WO1998025905A2 - Therapeutic antioxidants for alzheimer's disease - Google Patents

Abstract

The invention discloses therapeutic antioxidant compounds of formula (I), (II), (III); wherein Class I Compounds: X = S or O, Y = OR1 or SR1, R1 = hydrogen, lower alkyl, W and V selected from the following = OH, NO2, CF3, halogen, OR2, CONHR2 NHCOR2 (R2 = H, lower alkyl), CN, H, lower alkyl, -(CH2)nSH, -(CH2)nCONH(CH2)mSH, -(CH2)nNHCO(CH2)mSH, (a); Class II and III compounds: X = S or O, Y = OR1 or SR1, R and R1 selected from hydrogen, lower alkyl, -(CH2)nSH, -(CH2)nCONH(CH2)mSH, -(CH2)nNHCO(CH2)mSH, (a); additionally for class III compounds: P and Q selected from hydrogen, lower alkyl, aminomethyl, hydroxymethyl, CH2OCOR2, CH2NHCOR2 R2 = hydrogen, lower alkyl), -(CH2)nSH, -(CH2)nCONH(CH2)mSH, -(CH2)nNHCO(CH2)mSH, (a), wherein in general X = S or O and Y = OR1 or SR1. Compounds of the invention may be used as pharmaceuticals for therapy and prophylaxis of neurodegenerative disease such as Alzheimer's disease.

Description

THERAPEUTIC ANTIOXIDANTS FOR ALZHEIMER'S DISEASE

Technical Field

The present invention relates to therapeutic antioxidant compounds for use in treatment of Alzheimer's Disease (AD), and to pharmaceutical compositions for AD treatment. Where legally permissible the invention also relates to methods of treatment of AD using such compounds and compositions.

Alzheimer's disease is already a major problem and the problem will continue to grow as the average age of populations increases. Its cause is unknown: genetic predispositions have been identified by they do not, as yet, help in the treatment of patients. Work has already been earned out m:

• the study of neurodegenerative disease in human and animal models

• measurement of free radical damage in the human body and design of antioxidants to combat this

• chemical synthesis of biologically active molecules.

This work has already shown that Alzheimer's disease is accompanied by increased free radical damage m the bram to DNA and proteins. Damage increases with age m human bram anyway, but this is markedly accelerated in Alzheimer's disease. Hence antioxidant therapeutics should be beneficial.

Other evidence consistent with this view:

• free radical trappers improve cognitive function m old ammals

• toxicity of β-amyloid to cells in culture is blocked by antioxidants m some studied

• several antioxidants are neuroprotective m stroke models and transgemc animal models of Alzheimer's disease and motor neurone disease.

However,

• there is evidence that free radical damage is a common feature of all neurodegenerative disease so far studied including:

• Parkinson's disease

• motor neurone disease

• Huntmgton's chorea. Hence a neuroprotective antioxidant agent should have benefit in all neurodegenerative diseases and will have wide applicability (possibly including stroke also).

Background

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder affecting > 5% of the population over the age of 65. It is characteπsed pathologically by cortical atrophy, neuronal loss (Davies et al., 1987: DeKosky and Scheff, 1990), ghal proliferation, excessive formation of neurofϊbπllary tangles (Pearson et al 1985), and the deposition of β-amyloid in neuπtic plaques (Hardy and Alsopp, 1991). The mechanisms responsible for neuronal loss m AD remain unknown, but one hypothesis is that cellular events involving oxidative stress may lead to neurodegeneration (Martins et_al., 1986, Halhwell, 1992, Harman, 1993, Gotz et al, 1994, Menococci et_al., 1994, Frolich and Riederer, 1995, Gsell et al.. 1995). Indeed, reactive oxygen species (ROS) may be involved m the production, aggregation and toxicity of β- amyloid (Fπedhch and Butcher, 1993), which is thought to contribute to neuronal damage m AD (Nixon and Cataldo, 1994).

There is accumulating evidence consistent with the occurrence of oxidative stress from postmortem studies on AD bram (reviewed in Lyras et al.. 1997). Further evidence consistent with oxidative stress has come from investigations of oxidative damage to biomolecules m AD Several articles present data suggestive of increased peroxidation, but data are contradictory on the areas of the brain affected. Recently, Lyras et al. (1997) have performed a systematic examination of oxidative damage to biomolecules m AD and normal brain regions. They found no evidence for increased lipid peroxidation in any bram area in AD, whereas there was a significant increase in protein carbonyls (an index of oxidative modification of proteins in vivo) m the paπetal lobe in AD. There were also significant increases in the levels of certain oxidatively modified DNA bases in DNA isolated from AD bram in this bram area suggestive of increased attack by hydroxyl radical on the DNA.

When ROS and reactive nitrogen species (RNS) are generated m vivo they damage any local oxidisable substrate- a term which mcludes almost anything found m living cells including proteins, lφids, carbohydrates and DNA. Mechanisms of Antioxidant (AO) action may include

(I) Inhibiting the formation of ROS/RNS (n) Scavenging of ROS/RNS or their precursors

(m) Binding transition metal ions needed for catalysis of ROS generation (IV) Upregulation of endogenous antioxidant defences The relative importance of antioxidants as neuroprotective agents in AD depends on

(a) which ROS/RNS is generated

(b) how and where this is generated

(c) which target of damage is measured.

To evaluate the potential of an AO in vivo, these questions must be addressed. Simple m vitro expeπments can answer some of these questions and often allow one to dismiss the proposed AO since a compound which is a poor direct AO in vitro will not be any better m vivo. Direct AO activity should be measured using biologically relevant ROS/RNS and tested at concentrations achievable m vivo.

As a preliminary screen for prospective neuroprotective AO possibly of therapeutic use in AD, three biologically relevant m vitro assay systems were chosen. These allow estimation of the effect of an AO on hpid peroxidation, scavenging of peroxymtrous acid (PN- a cytotoxic product of the rapid reaction between a ROS, O2" and a RNS, NO) and scavenging of OH* a most damaging ROS produced by the action of a transition metal ion such as Fe(II) on H2O2 Lipid peroxidation may be assayed in simple dispersions of ox bram phospholipids which can be oxidized by incubation with mixtures of Fe(II) and ascorbic acid to produce malondialdehyde (MDA) which is then conjugated to thiobarbituπc acid at low pH to produce a chromogen absorbing at 532 nm (the TBA test, Qumlan et al. 1988). This test can also be used to quantitate the reaction which occurs between deoxyπbose (DOR) and OH* also producing a TBA-reactive species. In this assay, (the DOR assay), OH* is generated by a Fenton reaction between Fe(II) and H2O2 with added ascorbic acid recycling the Fe(III) formed m the reaction back to Fe(LI):

Fe(II) -r H2O2 → Fe(Lπ) + OH" + OH- The ability of a compound to intercept OH* may be measured in terms of its rate constant for OH- by varying the concentration of the compound (Hal well et al.. 1987). Fe(LI) is supplied to the assay as a chelate with ethylenediaminetetraacetate (EDTA). Omitting the EDTA allows the prospective AO access to the iron when its ability to inhibit Fe-dependent OH generation may be a consequence of its ability to bind Fe and trap the OH- formed from H2O2. Some AO have a dual nature, under certam conditions they may promote oxidative damage instead of inhibiting it (so called pro-oxidant actions, Laughton et al.. 1987). Performing the DOR assay m the absence of ascorbic acid assesses the ability of the AO to reduce Fe(m) and thus promote rather than inhibit DOR fragmentation by OH- PN is a highly cytotoxic species believed to be formed in inflammation from the lnflammory species O2" and NO (Huie and Padmaja, 1993). The scavenging effect of prospective AO for PN may be determined by the ability of the AO to attenuate tyrosine nitration by PN (Evans et al. 1997)

Summary of the invention

It is an object of the mvention to design a neuroprotective antioxidant for the treatment of Alzheimer's disease and other neurodegenerative diseases

Such a compound can.

• Delay the worsenmg of symptoms m Alzheimer's disease patients

• Delay the onset of Alzheimer's disease in persons at high genetic πsk of developing the disease

• Have to be taken dunng the patient's lifetime Such a compound should:

• Be non- toxic

• Enter the bram

• Protect against damage to DNA and proteins, major targets of free radical attack m Alzheimer's disease

• Scavenge the free radicals responsible for the damage, including oxygen radicals and reactive nitrogen species such as peroxynitnte

• Interact with antioxidants naturally present m the bram, maintaining or enhancing their levels (especially reduced glutathione, GSH)

• Bind metal ions that catalyse free radical formation and stop them doing this.

Compounds of the invention may have some or all of the following features:

• Iron binding activity, inhibiting free radical formation

• An -SH group (acetylated m the pro-drug) to interact with GSH and scavenge oxygen free radicals and peroxynitnte

• A basic nucleus (for example of the type that has already been used m the design of iron chelators for iron overload disease)

• General properties that will allow it to cross the blood-bram barπer.

The antioxidants used in animal models have been either dietary (e.g. vitamin E) or spin traps (stroke models, neuroprotection). Their partial effectiveness m animals illustrates the values of the concept.

But,

• Nutntional antioxidants have poor bram penetration and are not depleted m bram in Alzheimer's disease or Parkinson's disease. They protect against free radical damage to hpids not DNA or proteins. Lipid damage is not elevated m these diseases.

• Lazaroids (Upjohn) have been in clinical tnals for stroke for a long time. They are again targeted against lipid damage. Effectiveness m humans is as yet uncertain.

Chemical Structures

Compounds with free radical scavenging properties have been demonstrated to have an effect on the age related increases m products of free radical damage to proteins that are considered to affect bram function (Dubrey, A. et al 1995, Archives of Biochemistry and Biophysics, 324, 249-254) and to improve cognitive function m aged rats (Socci, D.J. et al, 1995, Brain Research, 693, 88-94). Unfortunately, many of the compounds so far employed have limits of use due to toxicity and/or potential carcinogenicity. This appears to be of particular relevance to the group of "spin-trapping" nitrones currently under investigation by several groups.

The present invention provides antioxidant compounds of general formulae (I), (II), (III):

Wherein:

Class I Compounds:

X = S or O, Y = OR[ or SR-, Ri = hydrogen, lower alkyl

W and V selected from the following = OH, NO,, CF₃, halogen, OR,, CONHR,

NHCOR, (R, = H, lower alkyl), CN, H, lower alkyl, -(CH^SH,

-(CH₂)_ncONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

.CONHCCH -

Class II and HI Compounds:

X = S or O, Y = OR] or SR_1; R and R_! selected from hydrogen, lower alkyl, -(CH,)_nSH, -(CH₂)_nCONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

Additioπally for Class HI compounds:

P and Q selected from hydrogen, lower alkyl,aminomethyl, hydroxymethyl. CH,OCOR,, CH₂NHCOR, (R₂ = hydrogen, lower alkyl, -(CH₂)_nSH, -(CH,)_nCONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

and wherein n=l-4, m=l-4; and wherein, with the exception of the case when the πng is substituted with a thiol moiety or two nngs are linked through respective thiol substituent moieties forming a disulphide link, both X is not O and Y is not OH together.

The invention further provides compounds of general formulae (I), (II), (III), m which at least one of P, Q, R, Y has a terminal thiol group which has been modified so as to form an S-S disulphide bond to another like compound, thereby forming a disulphide-hnked dimeπc compound.

Still further the invention provides dimeπc and tnmeπc amide-lmked o gomenc deπvatives in which the amide linkage is via substituent Q for class III compounds and via substituent R for Class II compounds. Preferably in such o gomers there are between about 9 and about 12 atoms m the length of the link between the heterocycles.

Examples of ohgomenc denvatives are shown below:

Tetradentates

For good oral bioavaiiability and effective permeation of the blood-bram-bamer the compounds should possess partition coefficients (Log P octanol/water) from +3 to -1 and preferably from 0 < Log P < +2. The vaπation in lipophilicity can be achieved via judicious selection of substituents notably W and V (class I), R (classll) and P, Q and R (classIII). Examples of such substituents (not limited to these alone) are mtro-, methoxy-, carboxy- substituents.

The invention also provides pharmaceutical compositions and medicaments containing the compounds as active ingredients, including known compounds when used m the manufacture of medicaments for a new therapeutic application (treatment of neurodegenerative disease)

The mvention also provides novel methods for making compounds of the invention, as well as certain useful intermediates.

Synthesis

Compounds 4, 6, 10 and 14 are known m the art. See for example GB 2118176, GB 2136807, GB 2146989 and GB 2146990, which disclose compounds for iron chelation applications

Starting Materials Aldrich Pfizer

1 2. Rj = CH2CH3 , Ethyl maltol 3. Rj = CH3 Maltol

Compounds synthesised for testing.

CH₃

14 19

1 4

Scheme 1: Synthesis of l-methyl-3-hydroxy-2-(lif)-pyndιnone.

Reference: Streater M, Taylor PD, Hider RC and Porter J, (1990), Novel 3-hydroxy-2(lH)- pyndmones. Synthesis, ιron(III)-chelatmg properties, and biological activity., J. Med. Chem.,

33. 1749-1755.

l-Methyl-3-hydroxy-2-(l.H)-pyridinone (4.) 3-Hydroxy-2(lif)-pyπdιnone (1) (7g, 63mmol) was placed in a thick walled glass tube (20cm x 1cm) and methyl iodide (21ml, 32mmol) was added. It was frozen m liquid nitrogen, evacuated and flame sealed. After thawing, the tube was placed in a protective steel chamber and heated at 140^0 for two days in an oven. The cooled tube was refrozen and reopened and the excess methyl iodide was decanted. Distilled water (10ml) and ethanol (10ml) were added to the resulting dark brown oil, warmed and transferred into a round bottomed flask. The solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in distilled water (200ml), sodium metabisulphite (2g) was added and pH of the solution was adjusted to 6 by adding 10M sodium hydroxide solution. The solution was extracted with dichloromethane (3 x 50ml), dned over anhydrous sodium sulphate, filtered and concentrated to dryness under reduced pressure. The crude product was recrystal sed from petroleum ether (b.p. 100 - 120^0 to yield colourless crystals (4.6g, 59%); MP 127-129^0 [Lit. Value, Streater et al, 1990, MP 126-1270C]. 90MI1_/ iHNMR (DMSO-d₆): δ 3.49(3H, s, N-CH3), 6.09(1H, t, 5-H), 6.72(1H, dd, 4-H), 7.15(1H, dil. 6-H), 8.93(1H, s, OH); Anal. Calcd. for C6H7O2N: C,57.59; H, 5.64; N, 11.19 Found C,57.61; H,5.88; N, 11.13%.

5 6

Scheme 2: Synthesis of l-carboxymethyl-3-hydroxy-2(li7)-pyπdιnone.

Reference: Rai BL, (1996), Design and synthesis of ιron(III) chelators for clinical use, Ph.D Thesis, King's College London, Manresa Road, London SW3 6LX, p 271-272.

l-[(Methoxycarbonyl)-methyl]-3-hydroxy-2(liϊ)-pyridmone (5.) Compound C was prepared from (1) by following the methodology as descnbed by Rai (1996). Yield 83%, MP 160.5-16lOC; IR(nujol): 3200 (OH), 1740 (ester CO), 1655 (pyndinone C=0), 1605 (πng C=C) cm-1; 90MHz IHNMR (DMSO-d₆): δ 3.7(3H, t, COOCH3), 4.79(2H, s, NCH₂), 6.15(1H, t, 5-H), 6.77((1H, dd, 4-H), 7.17(lH,dd, 6-H), 9.11(1H, s, OH).

l-carboxymethyI-3-hydroxy-2(l Ϊ)-pyridinone (6.) Concentrared hydrochlorc acid was added to water (60ml) until it reached pH 1. The methyl ester (3) (4g, 22mmol) was added to this solution and the mixmre was heated to reflux for 6h and cooled. The reaction mixture was boiled with activated charcoal (~ o.6g) for 5 minutes and filtered. The solvent was removed by rotary evaporation to give a colourless solid. Recrystallisation from hot water yielded colourless crystals (2.9g, 79%); MP 218-2190C [Lit. Value, Rai, 1996, 220-220C]; IR(nujol): 3200 (OH), 1700 (acid C=0), 1660 (pyndmone C=0), 1590 (nng C=C) cm-1; 90MHz iH NMR (DMSO-d₆): δ 4.63(2H, s, NCH₂), 6.09(1H, t, 5-H), 6.74((1H, dd, 4-H), 7.14(lH,dd, 6- H), 9.0(2H, br, s, OH). Anal. Calcd. for C7H7O4N: C.49.71; H,4.17; N,8.28 Found C,49.53; H,4.35; N, 8.14%.

r.t. CH₃NH₂

11 10

Scheme 3: Synthesis of l-(methylcarbamoyl)methyl-3-hydroxy-2(l//)-pyndmone.

Reference: Rai BL, (1996), Design and synthesis of ιron(m) chelators for clinical use, Ph.D.

Thesis, King's College London, Manresa Road, London SW3 6LX, p 271, 279.

Streater M, Taylor PD, Hider RC and Porter J, (1990), Novel 3-hydroxy-2(lH)-pyndmones.

Synthesis, iron(UI)-chelating properties, and biological activity., J Med. Chem., 33, 1749-

1755.

l-[(Ethoxycarbonyl)methyl]-3-hydroxy-2(lH)-pyridinone (7.) Compound (7) as prepared from (1) by following the methodology as descπbed by Rai (1996).

MP 150-1520Q IR(nujol): 3240 (OH), 1735 (ester CO), 1655 (pyndinone CO), 1605 (πng CO) cm-1; 90MHz IHNMR (DMSO-d₆): δ 1.2(3H, t, COOCH₂CH₃), 4.15(2H, q, COOCH2CH3), 4.72(2H, s, NCH2), 6.13(1H, t, 5-H), 6.75((1H, dd, 4-H), 7.15(1H, dd, 6-H), 9.07(1H, s, OH).

3-Beπzyloxy-l-carboxymethyI-2(lH)-pyridinone (8.) l-[(Ethoxycarbonyl) methyl]-3- hydroxy-2(lH)-pyndmone 7 (lOg, 50mmol) was dissolved in methanol (225ml) and water (45ml). The solution was adjusted to pH 12 usmg sodium hydroxide solution (10M) and benzyl chlonde (25 g, 200mmol) was added. The mixture was heated to reflux for 12h and 10M sodium hydroxide solution was added to achive pH 12. The resulting brown aqueous solution was extracted with dichloromethane (3 x 50ml), diluted with water (150ml) and acidified to pHl with concentrated hydrochloπc acid, whereupon the crude product was precipitated. Removal of the crude product by filtration and recrystal sation from 95% ethanol afforded colourless crystals (9.5g, 73%); MP 185-186°C [Lit. Value, Rai, (1996), 185-1860C], IR(nujol). 1740 (acid CO), 1645 (pyndmone CO), 1580 (nng CO) cm"¹; 90MHz ∑ΪNMR (DMSO-d₆). δ 4.63(2H, s, NCH2), 5.01 (2H, s, CH₂Ph), 6.13(1H, t, 5-H), 6.94((1H, dd, 4-H), 7 25(lH,dd, 6-H), 7 4(5H, , Ar).

3-Benzyloxy-l-[[(succinimidyloxy)carbonyl]methyl]-2-(lH)-pyridinone (90 To a solution of 3-benzyloxy-l-carboxymethyl-2(lH)-pyndmone (8) (5g, 19mmol) in dimethylformamide (100ml) were added solutions of N-hydroxysuccmimide (NHSu, 2.3g, 20mmol) and dicyclohexylcarbodnmide (DCCI, 4.13g, 20mmol) each m dimethylformamide (50ml) at O^C The reaction mixture was then allowed to warm slowly to ambient temperature and stirred for 16h m darkness. Glacial acetic acid (0.5ml) was added and after stirnng for lh the solution was filtered. The dimethylformamide was removed by rotary evaporation under high vacuum to yield the crude product. Recrystalhzation from hot ethyl acetate afforded colourless needles (2.9g, 40%); MP 170-1720C [Lit. Value, Streater et al, 1990, 174.5-1750C]; 60MHz iHNMR (DMSO-d₆): δ 2.75(4H, s, succmιmιdeCH2), 4.95(2H, s, NCH₂), 5.1(2H, s, CH Ph), 6.09(1H, t, 5-H), 6.86((1H, dd, 4-H), 7.0-7.6(6H,m, 6-H an Ar H).

l-(Methylcarbamoyl)methyl-3-hydroxy-2(LH)-pyridinone (110 To a solution of 3- benzyloxy-l-[[(succιmmιdyloxy)carbonyl]methyl]-2-(lH)-pyndmone (9) (1.8g, 5mmol) m dichloromethane (50ml) was added methylamme (1.2g, 15mmol, 40%> w/v m water) dropwise.

After stimng at room temperature for 16h the mixture was filtered and the dichloromethane layer was first washed with aqueous sodium hydroxide (5% w/v, 3 x 25ml) and then with water

(2 x 25ml). The organic layer was dned over anhydrous sodium sulphate, filtered and concentrated m volume into dryness under reduced pressure to afford the crude white solid of

10 (lg, 74%); 60MHz ^HNMR (DMSO-dg): δ 2.55(3H, d, NHCH3, coupled with NH),

4.47(2H, s, NCH₂), 4.99(2H, s, CH₂Ph), 6.03(1H, t, 5-H), 6.86((1H, dd, 4-H), 7.1(1H, dd, 6-

H), 7.34(5H,m, Ar), 7.98(1H, br, s, CONH). The crude product (10) was directly used for next step.

The crude product of (10) (lg, 4mmol) was dissolved in dimethylformamide (50ml) and 5% palladium on charcoal catalyst (120mg) was added followed by the addition of 2 drops of concentrated hydrochloπc acid. The reaction mixture was then stirred at room temperature for

16h under a constant stream of hydrogen. After removal of used catalyst by filtration dimethylformamide was removed under high vacuum. The oil given solidified on standing at room temperature. Recrystalhsation from absolute ethanol after treatment with activated charcoal afforded colourless crystals of (11) (260mg, 40%); MP 202-204^0 [Lit. Value,

Streater et al, 1990, 209-21 lOq; IR(nujol). 3290, 3110, 1650, 1590 cm-1, 60MHz iHNMR (DMSO-d₆): δ 2.53(3H, d, NHCH3, coupled with NH), 4.47(2H, s, NCH₂), 6.0 (1H, t, 5-H),

6.65((1H, dd, 4-H), 6.69(1H, dd, 6-H), 7.9 (1H, br, s, CONH), 8.78(1H, s, OH).

Anal. Calcd. for C₈Hιoθ3N₂: C,52.74; H.5.53; N.15.38 Found C,52.85; H,5.68; N, 15.30%.

12 13 14

Scheme 4. Synthesis of l-methyl-2-ethyl-3-hydroxy-4(lH)-pyndmone.

Reference: Diobbm PS, Hider RC, Hall AD, Taylor PD, Sarpong S, Porter JB, Xiao G and van der Helm D, (1993), Synthesis, physicochemical properties and biological evaluation of N- substιtuted-2-alkyl-3-hydroxy-4(lH)-pyndmones: Orally active iron chelators with clinical potential, J Med. Chem., 36, 2448-2458.

Compound l-methyl-2-ethyl-3-hydroxy-4(lH)-pyndmone (14) was prepared utilizing the methodology as descπbed by Dobbm et al (Scheme 4). Benzylation of ethyl maltol (2) in 90% aqueous methanol yielded compound (12) which was reacted with methylamme at reflux m 50% aqueous methanol with a catalytic amount of sodium hydroxide to give (13). Ηydrozenolysis reaction of (13) m the presence of Pd/C/cat afforded (14) in a yield of 72%; MP 217-2190C [Lit Value, Dobbm et al, 1993, 219-220°C]; 90MΗz ΪHΝMR (DMSO-d₆): δ 1.17(3H, t, CH₂CH₃), 2.95(2H, q, CH₂CH₃),4.1(3H, s, NCH3), 7 44(1H, d, 5-H), 8.33(1H, d, 6-H), 10.75(2H, br, s, OH);

Anal. Calcd. for CgHι₂0₂NCl : C.50.67; H,6.38; N,7.39, Cl.18.69 Found C,50.97; H,6.08, N,7.09; Cl, 18.84%. TFA

NH₂CH₂CH₂SH.HC1 + Ph₃COH *- NH2CH9CH7S.CPI13

_{1 C}. work-up

¹⁵ 16

(lM NaOH)

Scheme 5: 2-((Tnphenylmethyl)thιo)ethylamme.

Reference. Brenner D, Davison A, Lister- James J and Jones AG, (1984), Synthesis and characterization of a seπes of isomenc oxotechnetιum(v) diamido dithiolates, Inorg. Chem. 23,

3793-3797

Pearson DA, Blanchette M, Baker ML and Gumdon CA, (1989), Trialkylsilanes as scavengers for the tπfluoroacetic acid deblocking of protecting groups m peptide synthesis, Tetrahedron

Lett., 30, 2739-2742.

2-((Triphenylmethyl)thio)ethylamine (16). Tπfluoroacetic acid (50ml) was added to a mixture of 2-mercaptoethylamme hydrochlonde (5.68g, 50mmol) and tnphenylmethanol (13g, 50mmol) by stirnng under an atmosphere of nitrogen. After stirπng the reaction mixture for 30 minutes at room temperature, trifluoroacetic acid was removed by rotary evaporation to give a brown oil. The oil was triturated with diethyl ether (250ml) whereby complete colour discharge was noticed and kept stirπng at O^C for lh. The white precipitate of trifluoroacetate salt was separated by filtration and washed with ether. The washings were collected and concentrated in volume and cooled to give a second crop (14.9g, 69%).

The tπfluoroacetate salt (14.9g) was partitioned between aqueous sodium hydroxide (1M, 200ml) and diethyl ether (200ml) in a separating funnel with shaking and diethyl ether layer was collected. The aqueous layer was then extracted with diethyl ether (2x100ml). The diethyl ether fractions were combined, dried (Na₂SO_ι), filtered and evaporated to yield a colourless oil which was dissolved in diethyl ether (80ml), and hexane (200ml) was added. The free base was then allowed to crystallize slowly at O^C. The white floppy crystals were filtered and dried (11.5g, 72%), MP 94-95.60C [Lit Value, Brenner et al, 93-940Q, 60MHz iH MR (DMSO- dg): δ 1.9-2.65(6H, m, CH₂CH₂ and NH₂), 7.22(15H, m, Ar); 400MHz iHNM (DMSO-d6): δ 2.15(2H, t, CH₂), 2.42(2H, t, CH₂), 3.37(2H, br, s, NH₂), 7.21-7.33(15H, m, Ar).

17

EtOH/H,O/pH>12/reflux

18

TFA/Et₃SiH

19

Scheme 6 Synthesis of l-(2'-thιoethyl)-2-methyl-3-hydroxy-4(lH)-pyndmone.

Reference. Corey EJ, Grass J-L and Ulnch P, (1976), A new general method for protection of the hydroxyl function, Tetrahedron Lett., 11, 809-812.

Rai BL. (1996), Design and synthesis of ιron(ITf) chelators for clinical use, Ph.D. Thesis,

King's College London, Manresa Road, London SW3 6LX, p 239-240.

Mayargue J, Essamkaoui M and Moskowitz H, (1989), Tetrahedron Lett., 30, 6867-6870.

Brenner D, Davison A, Lister- James J and Jones AG, (1984), Synthesis and charectenzation of a senes of isomenc oxotechnetιum(v) diamido dithiolates, Inorg Chem., 23, 3793-3797. Compound l-(2'-thιolethyl)-2-methyl-3-hydroxy-4(lΛ)-pyπdonone (19) was synthesised from tntyl (tnphenylmethyl) protected amme (16) and MEM-protected maltol (17) as descπbed in the Scheme 6.

2-methyl -3-Methoxyethoxymethoxy-4(LH)-pyrone (17.)

Methoxyethoxymethyl chloπde (2.5g, 20mmol) m dry dichoromethane (5ml) was added dropwise to a solution of maltol 3 (3.8g, 30mmol) in dry dichloromethane (50ml) and dusopropylethylamine (5.2g, 40mmol) at O^C under nitrogen with stirπng. The mixture was allowed to warm to ambient temperature and stirred for 16h. The reaction mixture was then partitioned in water (50ml) and the organic layer was separated. The aqueous layer was extracted with dichloromethane (3 x 30ml). The organic fractions were combined, dried (Na₂Sθ4), filtered and concentrated to dryness under reduced pressure to give the crude solid. Recrystalhzation from ethyl acetate and petroleum ether (b.p. 40-60^0) gave colourless crystals (3 2g, 76%), Rf = 0 56 (silica, methanohdichloromethane; 6 94); MP 70-72°C. 60MHz iHNMR (CDCI3) δ 2.3(3H, s, CH3), 3.3(3H, s, CH3), 3.35-3.6(2H, m, CH₂), 3 7- 3 93(2H. m, CH₂), 5.14(2H, s, CH₂), 6.17(1H, d, 5-H), 7.47(1H, d, 6-H).

l-((2'-Triphenyimethylthio)ethyl)-2-methyl-3-methoxyethoxymethyl 4(l /)-pyridi-none

(180 0 M Sodium hydroxide solution was added to a suspension of MEM-maltol (17) (0.642g, 3mmol) and 2-((tnphenylmethyl)thιo)ethylamme (16) (lg, 3.3mmol) in ethanol/water (40 ^• 40) to attain pH 13. The mixture was then heated under reflux for 16h. Tic (silica, chloroform : methanol; 10 : 90) indicated three spots corresponding to the starting matenal 17 (Rf = 0 56), product (Rf = 0.37) and tntylamine 16 (Rf = 0.19). 2M Sodium hydroxide solution was then added to the reaction mixture (~ pH 8) to achieve pH ~13 and refluxed for 4 more hours. Tic (above solvent) indicated absence of the starting matenal. After coolmg the solvent was removed by rotary evaporation. Water (50ml) was added and pH was adjusted to 7 by adding 2M HC1, extracted with dichloromethane (3 x 20ml), dned (Na S04), filtered and concentrated m volume under reduced pressure to give yellow oil. The crude product (320mg) was purified by column chromatography (silica, EtOH ; DCM; 8 : 92) to give colourless oil (250mg) 60MHz HNMR (CDCI3); δ 2.08(3H, s, CH3), 2.51(2H, t, CH ), 3.3(3H, s, CH3), 3.2-3 6(4H, m, 2 x CH₂), 3.6-3.9(2H, m, CH ), 5.2(2H, s, CH₂), 6.15(1H, d, 5-H), 6.62(1H, d, 6-H). 7 2(15H, m, Ar).

l-(2'-ThiolethyI)-2-methyl-3-hydroxy-4(l.ff)-pyridinone (19.) The protected compound (18) (240mg, 0 47mmol) was dissolved m dry dichloromethane (1.5ml) anf tπfluoroacetic acid (3ml) was added and stirred at room temperature for lh. The yellow reaction mixture was then cooled at O^C over an ice bath. Tπethylsilane (75mg, 0.65mmol) was added. Immediately discharge of colour was noted and white precipitate was formed. The reaction mixture was stirred at O^C for 20 mm. and at room temperature for 20 nuns. Tπfluoroacetic acid was removed under reduced pressure (40^0), water (10ml) was added and the aqueous layer was extracted with diethylether (3 x 10ml) to remove the tntyl by-product. Water was removed under reduced pressure (40^C) to give colourless oil (80mg, 93%). The oil could not be crystallised, it was dissolved m water (15ml) and pH was adjusted to 6 by adding 10M sodium hydroxide solution, extracted with chloroform (6 x 10ml), dned (Na2Sθ4), and filtered. The solvent was removed by rotary evaporation to give the product. 60MHz IflNMR (CD3OD): δ 2.39(3H. s, 2-CH3), 2.95(2H, q, CH₂SH), 3.54(1H, br.s, SH), 4.15(2H, t, NCH₂), 6.32(111, d, 5-H). 7 32(1H, d, 6-H).

Scheme 7: Synthetic route to l,2-dιmethyl-3-methoxy-4(lH)-thιopyndone

3-Methoxy-2-methyl-4(lH)-pyrone (20)(l).

Potassium hydroxide (11.12 g, 0.198 mol) was dissolved in water (300 ml), and maltol (3) (25g, 0 198mol) was added. The solution was stirred at 0°C until all dissolved, then dimethylsulphate (24.97 g, 0.198 mol) was added dropwise over a 30 minute peπod. After the addition was complete, the reaction was stirred at 25°C for 24 hours checking the progress of the reaction using FeCl3 in methanol to detect any remaining starting matenal. (in the presence of 3 a charactenstically red colour is observed, due to the formation of an FeM complex) After 24 hours some starting matenal was still present and hence KOH (5 g, 0.89 mol) was added to dnve the reaction to completion. The resulting mixture was stirred at 25 °C for a further 24 hours and was monitored by tic (silica gel 60; eluent: ethylacetate, U.V. detec; FeCl3 test; Rf(20)=0.43).

The reaction completed, 10M aq. NaOH was added to get pH>l l. The compound was then extracted by dichloromethane (4 x 200 ml), the organic fractions pooled, dned over Na2Sθ4(_anh) filtered, and concentrated under reduced pressure. After drying under high vacuum a pale yellow oil was obtained (16.82 g, 67%). For greater punty, this oil was distilled under reduced pressure and was stored at low temperature (4°C) under N2 atmosphere. 3-Methoxy-2-methyl-4(lH)-thiopyrone (21).

3-Methoxy-2-methyl-4(lH)-pyrone (20) (28.38 g, 0.20 mol) and ground Lawesson's

Reagent(-) (41.26 g, 0.10 mol) were transferred to a dry flask under a N₂ blanket. Dry toluene

(100 ml) was syπnged into the septum stoppered flask which was then heated to 80°C for 30 minutes.the progress of the reaction was monitored by tic. (Silica gel 60 plates; eluent- ethylacetate; UV detec; Rf(20)=0.42, R (21)=0.74)

The reaction mixture was concentrated under reduced pressure and a crude brown oil was obtained This was in turn dissolved in ethanol and filtered through a hirsh funnel. The alcoholic solution was the boiled in the presence of activated charcoal, filtered whilst hot and concentrated under reduced pressure. The resultant red oil was obtained and was chromatographed on a silica gel 60 column (twice: first column using CHCI3 as eluent: secondly using ethylacetate as eluent). The required fractions from both columns were concentrated under reduced pressure, and the crude red solid obtained was recrystalhsed from toluene over a few days peπod m the fridge (ca 4°C).

Two crops of product were obtained. The first batch was composed of large transparent crystals which were dned under high vacuum, MP 64.0-64.9°C. The second batch of crystals was dried under high vacuum, MP 63.4-64.4⁰C. (Overall yield: 20.0 g, 63%)

400MHz lH N.M.R. (CDCI3): δ 7.41 (1H, d, 5-H), 7.19 (1H, d, 6-H), 3.88 (3H, s, 0-CH3),

2.36 (3H, s, 2-CH3);

Anal. Calcd. For C₇H8θ S: Q53.83; H, 5.16; S, 20.52 Found C, 53.82; H.5.13; S, 20.65;

Mass Spec. (F.A.B., matπx MNOBA+Na): M+l = 157.

l,2-Dimethyl-3-methoxy-4(lH)-thiopyridone (22).

3-Methoxy-2-methyl-4(lH)-thιopyrone (21) (1.0 g, 6.40 x 10"³ mol) was dissolved m tetrahydrofuran (10 ml) mside a thick- alled glass screw-stoppered tube. An excess of aqueous methylamme (10 ml, 11.85 M) was added; the tube was then sealed and brought to 70 °C where the contents were stirred for three hours. Completion of the reaction was assessed by tic.

(Silica gel 60; eluent: CHCtymethanol, 1:1; detec. U.V.; R 4)=0.90, Rf(22)=0.72)

The reaction being complete the mixture was evaporated to dryness under reduced pressure

(using ethanol azeotrope the water). The crude brown oil was the chromatographed on a silica gel 60 column using above eluent. After subsequent combination and concentration of the homogeneous fractions collected, an orange crystalline compound was obtained (0.75 g, 70%)

MP 115.5-118.5°C. This was in turn recrystalhsed from ethanol/diethyl ether, after treatment of ethanohc solution with activated charcoal. Orange crystals were obtamed MP 117.9-

120.0°C

400MHz lH N.M.R. (CDCI3): δ 7.33 (1H, d, 5-H), 7.09 (1H, d, 6-H), 3.90 (3H, s, O-CH3),

3.75 (3H, s, N-CH3), 2.42 (3H, s, 2-CH3);

Anal. Calcd. For CgHnOSN: C, 56.78; H,6.55; N, 8.28; S, 18.94 Found C, 56.92; H, 6.59; N.

8.24. S, 18.75;

Mass Spec. (F.A.B., matπx MNOBA+Na): M+l = 170.

I23J

Scheme 8: Synthesis of 3-hydroxy-l,2-dιmethyl-4(lH)-thιopyndone.

3-Hydroxy-l,2-dimethyl-4(lH)-pyridone (230

3-Hydroxy-l,2-dιmethyl-4(lH)-pyndone (23) was obtained from its dihydrochloπde salt(3) after neutralisation m water, recrystalhsation of the white solid from ehanol/diethyl ether, and drying under high vacuum, MP 272-275. (Compound (23) is also commercially available from Aldπch)

3-Hy dr oxy- 1 ,2-dimethyl-4(l H)-thiopyridone (24.)

3-Hydroxy-l,2-dιmethyl-4(lH)-pyndone (23) (0.63 g, 4.53 x 10"³ mol) was transferred to a dry flask and flushed with N₂ for a few mmutes. Dry toluene (ca 21 ml) was syringed into the flask and ground Lawesson's Reagent(2) (0.92 mg, 2.26 x 10^~3 mol) was added. The suspension was stirred overnight under reflux under N₂.

A cloudy yellow solution was obtained from which a white solid precipitated and a brown- yellow oil coated the walls of the vessel was observed. A tic (silica gel 60 plate; eluent: methanol/chloroform, 3:7; U.V. detec.) revealed the white solid to be the required compound. (This compound gave one spot on tic, which turned green upon treatment with FeCl3) The bulk of the solvent and the yellow-brown oil, showed several spots on tic, one of them was identical to that from the white solid, although this was a minor component. The white solid was treated with activated charcoal m hot toluene and was subsequently recrystalhsed from toluene. A pale yellow powder (0.140 g, 20%) was thus obtained MP 163.0- 163.8⁰C.

A small sample of this impure compound was sublimed under high vacuum (oil bath at 150°C) to afford an analytical sample as a whitish-pink solid (23 mg) PM 165.3-165.9°C. 400MHz lH N.M.R. (CD₃OD): δ 7.35 (1H, d, 5-H), 7.19 (1H, d, 6-H), 3.81 (3H, s, N-CH3), 2.47 (3H, s, 2-CH3);

13c N.M.R.(decoupled): δ 170.83 (OS), 155.25 (3-C), 133.83 (5-CH), 130.55 (2-C), 127.71 (6-CH), 45.08 (N-CH3), 14.63 (C-CH3); Anal. Calcd For C7H9OSN: C, 54.17; H, 5.84; N, 9.02; S, 20.66 Found C, 54.33; H, 5.86; N,

8.85; S, 20. ,9;

Mass Spec. (F.A.B.): M/Z+l=156.

Biological Data:

Abilitv to scavenge peroxynitrite (inhibition of tyrosine nitration) and inhibition of lipid peroxidation (LP)

N-Hydroxy-2(1 H)-thiopyridinone

This is a known compound but forms the minimum skeleton for a subtype of class I compounds. References:

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2069.

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6-phosphate dehydrogenase activity m AD may reflect oxidative stress. J. Neurochem. 46,

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Claims

Claims

1. Compounds of general formulae (I), (II), (III):

Wherein:

Class I Compounds:

X = S or 0, Y = OR, or SR,, Rj = hydrogen, lower alkyl

W and V selected from the following = OH, N0₂, CF₃, halogen, 0R₂, CONHR₂

NHCOR₂ (R₂ = H, lower alkyl), CN, H, lower alkyl, -(CH^SH,

-(CH₂)_nCONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

Class II and HI Compounds:

X = S or 0, Y = ORj or SRi, R and Rj selected from hydrogen, lower alkyl, -(CH SH, -(CH₂)_nCONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

Additionally for Class 1TJ compounds:

P and Q selected from hydrogen, lower alkyl,aminomethyl, hydroxymethyl. CH.OCOR₂, CH₂NHCOR₂ (R₂ = hydrogen, lower alkyl, -(CH₂)_nSH, -(CH₂)_nCONH(CH₂)_mSH, -(CH₂)_nNHCO(CH₂)_mSH,

and wherein n=l-4, m=l-4; and wherein, with the exception of the case when the πng is substituted with a thiol moiety or two πngs are linked through respective thiol substituent moieties forming a disulphide link, both X is not O and Y is not OH together.

2 Compounds of claim 1 in which at least one of P, Q, R, Y has a terminal thiol group which has been modified so as to form an S-S disulphide bond to another like compound, thereby forming a disulphide-lmked dimenc compound.

3 Dimenc or tnmeπc amide-lmked ohgomenc denvatives of compounds of claim 1 m which the amide linkage is via substituent Q for class III compounds and via substiment R for Class II compounds.

4 Ohgomers according to claim 3 in which there are between about 9 and about 12 atoms m the length of the link between the heterocycles.

5 Compounds according to any preceding claim having good oral bioavaiiability and effective permeation of the blood-bram-bamer, wherein the compounds possess partition coefficients (Log P octanol/water) from +3 to -1 and preferably from 0 ≤ Log P ≤+2.

6. A pharmaceutical composition compnsmg as active ingredient a compound accordmg to any preceding claim together with a physiologically acceptable excipient, adjuvant or earner.

7 Use of a compound according to any of claims 1 to 5, including all compounds m which both X is O and Y is OH together, m the manufacture of a medicament which is a therapeutic antioxidant for treatment of neurodegenerative disease such as Alzheimer's disease

8 A method for the production of intermediate compound of formula 16 according to scheme 5

TFA

NH,CH₉CH₂SH.HC1 - Ph₃COH _*- NH₂CH₂CH₂S.CPh₃ work-up 1^ 16

(1M NaOH)

Scheme 5: 2-((Tπphenylmethyl)thιo)ethylamme.

A method for the production of the compound for formula 19 according to scheme 6:

17

EtOH/H₂O/pH>12/reflux

18

TFA/Et₃SiH

19

Scheme 6: Synthesis of l-(2'-thioethyl)-2-methyl-3-hydroxy-4(lH)-pyridinone.

10. A method for the production of the compound for formula 22 according to scheme 7:

Scheme 7: Synthetic route to l,2-dιmethyl-3-methoxy-4(lH)-thιopyndone

1 1. A method for the production of the compound for formula 24 according to scheme 8:

Scheme 8: Synthesis of 3-hydroxy-l,2-dιmethyl-4(lH)-thιopyndone.

12. A method for the treatment of neurodegenerative disease such as Alzheimer's disease in a patient having said disease which compnses admmistenng to said patient a therapeutically effective dose of a composition according to claim 6 and including such compositions m which both X is 0 and Y is OH together.

13. A method for delaying the worsenmg of symptoms or delaying the onset of symptoms of neurodegenerative disease such as Alzheimer's disease m a patient susceptible to said disease which compnses admmistenng to said patient a prophylactically effective dose of a composition according to claim 6 and including such compositions in which both X is O and Y is OH together.