KR101375074B1 - Neurologically-active compounds - Google Patents

Abstract

Neuronal activity comprising two condensed 6-membered rings having a nitrogen atom at positions 1 and 3, a carboxy group at position 4 and a hydroxy group at position 8, wherein one ring is aromatic The heterocyclic compound of this is disclosed. Methods of preparing such compounds and in particular their use as pharmaceutical or veterinary medicaments for the treatment of neurological diseases, more specifically neurodegenerative diseases such as Alzheimer's disease, are disclosed.

Neurological Disease, Neurodegenerative Diseases, Alzheimer's, Heterocyclic

Description

Neuroactive compound {NEUROLOGICALLY-ACTIVE COMPOUNDS}

The present invention relates to neuroactive compounds, methods for their preparation and in particular their use as pharmaceutical or veterinary medicaments for the treatment of neurological diseases, more specifically neurodegenerative diseases such as Alzheimer's disease.

All references, including any patents or patent applications, cited herein are hereby incorporated by reference. No reference is made to constitute a prior art. The discussion of these references states what the authors of the references claim and we have the right to challenge the accuracy and suitability of the cited references. Although a number of prior art publications are mentioned in the present invention, these references do not constitute an admission that none of these documents form part of the common and general knowledge in the art, Australia or any other country. I will definitely understand.

Lifespan is believed to be biologically defined for each species, and human lifespans are indeterminate but can be up to 120 years old. Because life expectancy is rising significantly in this century, the elderly population in our population is increasing and their health care needs will continue to grow for decades.

Normal aging is characterized by moderate reductions in the mass and volume of the human brain, which can be attributed to degeneration and / or death of brain cells, but these changes are even more severe in the brains of patients who have fallen over neurodegenerative. Most of these diseases are sporadic (ie, not due to gene mutations) and the cause is unknown, but hundreds of different mutations in many genes have been shown to cause familial (genetic) modifications of several neurodegenerative disorders. Many dozens or more of these genes with these mutations have been discovered in the last decade in an effort to determine the genetic basis for neurodegenerative abnormalities. Neurodegenerative abnormalities develop gradually after prolonged normal brain function, due to the progressive degeneration of certain brain regions (ie neuronal dysfunction and death). Since symptomatic manifestations of disease appear when neuronal loss exceeds the "threshold" for sustained function (e.g. memory, exercise) performed by a diseased brain region, the actual onset of cerebral degeneration does not manifest clinical manifestations. It may be up to several years.

Intellectual and higher-level integrated cognitive abilities are progressively impaired and cause dementia by disrupting daily life with neurological abnormalities. Although the exact prevalence of dementia in the elderly population is unknown, it may be 15% of older people aged 65 or older, of which 5% are severe and 10% are mild to moderate dementia. The prevalence of severe dementia increases from 1% at age 65 to 45% at age 85. There are a number of causes of dementia, but Alzheimer's disease (AD) accounts for 50% of people over age 65.

AD is a major degenerative brain disease. This is characterized by the gradual decline of cognitive functions such as memory, thinking, understanding, calculation, language, learning ability and judgment. Dementia is diagnosed when the decline is sufficient to harm the individual's daily life. AD has a latent onset that gradually worsens. The disease needs to be clearly differentiated from the age related normal decline in cognitive function. Normal decline is much less, much more gradual, and weaker than weaker. The onset of AD is usually after age 65, but earlier onset is not uncommon. With age, the incidence increases rapidly (roughly doubles every five years). This clearly includes the total number of individuals living with the disease as life expectancy increases in the population.

The pathogenesis of AD is not clear. There is considerable evidence of hereditary predisposition for some forms of AD (St George-Hyslop, 2000), and the expression of some isoforms of ApoE has also been associated with higher AD risks (Corder et al, 1993; Czech et al 1994 ). Toxic accumulation of aluminum has been suggested as a causative agent of AD, but the hypothesis has now been discarded. The brain of AD patients shows abnormal deposits containing β-amyloid protein (Aβ).

Aβ is known to be present in the brain of individuals with some neurodegenerative diseases, but it is not known whether it is a sign of the underlying disease process or is actually related to the pathogenesis of the disease. For example, some authors believe that Aβ deposits may suggest normal brain defense mechanisms, where the brain is willing to sequester Aβ; Such deposits may be present in the brain of a normal individual. There is a mutation of the tau protein in which the nerve fiber knot except the amyloid plaque is present in the brain; This symptom is known as tauopathy.

The proposed approach to AD therapy is to inhibit the production of Aβ in the brain. Proteolytic cleavage of APP by BACE1 and γ-secretase produces Aβ of sufficient length, which is then released from the cells (Nunan and Small, 2000). Thus inhibitors of BACE1 or γ-secretase may be of therapeutic value. On the other hand, a number of studies have demonstrated that cholesterol can affect Aβ release (Simons et al., 1998; Hartmann, 2001; Fassbender et al., 2001; Frears et al., 1999; Friedhoff et al. , 2001). However, some disagreements exist in the art regarding the value of lowering cholesterol levels, and some researchers consider cholesterol to be really beneficial. For example, Ji et al. (2002) suggested that binding of Aβ to cholesterol may prevent Aβ toxicity by inhibiting oligomerization.

In another approach, by elucidating the proteolytic process of amyloid precursor protein (APP) that produces Aβ amyloid monomers, it has been suggested that a number of possible therapeutic targets may be possible (Shearman et al., 2000; Sinha et al. , 1999); This approach is in the early stages of clinical development. Attempts to promote the elimination of Aβ from the brain via immunization with Aβ have been shown to have significant adverse effects, although it has been efficacious in the transgenic mouse model for AD (Schenk et al 1999) (Brower, 2002).

It has also been suggested that deposition of fibrils such as amyloid may be important in other neurodegenerative diseases. Such diseases include Parkinson's disease, Lewis body forming dementia, multiple systemic atrophy, haloboden-sparts disease, and diffuse Louis body disease.

One of the competing theories for the pathogenesis of AD is that the causative step (s) is within the pathway of brain biogeneration and accumulation of Aβ amyloid protein (see Selkoe, 2001; Beyreuther et al., 2001; Bush, 2001). However, to date any drug or agent that targets the pathway has a lasting effect on altering the clinical manifestations of the disease or preventing or ameliorating the decline in cognitive function associated with neurodegenerative diseases including Alzheimer's disease. Not proven.

A further hypothesis is that AD is caused by the toxic accumulation of Aβ amyloid, in part due to the excessive binding of copper and zinc, which are abundant metal ions in most diseased regions. Furthermore, Zn ^{2 +} and Cu ^{2 +} Aβ has been proposed being aggregated into fibrils and the plate if the ions interact with Aβ (Atwood et al, 1998; . Recent data from the synaptic Zn ^{2 +-deficient} animals (Lee et al., 2002). Redox-active that the Cu ^{+ 2} -Aβ interactions can generate H ₂ O ₂ from O ₂ was also present (Huang et al, 1999.) . Zn ^{2 +} and Cu ^{2 +} all appeared to affect the interaction Aβ- lipid membrane (Curtain et al., 2001) .

The brain is an organ that concentrates metal ions and recent evidence suggests that destruction of metal homeostasis plays a decisive role in various age-related neurodegenerative diseases. Common features of these diseases include the deposition of misfolded proteins (each disease has its own specific amyloid protein) and significant cellular damage as a result of oxidative compression. Indeed, current metalchemical reactions include amyloidogenic neurological diseases such as Alzheimer's disease, amylotropic lateral sclerosis (ALS), prion diseases such as Creutzfeldt-Jakob disease (CJD), infectious spongiform encephalopathy (TSE), Data is rapidly accumulating that it may appear as a fundamental common factor in cataracts, mitochondrial disease, Parkinson's disease and Huntington's disease. In this case, pathological aggregation of certain proteins is facilitated by abnormal redox activity under physiological environments, typified by the presence of transition metals and available reducing agents [Bush, 2000 (Curr Opin Chem Biol. 2000 Apr; 4 (2): 184-91).

Methods of treating AD using the antibiotic iodochlorohydroxyquinoline (also known as clioquinol (CQ)) are described in US Pat. Nos. 5,994,323 and 6,001,852 (PN Geromylatos SA) and US Patent Application No. 09 / 972,913 And patented in Bush et al. CQ was recovered as an antibiotic in 1970 because of its association with subacute myeloid-visual neuropathy (SMON), the only rare neurological syndrome observed in Japan in the 1960s. It is presumed to have been administered for a longer period of time than the recommended amount at that time (Shiraki, 1975). However, recent evidence suggests that SMON was caused by abuse-related vitamin B12 deficiency in exceptionally vulnerable populations and thus could be able to receive rehabilitation for research under clinical settings (Yassin et al., 2000; Bush and Masters, 2001).

However, in vivo results in animal models or humans are not provided in the patents of Geromylatos and Bush. U. S. Patent 5,994, 323 discloses compositions comprising CQ and vitamin B12, and their use for the treatment of “diseases or diseases responsive to CQ administration while inhibiting harmful side effects”. These diseases include AD. US Pat. No. 6,001,852 discloses a method of treating AD using CQ, preferably in combination with vitamin B12. Both US Pat. Nos. 5,994,323 and 6,001,852 both suggest doses of 10 to 750 mg per day; US Pat. No. 5,994,323 recommends that if treatment is performed over an extended period of time, CQ should be provided intermittently for up to three weeks at a time, followed by a “wash” period of one to four weeks.

In US patent application Ser. No. 09 / 972,913, CQ is mentioned exclusively in terms of its ability to degrade Aβ deposits. Other neurotoxic mechanisms are not discussed. PCT / US99 / 05291 of General Hospital Corporation discloses the use of CQ in combination with certain copper and zinc chelators to promote the dissolution of amyloid plates and the inhibition of amyloid plate formation and / or ROS production by Aβ. .

US Pat. No. 6,001,852 also shows that compositions comprising CQ and vitamin B12 can be used to treat Parkinson's disease; In this regard, CQ suggests that it mainly acts through the removal of iron from the black matter.

The efficacy of CQ in the treatment of AD depends on its ability to enter the CNS and sequester the transition metals Cu, Zn and Fe from the presence of various Aβ to reduce Aβ toxicity and liberate it for removal. The effectiveness of CQ is limited by its poor water solubility which limits its oral bioavailability. CQ is also known to undergo significant conjugation mechanisms and has a history of toxicity as discussed above. The fact that CQ is a bidentate metal ligand requires the confinement of two or more molecules for every metal ion captured.

Summary of the Invention

The present invention provides a means of treating neurological diseases, including diseases characterized by abnormal reactions between proteins and metals.

International Patent Publication WO2004 / 031161 discloses a heterocyclic compound having two condensed 6-membered rings having nitrogen at position 1 and having a hydroxy or mercapto group at position 8, wherein the at least one ring Is aromatic. The compounds are particularly useful as pharmaceutical or veterinary medicaments for the treatment of neurological diseases, more specifically neurodegenerative diseases such as Alzheimer's disease.

By collectively optimizing one or more of the following properties, two condensed 6 having nitrogen atoms at positions 1 and 3, a carboxyl group at position 4 and a hydroxy group at position 8 Heterocyclic compounds have been developed having a circular ring, wherein both rings are aromatic:

(a) metal chelation (as defined herein);

(b) water solubility;

(c) reduced cytotoxicity;

(d) amyloid dispersion properties;

(e) membrane permeability suitable for CNS penetration; And

(f) metabolic stability.

The compound is within the general scope of International Patent Publication No. 2004/031161, but is not specifically disclosed above, and includes examples of therapeutic agents that are concentrated in the CNS through active transport, and in some cases, the metal in addition to the metal chelation properties. Known esters that contain antioxidant activity that enhances chelation properties and represent prodrug strategies that block 8-hydroxy residues to favor CNS penetration and are present on the inner surface of the blood brain barrier (BBB) The first activity is used.

While not wishing to be bound by any particular theory, it is believed that the nature of the substituents at positions 2 and 3 may be important for increasing plaque degradation. It may be desirable for these substituents to be planar in 3D terms. Planar substituents on the ring system allow both free ligand and metal chelate to more effectively interact with the plaque to degrade the plaque.

According to the present invention there is provided a compound of formula I, salts, hydrates, solvates, derivatives, prodrugs, tautomers and / or isomers thereof:

Where

R ² is H or CH ₂ NR ¹ R ^4, wherein R ¹ and R ⁴ is H, optionally substituted C _{1 -6} alkyl, and optionally substituted C _{3 -6} is independently selected from cycloalkyl;

R ³ is H; Optionally substituted C _1-4 alkyl; Optionally substituted C _2-4 alkenyl; Optionally substituted C _3-6 cycloalkyl; Optionally substituted 6-membered aryl or optionally substituted 6-membered aryl optionally condensed with heteroaryl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ⁶ where n is an integer from 1 to 6, R ⁶ is optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- Or 6-membered N-containing heterocyclyl or optionally substituted 6-membered aryl); NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are H, optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing hetero Independently selected from cyclyl and optionally substituted 6-membered aryl); NHCOR ¹⁰ wherein R ¹⁰ is optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl or optionally substituted 6 -Raw aryl); CH ₂ CONR ¹¹ R ¹² , wherein R ¹¹ and R ¹² are H, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkynyl and optionally substituted 6-membered aryl, optionally substituted with optionally substituted Independently selected from 5- or 6-membered N-containing heterocyclyl); And (CH ₂ ) _m NHR ¹³ where R ¹³ is selected from optionally substituted C _1-6 alkyl and SO ₂ R ¹⁴ , wherein R ¹⁴ is optionally substituted C _1-6 alkyl and optionally substituted 6-membered Aryl, m is 1 to 6);

R ⁵ and R ⁷ are independently selected from H and halo;

X is O or S

(i) at least one of R ² and R ³ is other than H;

(ii) at least one of R ⁵ and R ⁷ is halo;
(iii) when X is O, R ⁵ and R ⁷ are Hl and R ² is H, R ³ is not cyclopropyl or parafluorophenyl;
(iv) when X is O, R ⁵ is H, R ⁷ is I and R ² is H, then R ³ is not C _2-4 alkyl.

The invention also provides the use of a compound of formula I as a medicament, preferably a neurotherapeutic or neuroprotective agent, more preferably an amyloidogenic inhibitor. Preferably, the neurological disease is a neurodegenerative disease, more preferably neurodegenerative amyloidosis, for example Alzheimer's disease or Parkinson's disease.

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The compound of formula (I) is advantageously administered in the form of a pharmaceutical or veterinary composition together with a pharmaceutically or veterinary acceptable carrier.

Accordingly, the present invention also provides a pharmaceutical or veterinary composition comprising a compound of formula (I) and a pharmaceutically or veterinary acceptable carrier.

According to the present invention, there is also provided a method for treating, ameliorating and / or preventing a disease comprising administering to a patient in need thereof an effective amount of a compound of formula (I).

Further according to the invention there is also provided the use of a compound of formula I in the manufacture of a medicament for the treatment, amelioration and / or prevention of neurological diseases.

The invention also provides the use of a compound of formula (I) for the treatment, amelioration and / or prevention of neurological diseases.

The invention also provides a compound of formula (I) for use in the treatment, amelioration and / or prevention of neurological diseases.

The present invention furthermore

(a) reacting an optionally protected compound of formula V with H ₂ NR ^3, wherein R ³ is as defined above to form an optionally protected compound of formula VII:

In which R ⁵ and R ⁷ are as defined above;

Reducing the compound of Formula VII to form an optionally protected compound of Formula VIII:

Cyclizing the compound of Formula VIII to form an optionally protected compound of Formula I wherein R ² is H; or

In the presence of R ² CHO, R ² CO ₂ H or R ² C (OR ^x ) wherein R ^x is optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl and R ² is as defined above Cyclization of the Compound of Formula VIII

It provides a method of preparing a compound of formula (I) as defined above.

The present invention also

(a) Aminating an optionally protected compound of formula VI to form an optionally protected compound of formula IX:

In which R ⁵ and R ⁷ are as defined above;

(b) reacting the compound of Formula IX with R ³ -L or R ³ OSO ₂ R ^x , wherein L is a leaving group, R ³ is as defined above and R ^x is as defined above

Provided is a process for the preparation of compounds of formula (I) as defined above, wherein R ² is H.

The present invention also

(a) reacting an optionally protected compound of formula (VI) as defined above with a formylating agent to form an optionally protected compound of formula (X) or optionally protected compound of formula (XI):

(b) reacting the compound of formula X or XI with an acylating agent containing R ² to form an optionally protected compound of formula VII or a compound of formula XIII:

(Wherein R ² is as defined above)

(c) reacting the compound of formula XII or XIII with H ₂ NR ^3, wherein R ³ is as defined above

Provided is a process for the preparation of compounds of formula (I) as defined above, wherein R ² is H.

It will be appreciated that the protecting groups, if present, may be removed at any suitable stage of the methods described above.

The inventors also found a less complex method of producing precursors of intermediates of Formulas V and VI described later, in high yield, when both R ⁵ and R ⁷ are halo.

Accordingly, there is also provided a process for the preparation of a compound of formula IV comprising the step of diazotizing a compound of formula III according to the process of the invention:

(상기 식에서, R⁵ 및 R⁷은 할로 중에서 독립적으로 선택된다)

(Wherein R ⁵ and R ⁷ are independently selected from halo)

Wherein R ⁵ and R ⁷ are as defined in Formula IV above.

The compound of formula III is prepared by reducing the compound of formula II for convenience:

Wherein R ⁵ and R ⁷ are as defined in Formula IV above.

The compound of formula IV is a precursor in the preparation of intermediates of formulas V and VI which can be used to prepare compounds of formula II:

(V)

(VI)

Wherein R ⁵ and R ⁷ are as defined in Formula IV above.

Thus, the present invention also provides

(a) diazotizing a compound of formula III as defined above to form a compound of formula IV as defined above;

(b) nitriding the compound of formula IV

Provided is a process for the preparation of the compound of formula V as defined above comprising the steps.

The present invention also

(a) diazotizing a compound of formula III as defined above to form a compound of formula IV as defined above;

(b) nitriding the compound of formula IV to form a compound of formula V as defined above;

(c) reducing the compound of formula IV as defined above

Provided is a process for preparing a compound of formula VI as defined above, wherein R ⁵ and R ⁷ are as defined in formula IV above, comprising the steps.

As used herein, unless otherwise indicated, the term "comprises" or variations thereof, such as "comprising" and the like, are used to mean inclusion, i.e., to specify the presence of the described features, but Various embodiments of the invention do not exclude the presence or addition of additional features.

As used herein, the singular forms "a" and "the" are intended to include the plural aspects unless otherwise indicated. Thus, for example, reference to "a compound" includes not only a single compound but also two or more compounds.

Preferred compounds of formula (I) are those of formula (IA):

Where

R ⁵ , R ⁷ and X are as defined in formula (I) above;

R ^3A is optionally substituted C _1-4 alkyl; Optionally substituted C _2-4 alkenyl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ⁶ where n is 1-3 and R ⁶ is optionally substituted C _3-6 cycloalkyl or optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl ; NR ⁸ R ⁹ , wherein R ⁸ is H and R ⁹ is H or optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl; NHCOR ¹⁰ , wherein R ¹⁰ is optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl.

Preferably, both R ⁵ and R ⁷ are halo, more preferably chloro.

Illustrative examples of the compounds of formula (IA) are shown below.

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In illustrative examples of Formula IA compounds, compounds 1076, 1077, 1082, 1083, 1084, 1085, 1087, 1088, 1089, 1091, 1092, 1093, 1097, 1098, 1099, 1100, 1101, 1107, 1108, 1109, 1110, 1112, 1115 and 1126 are planar substituent in position 3, for example, an optionally substituted C _{1 -4} alkyl; Optionally substituted C _{1 -4} alkenyl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl; (CH _{2) n} R ⁶ (where n is from 1 to 3, R ⁶ is an optionally substituted C _{3 -6} cycloalkyl or optionally containing saturated or unsaturated optionally substituted 5- or 6-membered N-heterocyclyl) ; And NR ⁸ R ⁹ (where R ⁸ is H, and R ⁹ is H or an optionally substituted C _{1 -4} alkyl or optionally substituted 6-membered aryl) has a. Of these planar compounds of formula (IA), compounds 1100 and 1101 also show very good degradation.

Another preferred compound of formula I is a compound of formula IB:

Where

R ² , R ⁵ , R ⁷ and X are as defined in formula (I) above.

Preferably R ² is CH ^₂ NR ¹ R _2, wherein R ¹ and R ⁴ are independently selected from H, optionally substituted C _{1 -6} alkyl, and optionally substituted C _{3 -6} cycloalkyl.

Preferably both R ⁵ and R ⁷ are halo, more preferably chloro.

Exemplary compounds of formula (IB) are shown below.

Illustrative examples of the general formula IB are both in position 2 plane has a substituent, for example, CH ₂ NR ¹ R ^4, wherein R ¹ and R ⁴ are independently selected from optionally substituted C _{1 -6} alkyl. Compound 1128 also shows very good degradation.

A further subclass of compounds of Formula I are compounds of Formula IC:

Where

R ⁵ , R ⁷ and X are as defined in formula (I) above;

R ^1C is CH ₂ NR ¹ R ⁴ (wherein R ¹ and R ⁴ are independently selected from H and optionally substituted C _{1 -6} alkyl);

R ^3C is an optionally substituted C _{1 -4} alkyl.

Preferably both R ⁵ and R ⁷ are halo, more preferably chloro.

Exemplary compounds of formula IC are shown below.

Blocking 8-hydroxy groups on compounds of formula I can form prodrugs, particularly ester prodrugs. The 8-hydroxy represents the major metabolic site of the compound of formula I: it provides hydrophilic species which are secreted directly with glucuronic acid or sulfate. Such conjugates presumably do not cross the blood brain barrier layer. The ester prodrug may protect the compound of formula (I) from binding. The esterase incorporated into the blood brain barrier layer may then release C8-hydroxy as it passes through the barrier layer to activate the compound for its role in the CNS.

Without wishing to be bound by theory, it is believed that the substituents R ³ and R ⁵ have an electronically or stericly limited effect on the chelating properties of the compounds of the invention. Thus, substitutions can be used to control other variables such as cytotoxic and physicochemical properties such as the number, lipophilicity (ClogP, ElogP and LogD), solubility and polar surface area of the hydrogen bond donors and receptors. Modulation of these variables contributes to the optimization of the pharmacokinetic profile of the compound. It is also hypothesized that substituents R ² and R ⁷ may also affect activity if the substituents provide chelating properties in addition to control of cytotoxicity and physicochemical properties.

Alone or used in compound terms such as "optionally substituted C _{1 -4} alkyl,""C _{1 -6} alkyl" or "C _{1 -4} alkyl" refers to straight chain, each having 1 to 6 carbon atoms and 1 to 4 or Refers to a branched chain hydrocarbon group. Such alkyl groups are exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary-butyl, tert-butyl, pentyl, neopentyl or hexyl, preferably methyl, ethyl or propyl.

The term "CH ₂ ) _n " or "(CH ₂ ) _m " as used herein includes both straight and branched chains.

Alone or in the "optionally substituted C _{2 -6-alkynyl"} and the "C _1-6 alkynyl" refers to 2 to 6 carbon atoms, and also linear or branched hydrocarbon group having one triple bond such as the compounds used in the term Refers to. Such groups include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, Illustrated by 3-hexynyl, 4-hexynyl and 5-hexynyl.

Alone or used in compound terms such as "optionally substituted C _{3 -6} cycloalkyl", "C _{3 -6} cycloalkyl" refers to saturated cyclic group between the carbonyl having 3 to 6 carbon atoms. Such groups are exemplified by cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, preferably cyclopropyl.

"Optionally substituted 6-membered aryl" used alone or in a compound term such as "optionally substituted unsaturated or saturated 5- or 6-membered N-containing heterocyclyl group optionally condensed with optionally substituted 6-membered aryl" And optionally condensed saturated or unsaturated 5- or 6-membered N-containing heterocyclyl groups "means monocyclic or polycyclic heterocycles containing one or more nitrogen atoms and optionally other heteroatoms selected from sulfur and oxygen. Refers to a click group.

Suitable heterocyclic groups include N-containing heterocyclic groups, for example

Unsaturated 5- or 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyridinyl, pyrimidinyl, Pyrazinyl, pyridazinyl, triazolyl or tetrazolyl;

Saturated 5- or 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms such as pyrrolidinyl, imidazolidinyl, piperidino or piperazinyl;

Unsaturated, condensed heterocyclic groups containing 1 to 5 nitrogen atoms, for example indolyl, isoindoleyl, indolinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotria Zolyl or tetrazolopyridazinyl;

Unsaturated 5- or 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as oxazolyl, isoxazolyl or oxdiazolyl;

Saturated 5- or 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example morpholinyl;

Unsaturated 5- or 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example thiazolyl or thiadiazolyl; And

Saturated 3 to 6-membered heteromonocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example thiazolidinyl.

Preferably, the heterocyclyl is an unsaturated 5 or 6 membered heteromonocyclic group containing 1 to 3 nitrogen atoms, for example pyrazolyl, pyridinyl or pyrimidinyl; Saturated 5 or 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example pyrrolidinyl or piperazinyl; Unsaturated, condensed heterocyclic groups containing 1 to 5 nitrogen atoms, for example benzimidazolyl; Saturated 5 or 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example morpholinyl; Or an unsaturated 5- or 6-membered heteromonocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 oxygen atoms, for example thiazolyl.

The term "6-membered aryl" used alone or in compound terms such as "optionally substituted 6-membered aryl" refers to a 6-membered carbocyclic aromatic group. Such aryl groups are exemplified by phenyl. Preferably the aryl is optionally substituted phenyl, for example 4-halophenyl, more preferably 4-fluorophenyl.

The term "6-membered heteroaryl" used alone or in a compound term such as "optionally substituted 6-membered heteroaryl" refers to a 6-membered aromatic heterocycle containing one or more heteroatoms. Examples include pyridyl pyrazinyl, pyrimidinyl and pyridazinyl, which may each be optionally substituted by methyl or methoxy.

The term "halo" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, iodine or chlorine, more preferably chlorine.

The term "optionally substituted" means alkyl, alkenyl, alkynyl, aryl, aldehyde, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy , Haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino , Arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, arylsulphenyloxy, heterocycle In the aryl, heterocycleoxy, heterocycleamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, benzylthio, acylthio, phosphorus-containing groups and the like In addition to one or more groups selected refers to a group that may or may not be substituted or unsubstituted. Preferably, the optional substituents are C _1-4 alkyl, hydroxy, fluorine, C _{1 -4} alkoxy, or C _{1 -4} acyl.

The term "protecting group" refers to an introduced functional group that can render a particular functional group, such as a hydroxy, amino, carbonyl or carboxy group, unreactive under certain conditions and later optionally removed to reveal the functional group. A hydroxy protecting group is a group that can make a hydroxy group temporarily non-reactive. A hydroxy protecting group refers to a hydroxy group which is made temporarily non-reactive by the hydroxy protecting group. By protected phenyl group is meant a group in which a bound reactive substituent such as OH, NH ₂ is protected by a protecting group. Suitable protecting groups are known in the art and are described in TW Greene and PG White, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, Inc., 1999 (incorporated herein by reference). A method of installation and removal is disclosed. Examples of protecting groups that can be used to protect hydroxy groups include, but are not limited to, silyl groups (eg trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), benzyl groups (eg benzyl, Methoxybenzyl, nitrobenzyl), alkyl groups (eg methyl, ethyl, n- and i-propyl, and n-, secondary- and t-butyl) and acyl groups (eg acetyl and benzoyl) .

Leaving groups may be of any suitable known type, for example as described in J. Departure groups disclosed in March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure" 4th Edition, pp 352-357, John Wiley & Sons, New York, 1992, incorporated herein by reference. Preferably, the leaving group is a halogen.

The term "metal chelator" is used herein in the broad sense and refers to a compound having at least two donor atoms capable of bonding to a metal atom, preferably Cu, Zn or Fe, wherein said donor atom At least two of them may be bonded to the metal atom at the same time, the resulting metal complex; Biological ligand complexes have thermodynamic stability equal to or greater than the metal ion. The use of such metal chelators in the treatment of neurological diseases in accordance with the present invention is distinguished from the known concept of "chelation therapy". "Chelation therapy" is a term that is clinically related to the removal of bulk metals, such as in Wilson's disease, thallesemia and hemochromatosis. The destruction of metal homeostasis in the disease can be described as a catastrophic event such as dam destruction causing overwhelming flooding of problem metals. The mechanism of action of such compounds is to isolate the bulk metal by chelator and remove it by secretion. In comparison, the breakdown of the metal homeostasis associated with the neurological anomalies of the present invention is more like a constant drip of the leaking tab, which will eventually cause local damage over a long period if left long enough. The purpose of the "metal chelator" of the present invention is to destroy abnormal metal-protein interactions to achieve a tight distribution of metals, and then once the toxicity cycle is stopped, endogenous removal processes will more effectively cope with the accumulation of amyloid-forming proteins. The aim is to normalize the metal distribution.

Salts of compounds of formula (I) are preferably pharmaceutically acceptable, but pharmaceutically unacceptable salts are also within the scope of the present invention because they are useful as intermediates in the preparation of pharmaceutically acceptable salts. to be. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; Pharmaceutically acceptable inorganic acids such as acid addition salts of hydrochloric acid, orthophosphoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid and hydrobromic acid; Or pharmaceutically acceptable organic acids, such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, mucinic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfuric acid Phonic acid, trihalomethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and There is a salt of valeric acid.

In addition, some of the compounds of the present invention may form solvates with water or conventional organic solvents. Such solvates are included within the scope of the present invention.

Preferably the derivative is a "pharmaceutically acceptable derivative". The term "pharmaceutically acceptable derivative" refers to any pharmaceutically acceptable salt, hydrate, ester, amide, active metabolite that is biologically or otherwise undesirable and induces the desired pharmacological and / or physiological effect. , Homologue, residue or any other compound.

The term “prodrug” is used in the broad sense of the present invention and includes compounds which are converted into the compound of formula I in vivo. The use of this prodrug strategy optimizes delivery of the drug to the site of action, for example the brain. In one embodiment, the term means that the prodrug crosses the BBB, where the esterase on the inner surface of the BBB acts to hydrolyze the ester to liberate the C8 hydroxyl of the compound of formula (I). It refers to a C _{1 -6} alkyl, or the presence of an aryl ester moiety designed to resist hydrolysis. In a second aspect, the term refers to the attachment at position 2 of the 8-hydroxyquinoline core of an antioxidant group, in particular a 3,4,5-trimethoxyphenyl residue or derivative thereof. Exposure of the brain to the preoxidative environment will then hydroxylate the 3,4,5-trimethoxyphenyl group to produce a 2-hydroxy-3,4,5-trimethoxyphenyl substituent, the substituent The hydroxyl group of acts to enhance the chelation properties of the compound of formula (I).

The term "antioxidant" is used in the broad sense of the present invention and refers to a group having the ability to react with reactive oxygen species, such as hydroxyl radicals, in a manner that produces non-toxic products. Examples are phenols such as 3,4,5-trimethoxyphenyl and 3,5-di-t-butyl-4-hydroxyphenyl, indole amines such as melatonin and flavonoids. Other examples can be found in the literature (Wright, 2001; Karbownik, 2001; Gilgun-Sherki, 2001).

The term "tautomer" is used herein in the broad sense and includes compounds of formula I which may exist in equilibrium between two isomeric forms. Such compounds may differ in the position at which they bond and link the two atoms or groups in the compound.

The term "isomer" is used herein in the broad sense and includes structures, geometries and stereoisomers. Since the compound of formula (I) may have one or more chiral centers, it may exist in enantiomeric form.

The compositions of the present invention comprise at least one compound of formula (I) together with at least one pharmaceutically acceptable carrier and optionally other therapeutic agents. Each carrier, diluent, adjuvant and / or excipient should be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and harmless to the patient. The compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may be presented in unit dosage form for convenience and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and tightly associating the active ingredient with the liquid carrier, diluent, adjuvant and / or excipient or finely divided solid carrier, or both, and optionally molding the product.

The term "nerve disease" is used in the broad sense of the present invention and refers to a condition in which various cell types of the nervous system are degenerated and / or damaged as a result of neurodegenerative disease or injury or exposure. In particular, the compounds of formula (I) can be used to treat symptoms produced by damage to nervous system cells resulting from surgical intervention, infection, exposure to toxic agents, tumors, nutritional deficiencies or metabolic diseases. Compounds of formula (I) may also be used for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amylotropic lateral sclerosis, epilepsy, drug abuse or drug addiction (alcohol, cocaine, heroin, amphetamines, etc.), spinal cord disease, and And / or for the treatment of sequelae of peripheral neuropathy caused by injury, malnutrition or degeneration of the retina (retinopathy) and peripheral neuropathies such as diabetic neuropathy and / or toxicity.

As used herein, the term "neurodegenerative disease" refers to an abnormality that threatens nerve preservation. Nerve conservation can be threatened if neurons exhibit reduced survival or neurons can no longer transmit signals.

Neurological diseases that can be treated by the compounds of the present invention include acute intermittent porphyria; Adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 induced neurotoxicity; Alzheimer's disease; Amylotropic lateral sclerosis; Atherosclerosis; Cataract; Cerebral ischemia; Cerebral palsy; Brain tumors; Chemotherapy-induced organ damage; Cisplatin-induced nephrotoxicity; Coronary artery bypass surgery; Its new variant associated with Creutzfeldt-Jakob disease and "mad cow" disease; Diabetic neuropathy; Down syndrome; Drowning; Epilepsy and post-traumatic epilepsy; Friedrich's disability; Forehead dementia; glaucoma; Glomerulopathy; Hemochromatosis; Hemodialysis; Hemolysis; Hemolytic uremic syndrome (fruit disease); Hemorrhagic seizures; Haloboden-sparts disease; Heart attack and reperfusion injury; Huntington's disease; Lewy body disease; Intermittent claudication; Ischemic attack; Inflammatory bowel disease; Macular degeneration; malaria; Methanol induced toxicity; Meningitis (aseptic and tuberculosis); Motor neuron disease; Multiple sclerosis; Multiple systemic atrophy; Myocardial ischemia; tumor; Parkinson's disease; Choking before and after birth; Pick disease; Jin planetary nuclear paralysis; Radiotherapy-induced organ damage; Restenosis after angioplasty; Retinopathy; Senile dementia; Schizophrenia; blood poisoning; Septic shock; Cavernous encephalopathy; Subarachnoid hemorrhage / cerebral vasospasm; Subdural hematoma; Surgical trauma including nerve surgery; Thalassemia; Transient ischemic attack (TIA); Traumatic brain injury (TBI); Traumatic spinal injury; transplantation; Vascular dementia; Viral meningitis; And viral encephalitis.

In addition, the compounds of the present invention can also be used to enhance other therapeutic effects, for example to enhance the neuroprotective effects of brain derived nerve growth factors.

The present invention particularly relates to oxidative damage of the central nervous system, such as acute and chronic neurological diseases such as traumatic brain injury, spinal cord injury, cerebral ischemia, seizures (ischemic and hemorrhagic), subarachnoid hemorrhage / cerebral vascular spasm, cerebral trauma. , Alzheimer's disease, Creutzfeldt-Jakob disease and its new variants related to "mad cow" disease, Sintington's disease, Parkinson's disease, Friedrich's dysfunction, cataracts, dementia associated with Louis body formation, multiple systemic atrophy, Haloboden-Sparts disease, Regarding symptoms causing hereditary cerebral bleeding with diffuse Lewis body disease, amylotropic lateral sclerosis, motor neuron disease, multiple sclerosis, fatal familial insomnia, Gerzmann Straussler Scheinker disease and Dutch-amyloidosis will be.

More particularly, the present invention relates to the treatment of neurodegenerative amyloidosis. The neurodegenerative amyloidosis may be any symptom arising from nerve damage resulting from abnormal interactions between redox active metal ions and biological ligands such as proteins that promote the formation, radicalization and / or deposition of reactive oxygen species. have. The amyloid can be formed from various protein or polypeptide precursors, such as but not limited to Aβ, synuclein, huntingtin, or prion protein.

Thus the disease is preferably a sporadic or familial Alzheimer's disease, amiotropic lateral sclerosis, motor neuron disease, cataracts, Parkinson's disease, Creutzfeldt-Jakob disease and its new variant associated with "mad cow" disease, Huntington's disease, Louis Dementia with body formation, multiple systemic atrophy, haloboden-spartz disease and diffuse Louis body disease.

More preferably the neurodegenerative amyloidosis is dementia associated with Aβ-related symptoms, such as Alzheimer's disease, or one of autosomal dominant forms of Down's syndrome or several forms of familial Alzheimer's disease (St George-Hyslop, 2000). ). Most preferably the Αβ-associated disease is Alzheimer's disease.

In a particularly preferred embodiment of all aspects of the invention, prior to treatment, the patient has a cognitive function that is moderately or severely impaired as assessed by the Alzheimer's Disease Assessment Grade (ADAS) -cog test, eg, an ADAS-cog value of 25 or greater. .

In addition to slowing or stopping cognitive decline in a patient, the methods and compounds of the present invention may also be suitable for use in the treatment or prevention of neurodegenerative abnormalities or for use in alleviating symptoms of neurodegenerative abnormalities. The compound may provide at least partial reversal of cognitive decline experienced by the patient. When administered to a patient identified as having an increased risk of neurodegenerative disorders, or to a patient with preclinical signs of cognitive decline, such as mild cognitive impairment or minimal progressive cognitive impairment, the methods and compounds In addition to slowing or reducing the rate, the onset of clinical signs can be prevented or delayed.

Currently Alzheimer's disease and other dementia are usually not diagnosed until one or more warning signs appear. These signs constitute a syndrome known as mild cognitive impairment (MCI), which was recently defined by the American Neurological Society and has memory impairment but others function well and meet clinical dementia criteria (Petersen et al., 2001). Refers to the clinical condition of an individual who does not. Symptoms of MCI include:

(1) memory loss affecting professional skills

(2) Difficulties in carrying out familiar tasks

(3) language problems

(4) disorientation of time and place (lost)

(5) poor or reduced judgment

(6) the problem of abstract thinking

(7) misplacement of things

(8) change in mood or behavior

(9) personality change

(10) loss of initiative

MCI can be detected using conventional cognitive screening tests such as small scale mental state testing, and memory impairment screening, and neuropsychological screening comprehensive tests.

As used herein, the term "patient" refers to any animal having a disease or condition in need of treatment using a pharmaceutically effective agent. The patient can be a mammal, preferably a human or a domestic animal or a familiar animal. Although the compounds of the present invention are specifically contemplated as being suitable for use in human medical treatment, they are also veterinary treatments, for example familiar animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys. , Mules, llamas, alpacas, pigs, cows and sheep, or zoo animals such as primates, felines, canines, bovines, and ungulates.

Suitable mammals include members of the primates, rodents, rabbits, whales, carnivores, bases and cows. Members of the bases and cows are particularly preferred because of their familiar biological and economic importance.

For example, the cow worm includes approximately 150 live species through nine families: pigs (Suidae), peckerry (Tayasuidae), hippopotamuses (Hippotamidae), and camels (camelidae) , Deer (tragulidae), giraffes and okapis (girapidae), deer (servidae), pronghorn (antirocapidae) and cattle, sheep, goats and antelopes (bovidae). Many of these animals are used as edible animals in various countries. More importantly, many of the economically important animals, such as goats, sheep, cattle and pigs, have very familiar ecology and share a high degree of genomic homology.

Bases include horses and donkeys, all of which are economically important and closely related. In fact, horses and donkeys are widely known for their hybrid breeding.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the present invention effective to provide a desired therapeutic response, eg, for the prevention or treatment of neurological diseases.

A particular “therapeutically effective amount” clearly refers to such things as the particular disease being treated, the physical condition of the patient, the type of patient being treated, the duration of treatment, the nature of the accompanying therapy (if any) and the structure of the particular formulation and compound or derivative thereof used. Will change according to the argument.

The compounds of the present invention may also be combined with other agents to provide effective cooperation. It is intended to include combinations of any chemically suitable pharmaceutically effective agents unless such combinations eliminate the activity of the compounds of formula (I) or (II). It will be appreciated that the compounds of the invention and other agents may be administered separately, continuously or simultaneously.

Other agents include, for example, inhibitors of acetylcholinesterase active sites, such as penserine, galantamine, or tacrine, if the condition is β-amyloid related disease, especially Alzheimer's disease; Antioxidants such as vitamin E or vitamin C; Anti-inflammatory agents such as flurbiprofen or ibuprofen (for example NCX-2216 produced by NicOx) or estrogen generating agents, such as 17-β-estradiol, optionally modified to release nitrogen oxides. have.

Methods and pharmaceutical carriers for the preparation of pharmaceutical compositions are well known in the art and are listed, for example, in Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA.

As used herein, a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering a compound of Formula I or II to a patient. The carrier may be liquid or solid, and the chosen mode of administration is carefully selected. Each carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and harmless to the patient.

The compounds of formula (I) can be administered orally, topically or non-orally in unit dosage forms containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term nasal oral as used herein includes subcutaneous injection, aerosol for pulmonary or nasal administration, nasal, intramuscular, intradural, intracranial injection or infusion techniques. The invention also provides topical, oral and non-oral pharmaceutical formulations suitable for use in the novel therapies of the invention. The compounds of the invention can be administered orally as tablets, aqueous or oleaginous suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The oral composition may contain one or more agents selected from the group of sweetening, flavoring, coloring and preservatives for the manufacture of pharmaceutically aesthetic and refreshing preparations. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavoring agents include peppermint oil, winter green oil, cherry, orange or raspberry flavors. Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time retardants include glyceryl monostearate or glyceryl distearate. Tablets contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

Such excipients include, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such as corn starch or alginic acid; (3) binders such as starch, gelatin or acacia; And (4) lubricants such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to retard disintegration and absorption in the gastrointestinal tract and thereby provide sustained action over a longer period of time. For example, a time delay substance such as glyceryl monostearate or glyceryl distearate can be used. The coatings are also described in US Pat. No. 4,256,108 to prepare osmotic therapeutic tablets for controlled release; 4,160,452; And the techniques disclosed in US Pat. No. 4,265,874.

The compounds of formula (I) as well as pharmaceutically effective agents useful in the methods of the invention can be administered orally or independently by injection or by gradual perfusion over time for in vivo application. Administration can be effected by intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal or infusion, for example by infusion with an osmotic pump. For in vitro studies, the agents can be added or dissolved in suitable biologically acceptable buffers and added to cells or tissues.

Formulations for non-oral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohols / aqueous solutions, emulsions or suspensions such as saline and buffered media. Non-oral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, while lactated Ringer's vein vehicles are based on fluid and nutritional supplements, electrolyte supplements (e.g., based on Ringer's dextrose). We include thing). Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, growth factors and inert gases and the like may also be present.

In general, the terms "treating", "treatment", etc., as used herein, mean affecting a patient, tissue, or cell to achieve the desired pharmacological and / or physiological effect. The effect may be prophylactic in connection with completely or partially preventing the disease or its signs or symptoms and / or therapeutic in connection with the partial or complete cure of the disease. As used herein, “treating” encompasses any treatment, amelioration or prevention of a disease in vertebrates, mammals, especially humans, and (a) the disease may be predisposed to the disease but is still ailment. Prevent occurrence in undiagnosed patients; (b) inhibit the disease, ie, stop its progression; Or (c) alleviating or ameliorating the effects of the disease, ie regressing the effects of the disease.

The present invention includes various pharmaceutical compositions useful for ameliorating diseases. A pharmaceutical composition according to one embodiment of the invention may be prepared by the use of a carrier, excipients and additives or auxiliaries in combination with a compound of formula (I), a homolog, derivative or salt thereof, or a combination of a compound of formula (I) with one or more pharmaceutically active agents By making it into a form suitable for administration to a patient. Commonly used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, vitamins, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycols and solvents such as Sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluids and nutritional supplements. Preservatives include antibacterial agents, antioxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers are described, for example, in Remington's Pharmaceutical Sciences, 20th ed. Williams and Wilkins (2000) and The British National Formulary 43rd ed., British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http://bnf.rhn.net, the contents of which are incorporated herein by reference, include aqueous solutions, nontoxic excipients such as salts, preservatives, buffers and the like. The pH and precise concentrations of the various components of the pharmaceutical composition are adjusted according to conventional techniques in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics, 7th ed., 1985.

The pharmaceutical composition is preferably prepared and administered in dosage units. Solid dosage units can be tablets, capsules and suppositories. For the treatment of patients, daily doses that differ depending on the activity of the compound, the mode of administration, the nature and severity of the disease, the age and weight of the patient can be used. However, under certain circumstances, more or less daily doses may be suitable. Administration of the daily dose may be effected by single administration in individual dosage units or in the form of many other smaller dosage units, and by multiple administrations of the subdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may be administered locally or systemically at therapeutically effective doses. The amount effective for this use will of course depend on the severity of the disease and the weight and general condition of the patient. Typically, doses used in vitro provide useful guidance in amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective doses for the treatment of cytotoxic side effects. Various considerations are disclosed, for example, in Langer, Science, 249: 1527 (1990). Oral formulations may be in the form of hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules, wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions usually contain the active substance in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include (1) suspending agents, for example sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) (a) natural phosphatides such as lecithin; (b) condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate; (c) condensation products of ethylene oxide with long chain fatty alcohols such as heptadecaethyleneoxyethanol; (d) condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, for example polyoxyethylene sorbitol monooleate, or (e) ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides Condensation products, such as polyoxyethylene sorbitan monooleate, which may be a dispersing or wetting agent.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension. The suspension can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic orally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any mild nonvolatile oil can be used including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

The compounds of formula (I) can also be administered in the form of liposome delivery systems such as small monolayer vesicles, large monolayer vesicles and multilayer vesicles. Liposomes can be prepared from various phospholipids such as cholesterol, stearylamine or phosphatidylcholine.

Compounds of formula (I) may also be provided for use in the form of a veterinary composition, wherein the composition may be prepared, for example, by methods conventional in the art. Examples of such veterinary compositions are those suitable for:

(a) oral administration, external application, eg a potion (eg an aqueous or non-aqueous solution or suspension); Tablets or ingots; Powders, granules or pellets for mixing with the feed; Paste for application to the tongue;

(b) subcutaneous, intramuscular or intravenous injections, eg as sterile solutions or suspensions; Or (or, if appropriate), oral administration by intramammary injection, introducing a suspension or solution into the udder through the nipple;

(c) topical application, eg as a cream, ointment or spray applied to the skin; or

(d) In the vagina, for example as a pessary, cream or foam.

Dosage levels of the compounds of formula I of the present invention range from about 0.5 to about 20 mg / kg body weight, with a preferred dosage range of about 0.5 to about 10 mg / kg body weight per day (about 0.5 to about 3 gms per patient per day). to be. The amount of active ingredient that can be combined with a carrier material to produce a single dose will vary depending upon the host treated and the particular mode of administration. For example, formulations for oral administration to humans may contain about 5 mg to 1 g of active compound together with a suitable and convenient amount of carrier material (which may vary from about 5 to 95% of the total composition). Dosage unit forms will generally contain about 5 to 500 mg of active ingredient.

Optionally, a compound of the invention is administered on a divided dose schedule such that a total of two or more administrations are scheduled. Administration is preferably carried out at least every 2 hours for at least 4 hours; For example, the compound may be administered hourly or every 30 minutes. In one preferred embodiment, the divided dose regimen of the compound of the invention after a period from the first dose is long enough that the level of the active compound in the blood is reduced to about 5-30% of the maximum plasma level after the first dose. Including the second administration, an effective amount of the effective agent in the blood is maintained. Optionally, one or more subsequent administrations can be performed at corresponding intervals from each preceding administration, preferably when the plasma level is reduced by about 10-50% of the immediately preceding maximum value.

However, the specific dosage level for any particular patient will depend on the activity, age, weight, general health, sex, diet, time of administration, route of administration, rate of secretion, drug combination and severity of the particular disease being treated for the particular compound used. It will, of course, vary with the various factors involved.

The present invention will now be described in detail with reference only to the following non-limiting examples.

For clarity, the compounds of the present invention are indicated by numbers, for example 1-4 and 2-3. The structures of the example compounds referred to above are shown in Tables 1-10.

In Examples 1-6, the following references are cited:

$

General Experiment Details

2,4-Dichlorobenzoic acid (1-1) and 2,4-dichloro-6-nitrophenol (2-1B) were purchased from Aldrich. All reagents / reactants were supplied from Aldrich unless otherwise indicated. 4-amino-1,3,5-trimethylpyrazole, 2-amino-4,6-dihydroxypyrimidine, 4-chloromethyl-3,5-dimethylisoxazole and 2-chloromethyl-4 Methylthiazole hydrochloride was purchased from Lancaster. (2-aminomethyl) thiazole was prepared according to document ¹ . The solvent was analytical grade and used as supplied. THF was distilled from sodium and benzophenone under argon. ¹ H NMR spectra (δ, relative to TMS) were recorded on a Varian Inova 400 spectrometer unless otherwise indicated; J-values are expressed in Hertz. Mass spectral data was recorded on a Micromass Quattro II mass spectrometer.

4,6-dichloro-3-hydroxy, which is the main intermediate used in the preparation of a range of 8-hydroxy-3H-quinazolin-4-one and specifically in the synthesis of 5,7-dichloro-substituted derivatives A practical and concise synthesis of 2-nitrobenzoic acid (1-6) is shown in Scheme 1A.

Thus, according to Goldstein and Schaaf ² , 2,4-dichloro-5-nitrobenzoic acid (1) was nitrided with commercially available 2,4-dichlorobenzoic acid (1-1). -2) is obtained. Compound 1-2 is converted to 3-acetamido-4,6-dichloro-2-nitrobenzoic acid (1-5) via amines 1-3 and acetamide 1-4. Subsequent base hydrolysis of 1-5 gives post-treatment with acid followed by 2-nitrobenzoic acid 1-6. All steps can be performed at high yields (approximately 90%) and scaled up. Compounds 1-6 can also be prepared according to the route shown in Scheme 1B. Thus, diazotization of 1-3 through standard or customary conditions ³ affords alcohol 1-7. Compound 1-7 is then nitrided according to the conditions previously disclosed in Document ⁴ to give 1-6. The synthetic route shown in both Schemes 1A and 1B for the preparation of 1-6 represents an improvement over the document ⁴ method, wherein all steps appear to proceed in good yield. Reduction of 1-6 with iron powder in HOAc at 80 ° C. for 45-50 minutes gives the corresponding amines 1-8 in 94% yield after standard workup. It is noteworthy that the reduction of 1-5 through catalytic hydrogenation ⁴ gave only 14% amine 1-8 yield.

A range of novel 8-hydroxy-3N-substituted quinazolin-4-ones of the present invention can be synthesized according to the routes schematically depicted in Scheme 2A.

In process A (Scheme 2A), 2-nitrobenzoic acid 1-6 may first be converted to anthranilic acid 2-4 via 2-1 or 2-2 and 2-3.

Another synthesis of 2-4 using standard procedure ⁶ is shown in Scheme 2B. Thus, 2,4-dichloro-6-nitrobenzoic acid (2-1B) is treated with dimethyl sulfate to provide 2-2B according to the conditions described above for the preparation of 2-2 from 1-6. Typically, tin (II) chloride or iron powder and glacial acetic acid / hydrochloric acid are used to reduce nitro compound 2-2B under standard conditions to provide anisidine 2-3B. The anisidine 2-3B is treated with chloral hydrate in the presence of hydroxylamine hydrochloride followed by acid hydrolysis to give isatin intermediate 2-4B. Subsequent treatment with 2-4B hydrogen peroxide under basic conditions gives anthranilic acid 2-4.

Treatment with formamide of 2-4 at elevated temperature, typically 150 ° C., provides 3H-quinazolin-4-one 2-5. Compound 2-5 is then reacted with a suitable alkyl halide in the presence of a base such as potassium carbonate to give 2-6. Examples of alkyl halides that can be used in Process C include, but are not limited to, 2- (chloromethyl) pyridine, (chloromethyl) cyclopropane, 1- (2-chloroethyl) pyrrolidine, 2- (2-chloroethyl)- 1-methylpyrrolidine, 4- (2-chloroethyl) morpholine, 2-chloromethyl-4-methylthiazole hydrochloride, 2,6-bis (chloromethyl) pyridine, 2-bromopropane, 1- Chloropropane, 1-chloro-2-methylpropane, 2-chloroethyl ethyl ether, (2-diethylamino) ethyl chloride hydrochloride, 1-chlorobutane, 2-chlorobutane, crotyl chloride and 4-chloromethyl- 3,5-dimethylisoxazole. The results are shown in a table (Table 1). Suitably with BBr ₃ or aqueous HBr at elevated temperature, subsequent removal of the protecting group from 2-6 results in the corresponding 3-N- (substituted) -8-hydroxy-3H-quinazolin-4-one 2- Provide 7 In the case of the ethyl ether derivative 2-6M, the hydrobromide salt of alcohol 2-7M1 was obtained (Table 2). Treatment of alkenes 2-6P with aqueous HBr resulted in hydrobromination of the alkene double bonds, at the same time removing the methoxy protecting group to give compound 2-7P1.

In processes B and C (Scheme 2A), anthranilic acid 1-8 was treated with formic anhydride to give formylamino compound 2-8 or benzo [d] [1,3] oxazin-4-one 2-9 can do. Compound 2-8 (or 2-9) is then reacted with a suitable amine at room temperature in the presence of a condensing agent such as phosphorus trichloride or triethyl orthoformate with toluene or xylene, typically near reflux temperature 8-hydroxy-3H-quinazolin-4-one 2-7 is obtained. Examples of amines that can be used in Processes B and C include, but are not limited to, 2-amino-5-methylthiazole, 2- (2-aminoethyl) pyridine, 3-aminopyridine, 4-aminomorpholine, 1-amino- 4-methylpiperazine, 1-aminopyrrolidine, 4- (aminomethyl) piperidine, 1-aminopiperazine, 5-amino-1-ethylpyrazole, 5-amino-2-methoxypyridine, 4 -Amino-1,3,5-trimethylpyrazole, 2-amino-1-methylbenzamide, 2-amino-5-methylpyridine, 2-amino-5-chloropyridine and 4-aminopiperidine . The results are shown in a table (Table 3). Other 8-hydroxy-3H-quinazolin-4-ones which can be prepared according to this procedure are shown in Table 4.

In process D (Scheme 2A), 2-nitrobenzoic acid 1-6 may first be treated with a suitable amine in the presence of an activator, for example CDI, to prepare a suitable N- (substituted) benzamide 2-10. Subsequent reduction of the nitro group with formic acid or formamide and coupling of the resulting amines 2-11, typically in the presence of a condensing agent such as CDI or triethyl orthoformate, with the corresponding 3-N-substituted Provides Derivatives 2-7.

Procedures B and C (Scheme 2A) can be repeated using suitably O-protected anthranilic acid, such as 2-4 (Scheme 3). In this case, the 8-hydroxy-3H-quinazolin-4-one 2-7 is obtained following the removal of the O-protective group.

2-substituted 8-hydroxy-3H-quinazolin-4-one can be prepared according to the route disclosed in Scheme 4. Thus, according to process E, anthranilic acid 1-6 is treated with an activator such as thionyl chloride and the intermediate acid chloride is then reacted with ammonia to give the corresponding benzamide 4-1. Typically the reduction of nitro compound 4-1 with SnCl ₂ or iron powder / HOAc provides the corresponding amine 4-2. Amine 4-2 in turn was treated with chloroacetyl acetic acid or 2-chloro-1,1,1-trimethoxyethane to give 5,7-dichloro-2-chloromethyl-8-hydroxy-3H-quinazolin-4 -On (4-3A) may be provided. The 2-chloromethyl compound can also be prepared from methoxy anthranilic acid derivatives 2-4 via process F. Thus, 2-4 is treated with chloroacetonitrile ^{7 in} the presence of a base, typically sodium methoxide, to give 4-3B. Many new 2-substituted derivatives 4 are further treated with a range of amines, such as, but not limited to, dimethylamine, methylamine and ethylamine, to 4-chloromethyl derivative 4-3A (or 4-3B). -16A-Provides 4-16D.

A number of 2,3-disubstituted 8-hydroxy-3H-quinazolin-4-ones 4-9 can be prepared via acid 2-1 according to procedure G, as shown in Scheme 4. Thus, 2-1 is treated with a suitable amine in the presence of an activator to give the corresponding benzamide 4-5, which is then typically reduced to 4-6 using SnCl ₂ or iron powder / HOAc. Compound 4-6 is subsequently converted to 2-chloromethyl derivative 4-7 which in turn is converted to 4-8 using reaction conditions similar to those described in Procedures E and F above. Following deprotection, 4-8 typically provides the corresponding 2,3-disubstituted derivatives 4-9 using aqueous HBr near reflux temperature.

2,3-disubstituted 8-hydroxy-3H-quinazolin-4-one 4-9 can also be obtained via processes H and I (Scheme 4). In process H, anthranilic acid 1-8 is suitably acylated with 2-azidoacetyl chloride, phthalyl-glycyl chloride and [(phenylmethyl) amino] acetyl chloride. Thus, examples of NP groups include azido, phthalimido and benzylamino. At elevated temperature, subsequent condensation of intermediate 4-10, typically in the presence of acetic anhydride near reflux temperature, gives benzo [d] [1,3] oxazin-4-one 4-11. 4-11 in the presence of a suitable amine (R ₁ NH ₂ ) gives 4-12; 4-12 in turn provides 4-13 through condensation. Suitable condensing agents include PCl ₃ , triethyl orthoformate, CDI and Ac ₂ O. Conditions for converting -NP residues to amino groups will depend on the particular NP group; In the case of the abovementioned groups, these are reduction, dimethylamine and catalytic hydrogenation, respectively (more examples of condition ⁵ can be found elsewhere for the latter two conversions). In process I, condensation of anthranilic acid 1-8 with a suitable amine (NR ₁ R ₂ ) affords amide 4-14. Subsequent successive treatments with 4-14 chloroacetyl chloride and amine give 4-9.

3-amino-5,7-dichloro-8-hydroxy-3H-quinazolin-4-one (5-4) and some 3- (substituted) amino-3H-quinazolin-4-one in Scheme 5 It can be prepared according to the route shown. Typically methyl ester 2-3 is N-formylated using acetic anhydride to give 5-1. On the one hand, 2-3 is treated with orthoesters, for example triethyl orthoformate, to give imidate ester 5-2. When treated with hydrazine, 5-1 or 5-2 provides 3-amino-3H-quinazolin-4-one 5-3. Typically at 120 ° C., the protecting group in 5-3 is removed by aqueous HBr to give 5-4. Further treatment of 3-amino compound 5-4 with suitable acid halides provides the corresponding 3-substituted acylated derivatives 5-5A-5-5C. Derivatives such as 5-7A-5-7C can be used to substitute hydrazine hydrates for suitably substituted hydrazines such as 4-fluorophenylhydrazine, 4-methoxyphenylhydrazine or 2,4-difluorophenylhydrazine. It can then be obtained from 5-2 via deprotection. Alkyl halides in a suitable solvent, typically ethanol, are used followed by deprotection to give 5-7D-5-7G from 5-3. On the one hand, compounds 5-7D-5-7G can also be prepared via the treatment of 5-2 with suitable alkylated hydrazines such as ethylhydrazine, propylhydrazine and (cyclopropyl) methylhydrazine.

Amines 2-7F2, 2-7Q2, 2-7R2, 2-7S2 and 2-7X2 can be converted to a range of derivatives such as 6-1-6-6 through treatment with alkyl halides or acylation reagents. . The data for 6-1-6-6 are shown in a table (Table 9).

A number of 3-substituted-3H-quinazolin-4-thiones 7-2 can be prepared from the corresponding 3H-quinazolin-4-ones 2-6 following the procedure shown in Scheme 6. Thus, 2-6 is treated with P ₄ S ₁₀ or Lawson's reagent to give thioketone 7-1. Suitably BBr ₃ is subsequently used to remove the protecting group to give the desired 3-substituted-3H-quinazolin-4-thione 7-2.

4,6- Dichloro -3- Hydroxy -2- Of nitrobenzoic acid (1-6)  Produce

Tin (II) chloride hydrate (50 g, 0.29 mol) was added to a solution of 2,4-dichloro-5-nitrobenzoic acid (1-2) ² (10.0 g, 0.045 mol) in EtOH (200 mL). The mixture was stirred at 70 ° C. for 0.5 h, cooled and poured on ice. The pH of the mixture was adjusted to 8 (saturated NaHCO ₃ ). The suspension was stirred at rt for 5 h and reacidified to pH 5 (glacial acetic acid). The resulting white suspension was carefully extracted with ethyl acetate, the extracts combined, washed with brine, dried and concentrated to give the desired amine (1-3) as a gray solid (8.8 g, 96%).

5-Amino-2, 4-dichlorobenzoic acid (1-3): ¹ H NMR (CD ₃ OD): δ 7.30 (s, 1H), 7.27 (s, 1H).

Acetic anhydride (27 mL) was added to 5-amino-2,4-dichlorobene crude acid (1-3) (8.0 g, 0.041 mol) in glacial acetic acid (150 mL). The solution was stirred at rt for 0.5 h and concentrated to give the desired acetamide (1-4) as a white solid (9.6 g, 96%).

5-acetamido-2,4-dichlorobenzoic acid (1-4): ¹ H NMR (CD ₃ OD): δ 8.32 (s, 1H), 7.62 (s, 1H), 2.19 (s, 3H).

Stirred ice-cooling of 5-acetamido-2,4-dichlorobenzoic acid (1-4) (9.6 g, 0.039 mol) over 30 minutes with fuming nitric acid (1.8 mL, 0.043 mol) and concentrated sulfuric acid (120 mL) The solution was added portionwise. After the addition was complete, additional fuming nitric acid (17 mL) and concentrated sulfuric acid (180 mL) were added at 30 and 60 minute intervals. The reaction mixture was then stirred for an additional 2.5 hours at 0 ° C., warmed to 12-16 ° C. and at this temperature until all starting material was consumed (about 3 hours). The solution was poured onto ice and extracted with ethyl acetate (3 x). The organic extracts were combined, washed with brine, dried and concentrated to give 3-acetamido-4,6-dichloro-2-nitrobenzoic acid (1-5) as an orange solid (9.8 g, 86%).

3-Acetamido-4,6-dichloro-2-nitrobenzoic acid (1-5): ¹ H NMR (CD ₃ OD): δ 8.01 (s, 1H), 2.13 (s, 3H).

3-acetamido-4,6-dichloro-2-nitrobenzoic acid (1-5) (9.7 g, 0.033 mol) was added to a solution of KOH (18.7 g, 0.034 mol) in H ₂ O (85 mL). . The solution was heated to reflux for 18 hours and cooled to room temperature. Concentrated HCl was added to adjust the pH to zero. The mixture was diluted with ethyl acetate and H ₂ O and stirred at rt for 30 min. Separating the layers; The aqueous layer was extracted with ethyl acetate (3x), the extracts were combined with the original organic layer, washed with brine, dried and concentrated to give 4,6-dichloro-3-hydroxy-2-nitrobenzoic acid as a dark red solid ( 1-6) was obtained (7.4 g, 89%); Melting point 188-189 ° C. (document ⁴ melting point 186 ° C. (decomposition)).

4,6-dichloro-3-hydroxy-2-nitrobenzoic acid (1-6): ¹ H NMR (CD ₃ OD): δ 7.79 (s, 1H); Mass spectrum: m / z 250, 252, 254 (M ⁺ -1, 100%, 66%, 11%).

5,7- Dichloro -8- Methoxy -3H- Quinazoline Preparation of 4-one (2-5)

Dimethyl sulfate (40 mL) was added to a stirred mixture of 1-6 (15.0 g, 0.059 mol), potassium carbonate (66 g, 0.5 mol) and DMF (300 mL). The resulting mixture was stirred at 60 ° C. overnight and then at 120 ° C. for 2 hours. The reaction mixture was concentrated under vacuum. The resulting reddish brown residue was washed with water and dried. This gave 2-2 as an orange solid (14.6 g, 88%). ¹ H NMR (CDCl ₃ ): δ 7.65 (s, 1H), 4.01 (s, 3H), 3.93 (s, 3H).

Iron powder (18.2 g, 0.33 mol) was added to a 2-2 (13.3 g, 0.048 mol) solution in acetic acid (120 mL). The mixture was stirred at 55 ° C. for 1.5 h and then filtered when hot through celite (ethyl acetate). The filtrate was concentrated, ethyl acetate and saturated sodium carbonate were added and the mixture was filtered (celite). The organic layer was isolated, washed with water, dried (K ₂ CO ₃ ) and concentrated to give 2-3 as a gray solid (11.6 g, 97%). ¹ H NMR (CDCl ₃ ): δ 6.71 (s, 1H), 3.89 (s, 3H), 3.79 (s, 3H).

To a stirred solution of 2-3 (11.5 g, 0.046 mol) in methanol (250 mL) and water (70 mL) was added 2M NaOH (25 mL). The reaction mixture was heated to reflux for 1 hour, additionally 2M NaOH was added (25 mL) and the mixture was heated to reflux for an additional hour. The solution was cooled and concentrated to remove methanol. The concentrate was dissolved in water, extracted with ethyl acetate and the pH adjusted to 1-2 (concentrated HCl). The milky suspension was extracted with ethyl acetate (3 ×). The combined extracts were washed with brine, dried and concentrated to give 2-4 as a beige solid (10.4 g, 95%). ¹ H NMR (CD ₃ OD): δ 6.70 (s, 1 H), 3.80 (s, 3 H).

A stirred suspension of 2-4 (16.9 g, 0.072 mol) and formamide (150 mL) was heated at 150 ° C. for 8 hours and then cooled to room temperature. Water was added and the resulting precipitate was isolated via filtration, washed with water and dried under vacuum to give 2-5 (13.0 g, 73%) as a light brown solid. ¹ H NMR (CD ₃ OD): δ 8.08 (s, 1H), 7.60 (s, 1H), 3.98 (s, 3H).

Example  1-5,7- Dichloro -8- Methoxy -3H- Quinazoline Compound 2-7A-2-7 through alkylation of -4-one (2-5) and subsequent deprotection R2 Manufacturing

To a stirred solution of 2-5 (1.5 g, 6.1 mmol) and chloride (7.3 mmol) in anhydrous DMF (30 mL) was added K ₂ CO ₃ (9.3 mmol) and the resulting mixture was heated at 95 ° C. for 16 h, Cooled and concentrated. The residue was extracted with ethyl acetate or dichloromethane (3 ×), the extracts were combined, washed successively with water and brine and dried. Subsequent purification by polishing with a suitable solvent, recrystallization or by SiO ₂ -gel chromatography provided the corresponding 3-substituted-8-methoxy-3H-quinazolin-4-one (2-6).

Examples of chloride used: 1- (2-chloroethyl) pyrrolidine for 2-6A, 2 (chloromethyl) cyclopropane for 2-6B, 2- (2-chloroethyl) -1-methylpyrroli Dean is 2-6C, 2- (chloromethyl) pyridine is 2-6D, 4- (2-chloroethyl) morpholine is 2-6E, and 2-chloromethyl-4-methylthiazole hydrochloride is 2 -6F, 4-chloromethyl-3,5-dimethylisoxazole is 2-6G, 2-bromopropane is 2-6H, 1-chloropropane is 2-6I, 1-chloro-2 -Methylpropane is 2-6J, 1-chlorobutane is 2-6K, 2-chlorobutane is 2-6L, 2-chloroethyl ethyl ether is 2-6M, (2-diethylamino) ethyl chloride Hydrochloride 2-6N, 2-chloromethyl-3-methylpyridine hydrochloride 2-6O, crotyl chloride 2-6P, 2,6-bis (chloromethyl) pyridine 2-6Q, 1-chloroethane gives 2-6R. In the case of 1- (2-chloroethyl) pyrrolidine hydrochloride, 2-chloromethyl-3-methylpyridine hydrochloride and 2-chloromethyl-4-methylthiazole hydrochloride, 2.2 equivalents of K ₂ CO ₃ Used.

(2- Chloromethyl ) -3- Methylpyridine Of hydrochloride  Produce

To a stirred solution of 2,3-lutidine (5.00 g, 46.7 mmol) in chloroform (100 mL) at 0 ° C. was added in portions over 5 minutes by m-chloroperbenzoic acid (12.0 g of 77% maximum reagent). The reaction mixture was stirred for an additional 30 minutes at 0 ° C. and then warmed to room temperature. After 6 hours, the reaction mixture was concentrated to dryness, water (20 mL) was added and the pH of the mixture was adjusted to 8 (saturated NaHCO ₃ ). The mixture was concentrated and the residue was extracted with dichloromethane / methanol (4: 1). The extract was concentrated to a white solid. Subsequent column purification (SiO ₂ ; dichloromethane / methanol, 9: 1) provided 2,3-lutidine-N-oxide as a white solid (4.80 g, 83%).

A stirred solution of 2,3-lutidine-N-oxide (4.80 g, 39.0 mmol) in acetic anhydride (50 mL) was heated to reflux overnight, cooled and then concentrated to dryness as brown oil (6.34 g) (2- Acetoxymethyl) -3-methylpyridine was provided. A mixture of crude (2-acetoxymethyl) -3-methylpyridine and K ₂ CO ₃ (10.0 g, 72.4 mmol), methanol (60 mL) and water (30 mL) was stirred overnight at room temperature. The solid was filtered off and the filtrate was concentrated to dryness. The residue was subjected to column chromatography (SiO ₂ ; dichloromethane / methanol, 9: 1), followed by (2-hydroxymethyl) -3-methylpyridine as light brown oil (2.86 g, 59% over 2 steps). Provided.

(2-hydroxymethyl) -3-methylpyridine: ¹ H NMR (CDCl ₃ ): δ 8.41 (d, J = 4.9, 1H), 7.48 (d, J = 7.5, 1H), 7.16 (dd, J = 4.9 and 7.5, 1H), 4.69 (s, 2H), 4.00 (br, 1H), 2.22 (s, 3H).

To an ice cold solution of (2-hydroxymethyl) -3-methylpyridine (1.00 g, 8.1 mmol) in dichloromethane (30 mL) was added a solution of thionyl chloride (2.5 mL) in dichloromethane (6 mL) for 10 minutes. Dropwise over. The ice bath was removed and the reaction mixture was stirred at rt for 2 h, concentrated and then washed with diethyl ether. This gave (2-chloromethyl) -3-methylpyridine hydrochloride as a pale straw solid (1.44 g, 99%).

(2-chloromethyl) -3-methylpyridine hydrochloride: ¹ H NMR (CD ₃ OD): δ 8.72 (d, J = 5.9, 1H), 8.54 (d, J = 8.1, 1H), 8.00 (dd, J = 5.9 and 8.1, 1H), 5.05 (s, 2H), 2.64 (s, 3H).

The yield and spectral data of the prepared Compounds 2-6A-2-6 are shown in Table 1.

Deprotection

Method A: To a stirred ice cold solution of 8-methoxy derivative 2-6 (1.9 g, 5.6 mmol) in dichloromethane (15 mL) was added BBr ₃ (12 mL of 1M solution in dichloromethane, 12 mmol). It was. The solution was then stirred at 45 ° C. for 18 h, cooled and methanol (20 mL) was added. The mixture was concentrated. Excess borate was repeatedly removed by methanol addition and evaporation. The crude hydrobromide salt of the product was washed with ether (3 ×). Some compounds were isolated as free base: therefore saturated Na ₂ CO ₃ (20 mL) was added and the mixture was extracted with dichloromethane (5 ×). The combined extracts were washed with water, dried and concentrated. The residue was simply washed with a suitable solvent or purified by recrystallization or SiO ₂ -column chromatography to give the corresponding 8-hydroxy derivative 2-7.

Method B: 3-substituted-8-methoxy-3H-quinazolin-4-one (2-6) (5.0 mmol) solution in 48% hydrobromic acid (25 mL) was heated under argon atmosphere for 16-18 hours It was refluxed and cooled to room temperature. The reaction mixture was concentrated to dryness or the precipitate formed was isolated by filtration. The crude solid was then washed successively with diethyl ether, dichloromethane and acetonitrile to afford the corresponding 8-hydroxy compound (2-7) as a hydrobromide salt. Some compounds were isolated as free base (see Method A above for conditions).

Method C: A solution of 8-methoxy compound 2-6 (4.46 mmol) and 48% hydrobromic acid (23 mL) was heated at 120 ° C. for 2-10 hours and cooled to room temperature. Water (30 mL) was added and the pH adjusted to 5 (NaOH pellets). The resulting precipitate was isolated via filtration, washed with water and dried under vacuum.

In the case of the ethyl ether derivative 2-6M, the hydrobromide salt of alcohol 2-7M1 was obtained (Table 2). Alkene 2-6P was treated with BBr ₃ according to Method A to give 2-7S1. 2-6P was treated with aqueous HBr according to Method B to give bromide 2-7P1.

The yield and spectral data of 2-7A1-2-7S1 are shown in Table 2.

2-amino-4,6- Dichloro -3- Of hydroxybenzoic acid (1-8)  Produce

A mixture of 4,6-dichloro-3-hydroxy-2-nitrobenzoic acid (1-6) (700 mg, 2.78 mmol), iron powder (400 mg, 7.16 mmol) and glacial acetic acid (13 mL) at 80 ° C Heat for 50 minutes, cool and filter the solid. The filtrate was concentrated to a brown solid. Subsequent SiO ₂ -gel column chromatography (ethyl acetate / HOAc, 100: 1-100: 3) gave 2-amino-4,6-dichloro-3-hydroxy as a light brown solid (582 mg, 94%). Benzoic acid (1-8) was provided. ¹ H NMR (DMSO-d ₆ ): δ 6.68 (s); This is consistent with document ⁴ .

4,6- Dichloro -2- Formylamino -3- Of hydroxybenzoic acid (2-8)  Produce

A solution of formic acid (1.04 mL of 90% solution) and acetic anhydride (2 mL) was heated and cooled at 50-60 ° C. for 2 hours. 2-amino-4,6-dichloro-3-hydroxybenzoic acid (1-8) (425 mg, 1.91 mmol) was then added in portions to the stirred acetic formic anhydride at room temperature. After 2.5 hours, the reaction mixture is poured into a mixture of ice and water; The solid was isolated via filtration. This gave 4,6-dichloro-2-formylamino-3-hydroxybenzoic acid (2-8) as an orange solid (320 mg, 67%). ¹ H NMR (DMSO-d ₆ ): δ 8.76 (s, 1H), 8.29 (s, 1H), 8.13 (s, 1H); Mass spectrum: m / z 248, 250, 252 (M ⁺ -1, 100%, 66%, 11%).

Example  2-4,6- Dichloro -2- Formylamino -3- With hydroxybenzoic acid (2-8) With amines  PCl ₃ - Mediated Condensation

Toluene (1 mL) in a stirred mixture of 4,6-dichloro-2-formylamino-3-hydroxybenzoic acid (2-8) (200 mg, 0.80 mmol), amine (0.88 mmol) and toluene (5 mL). Solution of PCl ₃ (0.12 mL, 1.38 mmol) was added dropwise over 2 minutes. The resulting suspension was heated to reflux and cooled for 4-16 hours. Saturated NaHCO ₃ was added to pH 9. The pH of the mixture was then readjusted to 7 (5N HCl), extracted with dichloromethane (3 ×), the extracts combined and dried (Na ₂ SO ₄ ). The volatiles were removed to give 5,7-dichloro-8-hydroxy-3- (substituted) -3H-quinazolin-4-one (2-7). In some cases, the crude product was purified by washing with 5% methanol in a suitable solvent, typically diethyl ether or diethyl ether, or via SiO ₂ -gel chromatography or recrystallization; Characterization data for compounds 2-7A2 and 2-7M2 are shown in Table 3. Other compounds 2-7 prepared from 2-8 according to Example 2 are shown in Table 4.

Examples of amines used in Example 2: (C-thiazol-2-yl) methylamine is 2-7A2, 2- (2-aminoethyl) pyridine is 2-7B2, 3-aminopyridine is 2- 7C2, 4-aminomorpholine 2-7D2, 1-amino-4-methylpiperazine 2-7E2, 4- (aminomethyl) piperidine 2-7F2, 5-amino-1- Ethylpyrazole is 2-7G2, 5-amino-2-methoxypyridine is 2-7H2, 2-amino-1-methylbenzamidazole is 2-7I2, 2-amino-5-methylpyridine is 2 -7J2, 2-amino-5-chloropyridine provides 2-7K2, 1-aminopiperidine provides 2-7L2, and 1-aminopyrrolidine provides 2-7M2. In the case of the amine hydrochloride salt, a suitable equivalent base of triethylamine is added to the reaction mixture. Other compounds prepared according to Example 2 are 2-7O 2-2-7AE 2 (Table 4).

Example  3  2,3- Disubstituted -3H- Quinazoline Preparation of 4-one (4-9)

Step 1: To a solution of acids 1-8 (1.00 g, 4.50 mmol) in anhydrous benzene (8.3 mL) was added dropwise thionyl chloride (2.09 g, 17.6 mmol) under an argon atmosphere. The mixture was heated to reflux for 5 hours and after this time excess thionyl chloride and benzene were removed by evaporation. The residue was dissolved in anhydrous dichloromethane (8.3 mL) cooled to 0 ° C. and treated with n-propylamine (798 mg, 13.5 mmol). The mixture was stirred at 0 ° C. for 15 minutes, then warmed to room temperature and stirred for a further 16 hours. The crude residue was purified via evaporation and column chromatography (ethyl acetate / petroleum ether, 3: 7-1: 1) to give benzamide, 2-amino-4,6-dichloro-3-hydroxy-N- (n -Propyl) benzamide (4-14, R ₁ = n-propyl) was obtained as an orange solid (550 mg, 46%).

2-amino-4,6-dichloro-3-hydroxy-N-propylbenzamide: ¹ H NMR (DMSO-d ₆ ): δ 8.39 (t, J = 5.6, 1H), 6.67 (s, 1H) , 4.94 (br, 1H), 3.16 (m, 2H), 2.44 (m, 2H), 1.50 (m, 2H), 0.88 (m, 3H).

Step 2: To a solution of 2-amino-4,6-dichloro-3-hydroxy-N-propylbenzamide (1.00 g, 4.50 mmol) in glacial acetic acid (5.5 mL) under chloroacetyl chloride (718 mg, 6.36 mmol) was added. The mixture was heated to reflux for 2 hours and then stirred at room temperature for 1 hour. The reaction mixture was evaporated in vacuo and the residue was neutralized with 2M NaOH. The precipitate was isolated via filtration, washed with water and dried under vacuum. Dichloromethane (10 mL) was added to the resulting residue and the insoluble material was filtered off. The filtrate was concentrated to give chloride, 2-chloromethyl-5,7-dichloro-8-hydroxy-3-n-propyl-3H-quinazolin-4-one (4- as orange solid (600 mg, 89%). 15, R ₁ = n-propyl).

2-chloromethyl-5,7-dichloro-8-hydroxy-3-n-propyl-3H-quinazolin-4-one: ¹ H NMR (DMSO-d ₆ ): δ 7.86 (s, 1H), 5.15 (s, 2H), 4.27 (s, 2H), 1.52 (m, 2H), 0.93 (m, 3H); Mass spectrum: m / z 323, 325, 327 (M ⁺ +1, 100%, 66%, 11%).

Step 3: To a solution of 2-chloromethyl-5,7-dichloro-8-hydroxy-3-n-propyl-3H-quinazolin-4-one (285 mg, 0.886 mmol) in dry THF (1.3 mL). A methylamine solution (7.5 mL of 8.0M solution, 60 mmol) in ethanol was added dropwise under argon atmosphere. The mixture was stirred at rt for 18 h, then concentrated and 2M HCl (5 mL) was added to the resulting residue. The mixture was evaporated and additional 2M HCl (5 mL) was added. The residue was evaporated and the procedure repeated two more times. The mixture was triturated with dichloromethane and dried under vacuum to give 5,7-dichloro-8-hydroxy-2-methylaminomethyl-3-n-propyl-3H-quina as a yellow solid (157 mg, 50%). Zolin-4-one hydrochloride (4-9A) was obtained (Table 6).

Another 2,3-disubstituted-3H-quinazolin-4-one (4) prepared via the substitution of n-propylamine (step 1) and methylamine (step 3) of Example 3 for suitable amine (s) -9B-4-9E) are shown in Table 6.

Example  4  2-substituted-3H- Quinazoline Preparation of 4-one (4-16A-4-16D)

To an ice cold solution of sodium methoxide in methanol (2.8 mL of 0.32 M solution, 0.89 mmol) was added dropwise chloroacetonitrile (0.25 mL, 3.90 mmol) under argon atmosphere ⁷ . The reaction mixture was stirred for 30 min at RT and then recooled to 0 ° C. before adding 2-4 (0.80 g, 3.39 mmol) in anhydrous methanol (14 mL). The solution was stirred at RT for 20 h and heated to reflux for another 20 h. Evaporation of the resulting residue and purification via column chromatography (ethyl acetate / petroleum ether, 3: 7) gave chloride 4-3B as a white solid (225 mg, 23%).

4-3B was subsequently treated with a suitable amine according to the conditions set forth in step 3 of Example 3 to give a 2-substituted-8-methoxy-3H-quinazolin-4-one: ethylamine yielded 4-4A , 1-propylamine provided 4-4B, diethylamine provided 4-4C, and dimethylamine provided 4-4D. Subsequent removal of each of the 8-methoxy protecting groups according to the general deprotection procedure (Example 1, Method A) provided 4-16A-4-16D (Table 7).

3-amino-5,7- Dichloro -8- Hydroxy -3H- Quinazoline -4-one hydrobromide (5-4 or PB  1099)

A solution of 2-amino-4,6-dichloro-3-methoxybenzoic acid methyl ester (2-3) (6.35 g, 25.4 mmol) and triethyl orthoformate (60 mL, 361 mmol) was heated for 5 days It was refluxed. The solution was cooled to room temperature and evaporated under reduced pressure to give imidate, 4,6-dichloro-2-ethoxymethyleneamino-3-methoxybenzoic acid methyl ester (5-2) and starting material (7.80 g) as brown oil. ; 5-2: 2-3-9: 1 were obtained.

4,6-Dichloro-2-ethoxymethyleneamino-3-methoxybenzoic acid methyl ester (5-2): ¹ H NMR (DMSO-d ₆ ): δ 7.99 (s, 1H), 7.48 (s, 1H ), 4.22 (q, 2H), 3.79 (s, 3H), 3.62 (s, 3H), 1.28 (t, J = 7.2, 3H).

To an ice cold solution of imidate 5-2 (400 mg, 1.31 mmol) in ethanol (12 mL) was added hydrazine hydrate (1.8 mL, 57.8 mmol) under argon. After 15 minutes, the solution was warmed to room temperature and stirred for a further 2 hours. The thick suspension was diluted with ethanol and filtered. The solid was washed with cold ethanol and dried under vacuum to afford 3-amino-5,7-dichloro-8-methoxy-3H-quinazolin-4-one as a white, lint-free solid (289 mg, 85%). Provided.

3-amino-5,7-dichloro-8-methoxy-3H-quinazolin-4-one (5-3): ¹ H NMR (DMSO-d ₆ ): δ 8.48 (s, 1H), 7.74 ( s, 1H), 5.85 (s, 2H), 3.94 (s, 3H); Mass spectrum: m / z 260, 262, 264 (M ⁺ +1, 100%, 66%, 11%).

A solution of 5-3 (60 mg, 0.231 mmol) and 48% aqueous hydrobromic acid (2 mL) was heated at 120 ° C. for 2 hours. The solution was cooled to room temperature and the precipitate was isolated via filtration, washed successively with dichloromethane and diethyl ether and dried under vacuum to afford 3-amino-5,7-di as a white solid (44 mg, 58%). Chloro-8-hydroxy-3H-quinazolin-4-one hydrobromide (5-4) was provided.

3-amino-5,7-dichloro-8-hydroxy-3H-quinazolin-4-one hydrobromide (5-4): ¹ H NMR (DMSO-d ₆ ): δ 8.44 (s, 1H), 7.60 (s, 1 H); m / z 246, 248, 250 (M ⁺⁺ 1, 100%, 66%, 11%).

Example  5  3- (substituted) amino-5,7- Dichloro -8- Hydroxy -3H- Quinazoline -4-one

To an ice cold suspension of substituted phenylhydrazine hydrochloride (1.61 mmol) in ethanol (4 mL) was added triethylamine (185 mg, 1.83 mmol) under argon atmosphere. The mixture was stirred at 0 ° C. for 15 minutes before the solution of imidate 5-2 (180 mg, 0.59 mmol) in ethanol (3 mL). The resulting mixture was stirred at 0 ° C. for 40 minutes and then at room temperature for 4 days. The suspension was filtered and the white solid was washed with cold ethanol and dried under vacuum to afford 8-methoxy-3- (substituted) amino compound 5-6.

Examples of hydrazine used: 5-6 A for 2,4-difluorophenylhydrazine hydrochloride, 5-6 B for 4-methoxyphenylhydrazine hydrochloride, 5-6 C for 4-fluorophenylhydrazine hydrochloride to provide.

A solution of 8-methoxy compound (5-6A, 5-6B or 5-6C) (0.133 mmol) and 48% aqueous hydrobromic acid (3 mL) was heated at 120 ° C. for 6 hours and cooled to room temperature. The solid was isolated via filtration, washed with dichloromethane and diethyl ether and dried under vacuum to give 3- (substituted) amino compounds (5-7A, 5-7B or 5-7C) (Table 8 ).

Chemical shifts for # 8-OH and HBr (if applicable) were not included in the enumeration)

Example  6  3-substituted-3H- Quinazoline -4- Of thion (7-2)  Produce

A mixture of 3-substituted-3H-quinazolin-4-one (0.70 mmol), P ₄ S ₁₀ (0.93 mmol) and pyridine (5 mL) was heated to reflux. When the reaction was complete, as monitored by TLC analysis, the mixture was concentrated to dryness and the resulting residue was subjected to column chromatography (SiO ₂ ; ethyl acetate / methanol, eluted with 100: 1) and the corresponding 3H-quinazolin- 4-Tion 7-1 was provided.

The mixture of 3H-quinazolin-4-thione 7-1 was treated with BBr ₃ according to the conditions disclosed in Example 1 above. Post-treatment with methanol in a conventional manner gave the corresponding 3H-quinazolin-4-thione 7-2 (Table 11).

Example  7-Evaluation of Compounds of Formula (I)

The following assays were used to evaluate the compounds of formula I for suitability for use in the methods of the present invention.

Assay 1. By fluorescence measurement H ₂ O ₂  analysis

Fluorometric analysis was used to test the ability of test compounds to inhibit hydrogen peroxide generation by Aβ in the presence of copper based on dichlorofluorocene diacetate (DCF; Molecular Probes, Eugene OR). DCF solution (5 mM) (preceded with argon for 2 hours at 20 ° C.) in 100% dimethyl sulfoxide was deacetylated in the presence of 0.25 M NaOH for 30 minutes and neutralized to a final concentration of 1 mM at pH 7.4. . Horseradish peroxidase (HRP) mother liquor was prepared at 1 μM at pH 7.4. The reaction was performed in PBS, pH 7.4 in 96 well plates (total volume = 250 μl / well). The reaction solution was prepared by adding Aβ 1-42, copper-glycine chelate (Cu-Gly) (CuCl ₂ in a ratio of 1: 6 to glycine, in a concentration ranging from 50 nM to 1 μM, which was added to the β2 2Cu-Gly: 1 Aβ. ), Reducing agents such as dopamine (5 μΜ) or ascorbic acid, deacylated DCF 100 μΜ and HRP, 0.1 μΜ. 1-10 μM EDTA or another chelator may also be present as a control for free copper, but its action was not required for the assay. The reaction mixture was incubated at 37 ° C. for 60 minutes. Catalase (4000 units / ml) and H ₂ O ₂ (1-2.5 μM) in PBS pH 7.4 may be included as positive controls. Fluorescence was recorded using a plate reader with excitation and emission filters at 485 nM and 530 nM, respectively. H ₂ O ₂ concentration was established by comparing fluorescence with the H ₂ O ₂ standard. Inhibition of Aβ H ₂ O ₂ production was analyzed by including a predetermined concentration of test compound (s) in the test wells.

Analysis 2. Neurotoxicity  analysis

Primary Cortical Neuronal Cultures

Cortical cultures were prepared as previously described (White et al., 1998). The embryonic 14-day BL6Jx129sv mouse cortex was recovered, dissected to remove the meninges and dissolved in 0.025% (wt / vol) trypsin. Lysed cells were plated in 48 well culture plates at a density of 2 × 10 ⁶ cells / ml in MEM with 25% (vol / vol) FCS and 5% (vol / vol) HS and incubated at 37 ° C. for 2 hours. . The medium was then exchanged to Neurobasal media (Neurobasal media, Invitrogen Life Technologies) and B27 supplement (Invitrogen Life Technologies). Cultures were maintained at 37 ° C. in 5% CO ₂ . Prior to the experiment, the culture medium was exchanged with neurobased medium and B27-antioxidant (Invitrogen Life Technologies).

Primary cerebellar granule neuron culture

The cerebellum of mice 5-5 days old (P5-6) were harvested, dissected to remove the meninges and dissolved in 0.025% trypsin. Cerebellar granule neurons (CGN) were plated in 24 well culture plates at 350 000 cells / cm 2 in BME (Invitrogen Life Technologies) supplemented with 10% calf fetal serum (FCS), 2 mM glutamine and 25 mM KCl. Gentamycin sulfate (100 μg / ml) was added to all smear medium and the culture was maintained at 37 ° C. in 5% CO ₂ .

Assay 3. Analysis of Cell Viability

(a) for cell viability MTS  analysis

Cell viability is measured using the MTS assay. Replace the culture medium with fresh neurobased medium + B27 supplement-antioxidant. 1/10 volume of MTS solution (Cell Titre 96 Aqueous One, Promega Corporation) is incubated at 37 ° C. for 2 hours. 200 μl aliquots are measured spectrophotometrically at 560 nm.

(b) for cell viability LDH  analysis

Cell death is measured from the culture supernatant free of serum and cell debris using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Boehringer Ingelheim) according to the manufacturer's instructions.

(c) Aβ Neurotoxicity  And Aβ Neuroprotection

Neural cortical cells were incubated for 5 days according to assay 2. On day 6, NB medium (Invitrogen Life Technologies) and B27 supplement (Invitrogen Life Technologies) were exchanged with NB medium and B27 supplement (without antioxidant). On day 6, test compounds were individually added to neuronal cultures:

Test compounds were dissolved in 100% DMSO at 2.5 mM concentration (10 mM- then diluted 2.5 mM if excess compound per vial was weighed). 2.5 mM mother liquor was serially diluted to 1 in 10 to give 250 uM, 25 uM, 2.5 uM of experimental solution.

Aβ manufacture:

Aβ was initially dissolved in 20 mM NaOH at a concentration of 1 mM and sonicated for 5 minutes. Peptides were then diluted in H ₂ O and 10 × PBS to a concentration of 200 uM Aβ in 1 × PBS. The peptide was sonicated again for 5 minutes and then spun for 5 minutes at 14000 rpm and transferred to a new tube.

Test compounds were dissolved in 100% DMSO at 2.5 mM concentration (10 mM- then diluted 2.5 mM if excess compound per vial was weighed). The 2.5 mM mother liquor was serially diluted to 1 in 10 (in NB medium and B27 (without antioxidant)) to give 250 uM, 25 uM, 2.5 uM of experimental solution. Test compounds were not added directly to the cells, but instead were added to a 48 well 'drug plate' comprising:

Preparation of "drug plate":

48 well plates

Well 1: 515 μl NB + B27 (no antioxidant) ^* + 24 μl 25 uM test compound + 60 μl Aβ diluent ^**

Well 2: 515 μl NB + B27 (no antioxidant) + 24 μl 250 uM test compound + 60 μl Aβ diluent

Well 3: 515 μl NB + B27 (no antioxidant) + 24 μl test compound diluent ^*** + 60 μl Aβ1-42

Well 4: 515 μl NB + B27 (no antioxidant) + 24 μl 2.5 uM test compound + 60 μl Aβ1-42

Well 5: 515 μl NB + B27 (no antioxidant) + 24 μl 25 uM test compound + 60 μl Aβ1-42

Well 6: 515 μl NB + B27 (no antioxidant) + 24 μl 250 uM test compound + 60 μl Aβ1-42 diluent

Well 7: 515 μl NB + B27 (no antioxidant) + 24 μl test compound diluent + 60 μl Aβ1-42 diluent

Well 8: 600 μl NB + B27 (no antioxidant)

Were added.

N.B. 60 μl Aβ1-42 = 20 μl Aβ1-42 / well = 20 uM Aα1-42

The drug plate was incubated at 37 ° C. for 15 minutes. 200 μl of each well was added in triplicate to the corresponding cell plate. The cell plates were incubated at 37 ° C. for 4 days.

^* NB medium + B27 (no antioxidant)

^** Αβ diluent 2 mM NaOH, 1 X PBS

^*** 10% DMSO in PBT Diluent NB + B27 (without antioxidant)

End of analysis:

Four days after cell treatment, the assay is terminated by adding MTS to the cells.

(d) Assay for test compound cytotoxicity

Neural cortical cells were incubated for 5 days in NB medium and B27 supplement according to assay 2.

On day 6, test compounds were added to neuronal cell cultures in NB medium and B27 supplement-antioxidant.

Test compounds were dissolved in 100% DMSO at 2.5 mM concentration (10 mM- then diluted 2.5 mM if excess compound per vial was weighed). 2.5 mM mother liquor was serially diluted to 1 in 10 to give 250 uM, 25 uM, 2.5 uM of experimental solution. Test compounds were not added directly to the cells, but instead were added to a 48 well 'drug plate' comprising:

Preparation of "drug plate":

48 well plates

Well 1: 576 μl NB + B27 (no antioxidant) ^* + 24 μl 2.5 uM test compound

Well 2: 576 μl NB + B27 (no antioxidant) + 24 μl 25 uM test compound

Well 3: 576 μl NB + B27 (no antioxidant) + 24 μl 250 uM test compound

Well 4: 576 μl NB + B27 (no antioxidant) + 24 μl 2.5 uM test compound

Well 5: 576 μl NB + B27 (no antioxidant) + 24 μl 25 uM test compound

Well 6: 576 μl NB + B27 (no antioxidant) + 24 μl 250 uM test compound

Well 7: 576 μl NB + B27 (no antioxidant) + 24 μl test compound diluent ^**

Well 8: 600 μl NB + B27 (no antioxidant)

Were added.

The drug plate was incubated at 37 ° C. for 15 minutes. 200 μl of each well was added in triplicate to the corresponding cell plate. The cell plates were incubated for 4 days at 37 ° C. (test two compounds on each cell plate).

^* NB medium + B27 (no antioxidant)

^** 10% DMSO in PBT Diluent NB + B27 (no antioxidant)

At the end of the assay, 1/10 volume of MTS was added per well of plate (ie 25 μl / 250 μl). The plate was incubated at 37 ° C. for 2 hours, then the absorbance was read at 560 nm.

Analysis 4. Caspase  analysis

To measure caspase activity in neuronal culture, growth medium was removed, cells were washed twice with control salt solution (pH 7.4) and ice cold cell extraction buffer was added directly to the culture. The extraction buffer was 20 mM Tris, pH 7.4, 1 mM Sucrose, 0.25 mM EDTA, 1 mM Dithiothreitol (DTT), 0.5 mM PMSF, 1% Triton X-100 (Tx-100) and 1 μg / Consisting of ml of pepstatin and aprotinin. After 15 min incubation on ice, the extraction buffer is removed and centrifuged for 5 min at 4 ° C. in a microcentrifuge and 100 μl of supernatant is added to each well of a 96 well plate. 100 μl of 200 μM substrate (DEVD-pNA, VEID-pNA or IETD-pNA for Caspase 3, 6 and 8, respectively) was added to each well to obtain a final substrate concentration of 100 μM. Plates are incubated at 37 ° C. for 2, 4, 6 or 24 hours and absorbance is measured at a wavelength of 415 nm (Abs415). Absorbance readings are compared to known standards of pNA alone.

Analysis 5. Annexin  V analysis

To determine the annexin V binding level to the cells, the cultures were washed twice with control salt solution (pH 7.4) followed by annexin V at a concentration of about 0.5 μg / ml in control salt solution (pH 7.4). -FITC was added. Propidium iodide (10 μg / ml) is also added to the culture at the same time. Cells are incubated for 30 min at ambient temperature in the dark and then washed three times with fresh control salt solution. FITC fluorescence (ex. 488 nm, em. 510 nm) analysis is measured using a Leica DMIRB microscope. Use ASA400 color film to take pictures with the Leica MPS 60 camera accessory and scan negatives into Adobe Photoshop v2.0.1.

Analysis 6. Lipoprotein Oxidation Analysis

Two different assays for metal-mediated lipid peroxidation can be used. The first assay involves measuring the oxidative activity of the metallized protein. This is measured by mixing dialyzed metallized or negative proteins (at specified concentrations) with 0.5 mg / ml LDL for 24 hours (37 ° C.). Lipid peroxidation (LPO) is measured using the Lipid Peroxidation Assay Kit (LPO 486, Oxis International Inc. Portland, OR) according to the kit instructions. The level of LPO is measured by comparing the absorbance (486 nm) to LDL alone (100% LPO). A second assay is used to determine the LPO activity of negative proteins in the presence of non-protein-bound free Cu. This includes adding non-metalized peptide (140 μM) with 20 μM Cu-gly to 0.5 μg / ml LDL and analyzing the LPO for metallized protein. The level of LPO is measured by comparing the absorbance (486 nm) to LDL + Cu-gly (100% LPO). As a negative control, LDL is also exposed to a dialyzed Cu-gly solution comparable to the solution used to Cu-metalize the protein.

Analysis 7. Cu - Metallized  Protein-induced Cytotoxicity

The protein or synthetic peptide is mixed with the metal-glycine solution at equimolar or twice the metal to protein concentration. The metal-protein mixture is incubated overnight at 37 ° C. and then extensively dialyzed using a small-dialysis cup (Pierce, Rockford, IL) with a cutoff of 3,500 kilodaltons (2 dH ₂ O (3 L / exchange at room temperature). A) 24 hours for exchange). Protein dialysis against PBS (pH 7.4) produced metallized proteins with the same activity as dH ₂ O dialysis.

To determine the neurotoxic effect, metallized proteins, native proteins or peptides are added to the two-day primary cortical neuronal culture. The culture is also exposed to Cu-gly (5 or 10 μM) or LDL. Positive control cultures are treated with 4-hydroxy-nonenol (HNE, Sigma Chemicals), a Cu-gly + LDL or LPO product. Cultures are analyzed for cell death using the Lactate Dehydrogenase (LDH) Assay Kit (Roche Molecular Biochemicals, Nunawading, Australia) according to the manufacturer's instructions.

Analysis 8. Lysosom  Aβ- of acidification Mediated  Against loss Acridine  Orange analysis

Cultured mouse cortical neurons are treated with Aβ1-42 (20 μM) for 16 hours and then stained with acridine orange (AO) 5 mg / ml for 5 minutes at 37 ° C. and 15 minutes at 37 ° C. The AO-induced fluorescence is measured with a red filter on a fluorescence microscope. AO is a lysosomal weak base that accumulates in the endosome / lysosomal compartment and exhibits orange fluorescence during culture. AO is sequestered inside the lysosomal as long as there is a substantial quantum gradient for the lysosomal membrane. Treatment of cells with Aβ1-42 disrupts the lysosomal membrane quantum gradient, repositioning AO into the cytosol, as indicated by the loss of orange fluorescence within 16-24 hours.

Analysis 9. Human Brain Amyloid Solubilization  analysis

This assay was performed to assess the ability of test compounds to recruit Aβ from post-human AD brain tissue extracts from insoluble to soluble.

Homogenous cortex of 0.5 g or less was homogenized using DIAX 900 homogenizer (Heudolph and Co, Kelheim, Germany) or other suitable device for a period of 3 x 30 seconds at full speed in 2 ml of ice cold phosphate buffered saline (pH 7.4). I was. To obtain the phosphate buffered saline extractable fraction, the homogenate was centrifuged at 100,000 g for 30 minutes and the supernatant was removed. On the one hand, the tissue was lyophilized and then ground to form a powder which was then weighed into aliquots for extraction as above. 10 μl aliquots of the supernatant were removed after centrifugation and mixed with the same amount of 2 × Tris-Tyssen SDS Sample Buffer (pH 8.3) containing 8% SDS, 10% 2-mercaptoethanol. The sample was then heated at 90 ° C. for 10 minutes and separated by gel electrophoresis. An insoluble fraction of the cortical sample was obtained by resuspending the initial pelletized sample in 1 ml of phosphate buffered saline. 50 μl aliquots of the suspension were then boiled in 200 ml of sample buffer as above.

Tris-tricin polyacrylamide gel electrophoresis was performed by loading a suitably diluted sample onto a 10% to 20% gradient gel (Novex, San Diego, Calif.) Followed by a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules). , CA). Aβ was detected using monoclonal antibody W02, which detected residues 5-8, 17 (or another suitable antibody) with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Dako, Denmark) And is visualized by enhanced chemiluminescence (eg ECL; Amersham Life Science, Buckinghamshire, UK). Each gel contained three lanes containing 0.5, 1 and 2 ng of synthetic Aβ ₄₀ (Keck Laboratory, Yale University, New Haven, CT) as reference standards.

Blot membranes were read using a suitable imager, eg Fuji LAS3000, and density measurements were performed using a suitable software, eg Multigauge. A linear range of signal intensities for the quantitative analysis of monomeric and dimeric Aβ bands was established against known Aß standards. The percentages calculated in Table 13 represent mean readings from the treated mouse group for the vehicle treated mouse group.

All samples were analyzed several times and the gel load and dilution adjusted to fit within the quantifiable region of the standard curve. Insoluble Αβ comprising a pelletable fraction was derived from an insoluble amyloid plate from the cortical sample and a soluble fraction comprising monomer and / or oligomeric soluble Αβ.

Multiple gels were run on test compounds with PBS controls included on each gel. Each gel contained various concentrations of test compound. The Student's t 'test was used to compare the average of the highest value obtained by the test compound for each gel at any concentration to the average of PBS values taken from the plurality of gels. Thus, a determination can be made whether the solubility average solubility increase obtained by any test compound is significant compared to PBS alone. Test compounds with positive scores are compounds that achieve a statistically significant increase in plaque solubility compared to PBS alone. Test compounds with a negative score are those that have not achieved a statistically significant increase in plaque solubility compared to PBS alone.

Analysis 10. Metal Partitioning

To analyze the effect on the partitioning of various metals, including zinc and copper, soluble and insoluble fractions were prepared from human brain tissue extracts following extraction of brain tissue in the presence of test compounds, followed by amyloid solubilization assay. The metals in these two fractions are optionally analyzed by suitable pretreatment with nitric acid and / or hydrogen peroxide followed by inductively coupled plasma mass spectrometry.

Analysis 11. Transgenic  Aβ in animals On the sediment  Dosing Effects of Test Compounds on

Transgenic mouse models have been described in a number of neurological diseases, such as Alzheimer's disease (Games et al., 1995; Hsiao et al., 1996); Parkinson's disease (Masliah et al., 2000); Familial amylotropic lateral sclerosis (ALS) (Gurney et al., 1994); Huntington's disease (Reddy et al., 1998); And Creutzfeldt-Jakob disease (CJD) (Telling et al., 1994). APP2576 transgenic mice (Hsiao et al., 1996), one of the transgenic models for Alzheimer's disease, also have a high cataract incidence. These animal models were suitable for testing the methods of the present invention.

Transgenic mice of the APP2576 strain (Hsiao et al 1996) were used. Female mice, 8-9 months old, were selected and divided into treatment groups.

Mice were killed at regular intervals and their brains examined to determine if treatment with test compounds reduced brain amyloid formation and identified the most effective dosing protocol. Levels of soluble and insoluble Αβ in the brain and serum were measured using a Western blot scaled according to the method disclosed for Analysis 9. Brain Amyloid Solubilization Assay.

Different mice in each group were tested over a period of up to 8 months for recognition performance using a Morris water maze according to standard methods. The general health and well-being of the animals was also measured daily by blindfolded operators using a 5-point integer rating that subjectively assesses the combination of features including motor activity, attention and general health cues.

Analysis 12. Physical and chemical properties

Polar surface area calculation PSA )

Polar surface area values were calculated using a web-based program available through Molinspiration, a package for calculating molecular properties.

Turbidity measurement  Solubility measurement

Solubility assessments were measured at both pH 2.0 and pH 6.5. It is within the pH range that can be expected along the human close gastrointestinal tract.

Compounds were dissolved at a concentration suitable for DMSO and then dissolved in isotonic phosphate buffer at 0.01 M HCl (approximately pH-2.0) or pH 6.5 and the final DMSO concentration was 1%. Samples were then analyzed via turbidity measurement for determination of solubility range [D. Bevan and R.S. Lloyd, Anal. Chem. 2000, 72, 1781-1787].

cLog  P value

Theoretical Log P values were measured using ACD Log P software. Cited values are calculated from untrained database and are for associated species.

Analysis 13. Blood Brain Barrier Penetration

Test compounds were dissolved in DMSO and phosphate buffered saline (PBS) was added to obtain a 50 μM concentration solution in PBS containing 1.25 to 2.5% DMSO. Traces of ¹⁴ C-suscrose were assessed for the integrity of the blood-brain barrier (BBB) during each perfusion and remained inside the vascular lumen at the end of each perfusion volume (ie, at the end of each perfusion). Volume of blood) was added to each injection mother liquor (approximately 0.01 μCi / ml) to serve as the BBB-invasive marker.

Adult male Sprague Dawley rats (180-190 g) were anesthetized by intraperitoneal injection of 1.0 mL / 100 g body weight urethane (25% w / v). The right common carotid artery was surgically exposed and a cannula was inserted for perfusion of the cerebral circulation. The right outer carotid artery (which supplies tissue outside the skull) was connected far away at the bifurcation from the right common carotid artery so that all infusion solution entered the brain through the remaining right inner carotid artery. The heart was then exposed and transversal immediately before the initiation of the infusion. The infusion rate was adjusted to deliver 3.2 ml / min (approximately 85% of normal blood is supplied to the brain for this size rat) by the pump set. The infusion cannula contained 0.5 ml of prewashed heparinized PBS (10 IU / ml), which initially acts to flush blood vessels and prevent blood from clotting and clogging small vessels.

After 1.5 minutes, the infusion pump was automatically stopped, the cannula was withdrawn from the carotid artery, and the infusion solution sample (1-1.5 mL) was then collected from the tip of the infusion cannula. The brain was then freely dissected and divided into three parts: the right hemisphere with the right midbrain, the left hemisphere with the left midbrain and the cerebellum (cerebellum, pons and brain stem). Only the right part of the brain was used for subsequent measurements, because perfusion through the right inner carotid artery is preferentially supplied to the right hemisphere and the right midbrain (the left hemisphere and the posterior brain receive variable secondary perfusion). Brain tissue samples from each animal were frozen at −30 ° C., homogenized and weighed aliquots were analyzed by LC-MS to obtain total brain concentration. The analysis was performed using a Micromass Triple Quad instrument. The mobile phase consisted of acetonitrile / water with 0.05% formic acid and the column was Phenomenex Luna CN.

Small fractions from each brain tissue sample and the corresponding infusion solution were analyzed by liquid scintillation counting to determine the level of ¹⁴ C-Sucrose. Residual vascular space (RVS) in each brain tissue sample was calculated by dividing the measured concentration (dpm / mg) of sucrose in brain tissue by its concentration (dpm / μl) in the corresponding infusion solution. This is the volume of fluid remaining inside the vessel at the end of each perfusion. The RVS is multiplied by the concentration of the test compound in the infusion solution to determine the total amount of test compound present inside the blood vessel (ie, not passing BBB) in each brain tissue sample. Subtract this from the total brain concentration to determine the amount of drug in each brain tissue sample outside of blood vessels (ie, through the BBB). The brain absorption rate is obtained by dividing the RVS-corrected brain concentration (Equation 1).

Brain Absorption Rate = ([Brain ng.mg ^-1 ]-[RVS ng.μl ^-1 ]) / [infusion solution ng.μl ^-1 ]

A total of 5 to 6 brain perfusion experiments were performed for each test compound and the average brain uptake ratio was calculated.

At least 50% indicate a compound entering the brain very quickly; A ratio of 10 to 50% refers to compounds that enter the brain well; A ratio less than 10% (not observed) refers to compounds that enter the brain very slowly and are not suitable for therapeutic administration; Less than 1% (not observed) refers to compounds that are effectively excluded from the brain.

Analysis 14. Transgenic  Mouse Brain Immunohistochemistry

The APP2576 transgenic mouse (Hsiao et al., 1996) mentioned in Assay 11 was used for this analysis. Contralateral formalin-fixed mouse brain tissue was coronally cut. Sections (10 μm) were taken from the corresponding sites and treated with 80% formic acid for antigen recovery. The primary antibody used was monoclonal antibody 1E8, which recognizes an epitope between residues 18-22 of Aβ (SmithKline Beecham, UK). Immune reactivity was used with horseradish peroxidase (using 3,39-diaminobenzidinechromene) (Dako) and alkaline phosphatase (5-bromo-4-chloro 3-doxyl phosphatase and nitroblue tetrazolium chloride chromagen) By a secondary antibody linked to Dako. Plate abundance per section was evaluated by two blinded operators for treatments according to the following grades:

0 = plate is not clear

1 = plate exists but is very thin

2 = multiple plates exist

3 = You can see multiple plates in a limited area

4 = rich in plate and not limited to any specific area

A median value, for example 2.5, was designated as appropriate.

The Student 't' test was used for fertilizers between groups.

Analysis 15. Pharmacokinetic Profile

Intravenous infusion of test compound; 2 mg / kg in a suitable vehicle was administered to two rats and arterial blood was sampled up to 24 hours.

Oral administration of test compounds; 30 mg / kg in a suitable vehicle was administered to two rats via oral gavage and arterial blood was sampled up to 24 hours.

Plasma concentrations of the test compounds were determined by suitable analytical methods.

Calculation:

CL _totle = total plasma removal after IV administration

V _{d β} = volume of dispense during the elimination phase after IV administration

BA = oral bioavailability

Area under plasma concentration versus time profile from 0 hours to infinity after AUC _IV = IV administration

AUC _oral = area under the plasma concentration versus time profile from 0 hours to infinity after oral administration

β = terminal elimination rate constant after IV administration

Assay 16. Measurement of Mouse Plasma Levels of Test Compounds

PB  1075

PB 1075 30 mg / kg was orally administered to four mice by oral gavage as a suspension in Na-carboxymethyl cellulose (CMC). Two mice were killed 30 minutes after administration and two mice were killed 60 minutes after administration. Blood was collected by cardiac puncture and plasma was separated by centrifugation.

The concentration of PB1075 was measured by LC / MS using a triple quadrupole device. The mobile phase consisted of an acetonitrile (ACN) / water gradient (containing 0.05% formic acid) and the column was a Phenomenex Rooney 5 μm C8 (50 × 2 mm) column.

The supplied acutely toxic mouse plasma samples were injected directly following protein precipitation by ACN. Analytical methods for plasma were linear in the range of 10 to 10,000 ng / ml (R ² = 0.994). The recovery rate of PB1075 from plasma was -100%.

The concentration of PB1075 in mouse plasma samples is shown in Table 12.

In mouse plasma after 30 mg / kg oral administration PB1075 Concentration Example No. Time (minutes) Concentration (ng / ml) 2459 30 431.26 2470 30 298.46 2495 60 424.81 2781 60 519.56

PB  1076

PB 1076 30 mg / kg was orally administered to four mice by oral gavage as a suspension in Na-carboxymethyl cellulose (CMC). Two mice were killed 30 minutes after administration and two mice were killed 60 minutes after administration. Blood was collected by cardiac puncture and plasma was separated by centrifugation.

The concentration of PB1076 was measured by LC / MS using a triple quadrupole device. The mobile phase consisted of an acetonitrile (ACN) / water gradient (containing 0.05% formic acid) and the column was a Phenomenex Rooney 5 μm C8 (50 × 2 mm) column.

The mouse plasma samples were injected directly following protein precipitation by ACN. Analytical methods for plasma were linear in the range of 500 to 10,000 ng / ml (R ² = 0.999). The recovery rate of PB1076 from plasma was -85%.

Table 13 shows the concentration of PB1076 in mouse plasma after 30 mg / kg oral administration.

In mouse plasma after 30 mg / kg oral administration PB1076 Concentration Mouse ID Time (minutes) Concentration (ng / ml) 3036 30 2325.77 3041 30 1593.61 3014 60 2697.62 3015 60 1167.52

PB  1077

30 mg / kg PB 1077 was orally administered to four mice by oral gavage as a suspension in Na-carboxymethyl cellulose (CMC). Two mice were killed 30 minutes after administration and two mice were killed 60 minutes after administration. Blood was collected by cardiac puncture and plasma was separated by centrifugation.

The concentration of PB1077 was measured by LC / MS using a triple quadrupole device. The mobile phase consisted of an acetonitrile (ACN) / water gradient (containing 0.05% formic acid) and the column was a Phenomenex Rooney 5 μm C8 (50 × 2 mm) column.

The mouse plasma samples were injected directly following protein precipitation by ACN. The analytical method for plasma was linear in the range of 5 to 5,000 ng / ml (R ² = 0.999). The recovery of PB1077 from plasma was -92%.

The concentration of PB1077 in mouse plasma after 30 mg / kg oral administration is shown in Table 14.

In mouse plasma after 30 mg / kg oral administration PB1077 Concentration Mouse ID Time (minutes) Concentration (ng / ml) 3062 30 731.2 3060 30 984.7 3093 60 752.8 3095 60 495.1

Assay 17. Synthetic Amyloid Plaque Degradation Assay

This assay measures the ability of test compounds to dissolve aggregates of the A-beta 42 residue peptide of synthetic Alzheimer's formed by precipitation with zinc.

The synthetic plaque degradation assay is a thioflavin T fluorometer assay that measures the ability of test compounds to degrade synthetic aggregates produced by the cultivation of Alzheimer's amyloid A-beta protein (Aß) in the presence of zinc.

The most prevalent form of Alzheimer's amyloid plaques, the 42 residue A-beta, is precipitated by the addition of zinc salts to form beta sheet form aggregates that are physicochemically consistent with the crystalline amyloid plaque core. Thioflavin T is a reagent that exhibits specific fluorescence when inserted into the beta sheet structure and is incorporated into the A-beta / Zn aggregate during the aggregation process. The fluorescence will decrease as the beta sheet form is lost as a result of dissolution of the metal bound aggregate by the test compound. The activity of a compound in this assay is a combination of structural elements that affect the metal chelating properties, solubility, hydrophobicity and interaction with the amyloid mass of the compound.

This assay is an in vitro model of plaque degradation, in which the bound metal is tested to produce Aß (dissolved) which is either precipitated by zinc by competing with Aß or by displacing the metal by competitive binding on the one hand. Model how the compound works.

Analytical reagent

For convenience, aliquots of synthetic Aß peptides are prepared. Aß is dissolved in distilled water and peptide concentration is assessed by absorption at 214 nm against the identified standard curve. A beta / Zn aggregates only in the presence of solvent (DMSO) and control vehicle (PBS) are included in each assay as a negative control. The test compounds were dissolved in DMSO at a concentration of 5 mM. As appropriate, DMSO was diluted to 100 times the desired final concentration and added directly to A beta aggregates.

Way

A beta 1-42 is incubated for 24 hours at 37 ° C. on a rotating wheel in PBS at pH 6.6 with a molar ratio of ZnCl 2 and thioflavin T (ThT) and (1: 2: 2). Following incubation, the aggregates are incubated with the test drug for another 2 hours at 37 ° C. while rotating the aggregates. PBS blanks, untreated aggregates and DMSO controls were included in each experiment. After 2 hours of incubation, samples are measured for ThT fluorescence in cuvettes using an LS55 (Perkin Elmer) fluorometer.

Data is generated in FL Winlab software (Perkin Elmer) and analyzed using GraphPad Prism v4.0 software. Data is calculated as the average of several readings.

result

The results are tabulated as the concentration at which 50% degradation (IC ₅₀ ) is obtained (uM) and the percent degradation at 5 uM (expressed as a 5 /% reduction). The two values together provide the magnitude of the decomposition efficiency.

If the compound does not reach 50% degradation within the concentration range tested, the results are reported as> 20 μM, which corresponds to the maximum concentration at which the compound is tested. These results indicate that the test compound is relatively inadequate to degrade Aß 1-42 aggregates. Test compounds that can achieve IC ₅₀ below 20 μM and score above 20% degradation at 5 μM are considered 'good'. Test compounds that can achieve IC ₅₀ below 20 μM and score above 40% degradation at 5 μM are considered 'very good'.

The references cited in the description and examples are listed on the following pages, which are incorporated herein by reference.

references

Although the present invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art that various changes and modifications to the embodiments and methods disclosed herein can be made without departing from the scope of the inventive concept disclosed herein. It will be obvious to them.

This invention provides neuroactive compounds, methods for their preparation and especially their use as pharmaceutical or veterinary medicaments for the treatment of neurological diseases, more particularly neurodegenerative diseases such as Alzheimer's disease.

Claims (40)

Compounds of formula (I), salts, hydrates, solvates or isomers thereof Formula I

Where R ² is H or CH ₂ NR ¹ R ^4, wherein R ¹ and R ⁴ are independently selected from H, optionally substituted C _1-6 alkyl and optionally substituted C _3-6 cycloalkyl; R ³ is H; Optionally substituted C _1-4 alkyl; Optionally substituted C _2-4 alkenyl; Optionally substituted C _3-6 cycloalkyl; Optionally substituted 6-membered aryl or optionally substituted 6-membered aryl condensed with heteroaryl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ⁶ where n is an integer from 1 to 6, R ⁶ is optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- Or 6-membered N-containing heterocyclyl or optionally substituted 6-membered aryl); NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are H, optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing hetero Independently selected from cyclyl and optionally substituted 6-membered aryl); NHCOR ¹⁰ wherein R ¹⁰ is optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl or optionally substituted 6 -Raw aryl); CH ₂ CONR ¹¹ R ¹² , wherein R ¹¹ and R ¹² are H, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkynyl and optionally substituted 6-membered aryl, optionally substituted with optionally substituted Independently selected from 5- or 6-membered N-containing heterocyclyl); And (CH ₂ ) _m NHR ¹³ where R ¹³ is selected from optionally substituted C _1-6 alkyl and SO ₂ R ¹⁴ , wherein R ¹⁴ is substituted C _1-6 alkyl and optionally substituted 6-membered aryl Optionally selected from m, m is 1 to 6); R ⁵ and R ⁷ are independently selected from H and halo; X is O or S Wherein each optionally substituted group defined in R ¹ to R ¹⁴ is optionally substituted with any substituent selected from C _1-4 alkyl, hydroxy, halo, C _1-4 alkoxy and C _1-4 acyl, (i) at least one of R ² and R ³ is other than H; (ii) at least one of R ⁵ and R ⁷ is halo; (iii) when X is O, R ⁵ and R ⁷ are Cl and R ² is H, R ³ is not cyclopropyl or parafluorophenyl; (iv) when X is O, R ⁵ is H, R ⁷ is I and R ² is H, then R ³ is not C _2-4 alkyl. The method of claim 1, Is a compound of Formula (IA) ≪

Where R ⁵ , R ⁷ and X are as defined in claim 1; R ³ is optionally substituted C _1-4 alkyl; Optionally substituted C _2-4 alkenyl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ⁶ where n is 1-3 and R ⁶ is optionally substituted C _3-6 cycloalkyl or optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl ; NR ⁸ R ⁹ , wherein R ⁸ is H and R ⁹ is H or optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl; NHCOR ¹⁰ , wherein R ¹⁰ is optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl. The method of claim 2, C _1-4 alkyl optionally substituted with R ³ ; Optionally substituted C _2-4 alkenyl; Optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ⁶ where n is 1-3 and R ⁶ is optionally substituted C _3-6 cycloalkyl or optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl ; Or NR ⁸ R ⁹ , wherein R ⁸ is H and R ⁹ is H or optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl. The method of claim 1, The following compounds:

The method of claim 1, Is a compound of Formula IB: IB

Wherein R ² , R ⁵ , R ⁷ and X are as defined in claim 1. 6. The method of claim 5, R ² is CH ₂ NR ¹ R ^4, and wherein R ¹ and R ⁴ is H, optionally substituted C _{1 -6} alkyl, and optionally substituted C _{3 -6} compound is independently selected from cycloalkyl. The method of claim 1, The following compounds:

The method of claim 1, Is a compound of Formula IC: IC

Where R ⁵ , R ⁷ and X are as defined in claim 1; R ^2C is CH ₂ NR ¹ R ⁴ (wherein R ¹ and R ⁴ are independently selected from H and optionally substituted C _{1 -6} alkyl); R ^3C is an optionally substituted C _{1 -4} alkyl. 9. The method of claim 8, The following compounds:

The method of claim 1, R ⁵ and R ⁷ are both halo. The method of claim 1, R ⁵ and R ⁷ are both chloro. delete delete delete A pharmaceutical or veterinary composition for the treatment, amelioration or prevention of neurodegenerative diseases comprising a compound of formula (I) as defined in claim 1 and a pharmaceutically or veterinary acceptable carrier. 16. The method of claim 15, further comprising another agent selected from fluvirprofen or ibuprofen optionally modified to release penserine, galantamine, tacrine, vitamin E, vitamin C, nitric oxide, or β-estradiol. Composition. delete delete delete 16. The method of claim 15, The neurodegenerative disease is neurodegenerative amyloidosis. 21. The method of claim 20, Neurodegenerative diseases are related to sporadic or familial Alzheimer's disease, Amiotropic lateral sclerosis, cataracts, Parkinson's disease, Creutzfeldt-Jakob disease and its “mad cow” disease, Huntington's disease, dementia with Lewis body formation, multiple systemic Atrophy, Haloboden-Sparta's disease, diffuse Lewis body disease, fatal familial insomnia, Gerzmann Straussler Schwinger disease, hereditary cerebral hemorrhage with Dutch-amyloidosis, multiple sclerosis, tauopathy, A composition that is motor neuron disease or prion disease. 16. The method of claim 15, The neurodegenerative disease is Parkinson's disease. 16. The method of claim 15, Wherein the neurodegenerative disease is an Aß-related disease. 24. The method of claim 23, Wherein the Aß-related disease is dementia associated with Alzheimer's disease, or one of autosomal dominant forms of Down's syndrome or several forms of familial Alzheimer's disease. 16. The method of claim 15, A composition that slows, reduces or stops a patient's loss of cognition. delete delete delete delete delete A compound of formula (I) as defined in claim 1 for use in the treatment, amelioration or prevention of neurodegenerative diseases. (a) reacting an optionally protected compound of formula V with H ₂ NR ³ , wherein R ³ is as defined in claim 1 to form an optionally protected compound of formula VII: Formula V

Formula VII

(Wherein R ⁵ and R ⁷ are as defined in claim 1); (b) reducing the compound of formula VII to form an optionally protected compound of formula VIII: VIII

(c) cyclizing the compound of Formula VIII to form an optionally protected compound of Formula I wherein R ² is H; or (d) R ² CHO, R ² CO ₂ H or R ² C (OR ^x ) ₃ , wherein R ^x is optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl and R ² is Cyclization of the compound of formula VIII in the presence of A process for preparing a compound of formula I as defined in claim 1. (a) Aminating an optionally protected compound of formula VI to form an optionally protected compound of formula IX: VI

IX

(Wherein R ⁵ and R ⁷ are as defined in claim 1); (b) wherein the compound of Formula IX is R ³ -L or R ³ OSO ₂ R ^x wherein L is a leaving group, R ³ is as defined in claim 1, and R ^x is optionally substituted C _1-4 alkyl Or optionally substituted 6-membered aryl) A process for preparing a compound of formula I as defined in claim 1, wherein R ² is H. (a) reacting an optionally protected compound of formula (VI) as defined in claim 33 with a formylating agent to form an optionally protected compound of formula (X) or optionally protected compound of formula (XI): X

XI

(b) reacting the compound of formula X or XI with an acylating agent containing R ² to form an optionally protected compound of formula XII or a compound of formula XIII: XII

Wherein R ² is as defined in claim 1 XIII

(c) reacting the compound of formula XII or XIII with H ₂ NR ³ , wherein R ³ is as defined in claim 1 A process for preparing a compound of formula I as defined in claim 1, comprising: delete delete delete (a) diazotizing a compound of formula III to form a compound of formula IV; (III)

Formula IV

(b) nitrating the compound of formula IV to form a compound of formula V; Formula V

(c) reacting the compound of formula V with H ₂ NR ³ , wherein R ³ is as defined in claim 1 to form a compound of formula VII: Formula VII

(d) reducing the compound of formula VII to form a compound of formula VIII: VIII

(e) cyclizing the compound of formula VIII to form an optionally protected compound of formula I wherein R ₂ is H A process for preparing a compound of formula I as defined in claim 1, wherein R ² is H and R ⁵ and R ⁷ are independently selected from halo. (a) diazotizing a compound of formula III to form a compound of formula IV; (III)

Formula IV

(b) nitrating the compound of formula IV to form a compound of formula V; Formula V

(c) reacting the compound of formula V with H ₂ NR ³ , wherein R ³ is as defined in claim 1 to form a compound of formula VII: Formula VII

(d) reducing the compound of formula VII to form a compound of formula VIII: VIII

(e) R ² CHO, R ² CO ₂ H or R ² C (OR ^x ) ₃ , wherein R ^x is optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl, and R ² is Cyclization of the compound of Formula VIII in the presence of A process for preparing a compound of formula I as defined in claim 1, wherein R ² is H and R ⁵ and R ⁷ are independently selected from halo. (a) diazotizing a compound of formula III to form a compound of formula IV; (III)

Formula IV

(b) nitrating the compound of formula IV to form a compound of formula V; Formula V

(c) reducing the compound of Formula V to form a compound of Formula VI: VI

(d) amination of the compound of formula VI to form a compound of formula IX; IX

(In the above formulas, as defined in claim 1); (e) wherein the compound of Formula IX is R ³ -L or R ³ OSO ₂ R ^x wherein L is a leaving group, R ³ is as defined in claim 1 and R ^x is optionally substituted C _1-4 alkyl Or optionally substituted 6-membered aryl) Wherein R ² is H and R ⁵ and R ⁷ are independently selected from halo.