of: amino(3,4-dimethoxyphenyl)methyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 4-dimethylaminophenyl, pyridin-4-yl, 2- methoxypyridin-3-yl and 2-acetamidophenyl;
or R1 is -CH(R3)NR4R5 with ■ R4 = H or Ci-C4 alkyl;
Figure imgf000079_0002
and R6 is selected from the group consiting of: Ci-C6 alkyl, C5-C6 aryl, benzyl, dimethylaminomethyl, phenylethyl and diphenylmethyl; and ■ R3 is selected from the group consisting of: H, methyl, phenyl, benzyl, 2-chlorophenyl, 2-methoxyphenyl, 4- chlorophenyl, 4-methylphenyl, 4-isopropylphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-methoxyphenyl, 4-cyanophenyl, 4-dimethylamino-phenyl, 4-diethylamino-phenyl, 2,4-dichlorophenyl, 3,4-dimethoxyphenyl, 4- methoxycarbonylphenyl, 3-methoxycarbonylphenyl, N-methylbenzamido, N-(3-methoxypropyl)benzamido, N,N-dimethylbenzamido, 4-(morpholin-4- ylcarbonyl)phenyl, 4-(pyridin-2-yl)phenyl, 4-(1 H-pyrazol-1-yl)phenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 6-(morpholin-4-yl)pyridin-3-yl, 2-furyl, 3-furyl, 2-(morpholin-4-yl)pyridin-3-yl, 2-methoxypyridin-3-yl, 4- methoxypyridin-3-yl, 2-thienyl, 2-butyl-1 H-imidazol-4-yl, quinolin-3-yl, quinolin-4-yl, 8-hydroxyquinolin-2-yl, 1 H-indol-3-yl, 1 H-indol-4-yl and 1 H- indol-7-yl;
LJ or Ri is — c — N — R7 and R7 is -(CH2)n-R8 where R8 is a C5-C6 aryl optionally
Il o substituted by one or two methoxyl groups, and n= 0 or 1 ; or
ii) when X and R1 both represent a hydrogen atom, then R2 is selected from the group consisting of: pyridin-2-ylcarboxamido, pyridin-2-ylacetamido, 3- hydroxyphenylacetamido, 4-hydroxyphenylacetamido, ((2- methoxyphenyl)amino)methyl, ((3-methoxyphenyl)amino)methyl, ((3- methoxybenzyl)amino)methyl, 2-thienylacetamido and ((2- thienylmethyl)amino)methyl;
the compound of formula (B), when comprising at least one asymmetric centre, being in the form of one of its enantiomers or a mixture thereof. 9- The method of claim 8 wherein said compound of formula (B) is selected from the group of compounds identified in Table 1.
10- The method of claim 8 wherein said compound of formula (B) is selected from the group consisting of:
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 5,
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 6, N-[(5-chloro-8-hydroxyquinolin-7-yl)(3,4-dimethoxyphenyl)methyl]acetamide
Z.
N-[(5-chloro-8-hydroxyquinolin-7-yl)(2-furyl)methyl]acetamide 135,
((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate 136. ((7-((acetylamino)(2-furyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl disodium phosphate 137,
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate dihydrochloride 138, and
((7-((acetylamino)(3,4-dimethoxyphenyl)methyl)-5-chloroquinolin-8-yl)oxy)methyl dihydrogen phosphate hydrochloride 139.
11- The method of claim 8 wherein said disorder is an ischemia / reperfusion injury, or an inflammation.
12- A method for the prophylaxis and/or treatment of an age-related disorder or its associated symptoms, in an animal, comprising the following steps: a) identifying an animal having an age-related disorder; and b) administering to the animal a compound of formula (B) as defined in claim 8, a pharmaceutically acceptable salt or a pro-drug thereof, with the proviso that said compound of formula (B) is not one of the following compounds, a pharmaceutically acceptable salt or a pro-drug thereof: 5-chloro-7-iodoquinolin-8-ol 52, 5-chloro-7-(2-hydroxyphenyl)quinolin-8-ol 56, or 5-chloro-7-(4-(dimethylamino)phenyl)quinolin-8-ol 60.
13- The method of claim 12 wherein said age-related disorder is selected from the group consisting cardiovascular diseases, peripheral vascular disease, metabolic disorders, cancers, neurodegenerative disorders, dementia, bladder and kidney disorders, diabetes complications, eyes disorders, lung and respiratory disorders, musculoskeletal disorders and skin conditions.
14- The method according to claim 13 wherein said neurodegenerative disorder is Alzheimer's disease.
15- The method of claim 11 wherein said ischemia / reperfusion injury is a renal, heart, myocardium, lung, brain, or spinal cord ischemia / reperfusion injury.
16- The method of claim 11 wherein said inflammation is a lung inflammation or an inflammation of any other organ.
17- The method of claim 8 wherein said animal is a human.
18- The method of claim 12 wherein said animal is a human
19- A pharmaceutical composition comprising a compound of formula (A), as defined in claim 1 , a pharmaceutically acceptable salt or a pro-drug thereof, and at least one pharmaceutically acceptable carrier.
20- A pharmaceutical composition comprising a compound of formula (A), as defined in claim 2, a pharmaceutically acceptable salt or a pro-drug thereof, and at least one pharmaceutically acceptable carrier.
21- A pharmaceutical composition comprising a compound of formula (A), as defined in claim 3, a pharmaceutically acceptable salt or a pro-drug thereof, and at least one pharmaceutically acceptable carrier. 22- A pharmaceutical composition comprising a pro-drug of claim 4 and at least one pharmaceutically acceptable carrier.
23- A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula (A), a pharmaceutically acceptable salt or a pro-drug thereof, as defined in claim 1 , to effectively reduce and/or inhibit CLK-1 activity.
24- A pharmaceutical composition comprising a pharmaceutically effective amount of the compound of formula (B), a pharmaceutically acceptable salt or a pro-drug thereof as defined in claim 8, to effectively reduce and/or inhibit CLK-1 activity.
25- The pharmaceutical composition of claim 22, comprising a pharmaceutically effective amount of the pro-drug to effectively reduce and/or inhibit CLK-1 activity.
ANNEX 1
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
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