Cross References to Related Applications This, application claims the priority of European patent application no. 05012616.8, filed June 13, 2005 and of US provisional application no. 60/690,415, filed June 14, 2005, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

This invention relates to phenyl urea and phenyl thiourea compounds, in particular to such compounds in the treatment of neurodegenerative diseases, in particular Alzheimer's disease.

Background Art

Alzheimer's disease (AD) is the most common form of dementia among older people, and affects parts of the brain that control thought, memory and language. Susceptibility to Alzheimer's disease increases with age, but Alzheimer's disease is not a normal part of the ageing process. A characteristic of this disease is the presence of extracellular senile plaque, the major component of which is the β-amyloid peptide (Aβ) . The hydrophobic, 39-43-amino-acid-long Aβ peptide is excised from the amyloid precursor protein (APP) by sequential cleavage by the so-called β- and γ-secretases .

Known genetic predispositions for AD mostly affect genes involved in Aβ generation or Aβ deposition. Since the Aβ peptide seems to play an important role in the pathogenesis of AD, current therapeutic strategies often focus on inhibition of Aβ deposition and generation. Inhibition of β-secretase activity represents an attractive option to achieve this goal.

Despite major efforts to identify novel β- secretase inhibitors by applying in vitro high-throughput screening (HTS) assays with purified soluble BACE-I fragments and fluorogenic peptide substrates, the best progress towards efficient BACE-I inhibition has been achieved so far by the use of peptidic transition-state mimetic compounds. However, for efficient inhibition of β-secretase in cells, their molecular weight must be reduced and their structure modified so as to allow for permeation of cellular membranes, the blood-brain barrier and for activity in the natural cellular environment.

There also exist some assays for identifying low molecular weight inhibitors of secretases that can block these membrane-bound enzymes at the natural location within intracellular compartments. Cell-based

HTS assays, however, are generally faced with the problem that selection signals are often caused by compounds that interfere with cellular processes or pathways that are redundant with that of the target. For example, some compounds found by mammalian cell based assays impair the production of Aβ through the increase of the pH in intracellular compartments, or they function through protein phosphorylation, or they simply catalyze polymerization of Aβ, thus reducing the percentage of soluble peptide.

A method for the identification of modulators of a secretase activity is described in WO 03/087842 Al.

Some candidate compounds for inhibiting the production of Aβ peptide in a biological system have been proposed in US 5,814,646 and US 5,624,937. A review is found in Gumming J.N. et al . , "Design and development of BACE-I inhibitors", Current Opinion in Drug Discovery and Development 2004, 7 (4 ): 536-556.

Phenylurea type compounds are indicated as inhibitors of cyclin-dependent kinase 5/p25 and as a potential treatment of Alzheimer's disease (see Helal CJ. et al., "Discovery and SAR of 2-aminothiazole

inhibitors of cyclin-dependent kinase 5/p25 as a potential treatment for Alzheimer' ^' s disease", Bioorganic & Medical Chemistry Letters 14 (2004) 5521-5525).

Several diphenylurea and phenylurea type compounds have been described in literature (see e.g. Database Chemcats, Chemical Abstracts Service Columbus , Ohio, USA) .

Nevertheless, there is still a need for potent β-secretase inhibitors that directly inhibit β- secretase .

Disclosure of the Invention

Hence, it is a general object of the invention to provide compounds that directly act as β- secretase inhibitors .

Now, in order to implement these and still further objects of the invention, which will become more readily apparent as the description proceeds, the urea compounds for use in the present invention that have been found to be β-secretase inhibitors are manifested by the following formula I

(I)

wherein

R is independently from each other selected from H, C _] _-Cg-alkyl, Cχ-Cg-alkoxy, halo-C]_-Cg-alkyl, e.g. CF3, Cχ-Cg-alkylcarbonyl _Λ halogen, cyano or an -NR2R3 group, especially R is independently from each other selected from H, C^-Cg-alkyl, C^-Cg-alkoxy, halo-C]_-Cg- alkyl, e.g. CF3, C]_-Cg-alkylcarbonyl, halogen, or cyano,

Y = 0 or S, preferably 0, R _] _ is optionally substituted linear or branched C^-C4-alkyl, wherein the substituents are selected from optionally halogen substituted aryl, C1-C4- alkoxy, or morpholinyl,

R2 and R3 are independently from each other H, linear or branched Cl-C6-alkyl, in particular linear or branched Cl-C4-alkyl, or R2 and R3 form together with the nitrogen to which they are bound an aliphatic or aromatic 5- or 6-membered one or more heteroatoms comprising heterocycle, e.g. a piperidino, a piperazino or a morpholino group, and n independently from each other is 0, 1 or 2. In presently slightly preferred inhibitors R is independently from each other selected from H, C]_-C4-alkyl, Cχ-C4-alkoxy, halo-C _] _-C4-alkyl, C]_- C4~alkylcarbonyl, halogen, and cyano.

Alternatively, R is independently from each H, an alkyl, an alkoxy, a haloalkyl, an alkylcarbonyl, or a halogen as defined above.

In a preferred embodiment, R _] _ is optionally substituted linear or branched C _] _-C4-alkyl, wherein the substituents are selected from optionally halogen substituted aryl, C1-C4- alkoxy, or morpholinyl,

^R 2 ⁼ Cχ-Cg-alkyl, R3 = Cχ-Cg-alkyl,

or R2 and R3 form together with the nitrogen to which they are bound a 5-membered or a 6-membered aliphatic or aromatic ring, and n = 0, 1 or 2, preferably 0.

In a more preferred embodiment

R _] _ is optionally substituted linear or branched C^-^-alkyl, wherein the substituents are selected from optionally halogen substituted aryl, C1-C4- alkoxy, or morpholinyl,

^R 2 ⁼ C]_-C4-alkyl, and

R3 = C]_-C4~alkyl, and n = 0 or 1, especially 0.

In an also preferred embodiment R2 and R3 form together with the nitrogen to which they are bound a heterocycle selected from

In especially preferred embodiments, R]_ is CH ₂ CH ₂ CH ₃ , (CH ₂ ) 3-OCH3, (CH ₂ ) 2-OCH3, (CH ₂ ) 3-OCH2CH3, CH ₂ CH ₂ -C ₆ C ₅ , p-F-benzyl, CH(CH ₃ )CgH ₅ , or

In general R]_ is optionally substituted linear C]_-C4~alkyls . In a preferred embodiment

R is independently selected from H, CH3, CH ₂ CH3, OCH3, COCH3, Cl, Br, CF3, CN, more preferred selected from H, CH3, CH ₂ CH ₃ , OCH3, Cl, Br, CF ₃ , CN.

Alternatively, R is independently selected from H, CH ₃ , CH ₂ CH ₃ , OCH ₃ , COCH ₃ , Cl, Br, CF _3^ more preferred selected from H, CH ₃ , CH ₂ CH ₃ , OCH ₃ , Cl, Br,

^C F3.

In an also preferred embodiment

^R l ⁼ C ₃ -alkyl, C]_~C4-alkoxy-C2-C3-alkyl, an optionally p-fluoro substituted phenyl-C;i_-C4-alkyl, and/or R ₂ = R ₃ = CH ₃

In another preferred embodiment, the compound is a compound of formula (II)

wherein

Rg = H or halogen,

R7 = H, C]_-C4-alkyl, C _] _-C4-alkoxy, C1-C4- alkylcarbonyl, halo-Cχ-C4-alkyl, cyano or halogen,

Rg = H, C]_-C4-alkyl, cyano or halogen,

R9 = H, C _] _-C4-alkyl, C _] _-C4-alkoxy or halo- Cχ-C4-alkyl,

R]_0 = H or halogen, and

R]_, R2r ^ ₃ ^an d Y are as defined above.

In an alternative embodiment,

Rg = H or halogen,

R7 = H, C _] _-C4-alkyl, C _] _-C4-alkoxy, C1-C4- alkylcarbonyl, halo-C _] _-C4-alkyl or halogen,

Rg = H, C]_-C4-alkyl or halogen,

Rg == H, C]_-C4~alkyl, Cχ-C4-alkoxy or halo- C _] _-C4-alkyl,

R^O = H or halogen, and

R^, R ₂ , R ₃ and Y are as defined above.

In more preferred compounds of formula (II)

Rg = H or Br,

R7 = H, Cl, CH3, OCH3, CO-CH3, CF ₃ , CN

Rg = H, CH2CH3, CH ₃ , Cl, CN

Rg = H, CH ₃ , OCH ₃ , CF3, and

R ₁₀ = H, Br.

In an alternative embodiment,

Rg = H or Br,

R ₇ = H, Cl, CH ₃ , OCH ₃ , CO-CH ₃ , CF ₃ ,

R ₈ = H, CH ₂ CH ₃ , CH ₃ , Cl,

Rg - H, CH ₃ , OCH ₃ , CF ₃ , and

R ₁₀ = H, Br.

Especially preferred compounds of formula (I) or formula (II) are those wherein

R]_ is optionally substituted linear or branched C]_-C4-alkyl, wherein the substituents are selected from optionally halogen substituted aryl, Ci-C/p alkoxy, morpholinyl

R ₂ = C]_-C4-alkyl

R ₃ = Cχ-C4~alkyl, and n = 0 or 1.

Much preferred, R2 and R3 both are methyl, and also much preferred n=0.

A more preferred Ri is CH ₂ CH ₂ CH ₃ , (CH ₂ ) 3- OCH ₃ , (CH ₂ ) ₂ -OCH ₃ , (CH ₂ J ₃ -OCH ₂ CH ₃ , CH ₂ CH ₂ -CgC ₅ , p-F- benzyl, CH(CH ₃ )CgHs, or

All substituents disclosed as being preferred, more preferred, most preferred with regard to formula (I) are also preferred substituents with regard to formula (II) .

In a further embodiment of the present invention the β-secretase inhibitors have the following formula (III)

wherein

R and Y are as defined above, but preferably R is independently from each other selected from H, CH3, CF3, OCH3, Cl, CO-CH3, and preferably Y is 0,

D = 0 or S or N or NR4 or CR5, in particular S or N or NR4 or CR5, wherein

R4 is linear or branched Cχ-Cg-alkyl, in particular C _] _-C4-alkyl

R5 is independently from each other selected from C _] _-Cg-alkylthio, aryl-C _] _-Cg-alkylthio, aryloxy-C^- Cg-alkyl, arylthio-C;j_-Cg-alkyl, alkyloxy-C]_-Cg-alkyl, alkylthio-C]_-Cg-alkyl, C]_-Cg-alkyloxy, aryl-C^-Cg- alkyloxy and optionally substituted linear or branched C _] _-Cg-alkyl,

U = -CH ₂ -, C=O, - (CH ₂ ) _n S-, - (CH ₂ ) _n O-, -(CH ₂ ) _n NH-, or

-(CH ₂ )ET I- (CH ₂ ) _n -, wherein the heterocycle is op-

tionally substituted, in particular by one or two C]_-C4- alkyl groups or such that a bicycle is formed, and n independently from each other is 0, 1 or 2,

with the proviso that only one D can be 0 or S, in particular S, and at most two adjacent D can be other than C-R5, and with the proviso that in the case that n is 0 and

R5 is substituted linear or branched C] ₁ -Cg- alkyl, preferably linear or branched C]_-C4-alkyl, with the substituent being selected from O-R]_3 or S-R^3 or N- R13R13 'with R]_3 and R13' being independently selected from the group consisting of unsubstituted or substituted 5- or β-membered aryl, unsubstituted or substituted 5- or 6-membered heteroaryl, with the substituents of the aryl or heteroaryl group being as defined for R, linear or branched C^-Cg-alkyl and C5~Cg-cycloalkyl, in particular from the group consisting of O-R13 or S-R13 with R]_3 being selected from the group consisting of 5- or 6- membered aryl, and linear or branched C]_-C4~alkyl . In a slightly preferred embodiment R5 is independently from each other selected from C _] _-C4~alkylthio, aryl-C _] _-C4-alkylthio, aryloxy-C]_- C4-alkyl, arylthio-C;j_-C4-alkyl, alkyloxy-C]_-C4-alkyl, alkylthio-C2_-C4-alkyl, C _] _-C4~alkyloxy, aryl-C _] _-C4~ alkyloxy and optionally substituted linear or branched Cχ-C4~alkyl, preferably substituted linear or branched C _] _-C4-alkyl, with the substituent being selected from O-R13 or S-R]_3 with R]_3 being selected from the group consisting of 5- or β-membered aryl, 5- or β-membered

heteroaryl, linear or branched C^-C^-alkyl and C5-C5- cycloalkyl .

In a preferred embodiment R is independently from each other selected from H, CH3, CF3, OCH3, Cl, CO- CH3, Y is 0, D is S or N or NR4 or CR5, wherein R4 and R5 and the other groups are as defined above.

In another preferred embodiment

wherein

E]_ = NR4 or S or O, in particular NR4 or S, wherein

R4 is linear or branched C]_-C4-alkyl, E2 = CR5 or N, wherein

R5 is C]_-C4-alkylthio, aryl-C _] _-C4-alkylthio, aryloxy-C2_-C4-alkyl,

E3 = E2 with the proviso that if E2 is CR5, E3 is N and if E2 is N, E3 is CR5, and n is as defined above.

In a preferred embodiment

-

I S

wherein R4 = linear or branched C]_-C4-alkyl, in particular CH3 or -CH (0113)2

R5 = C _] _-C4-alkylthio, and n = 1 or 2,

or

wherein n = 0,

R5 = aryloxy-C _] _-C4 alkyl, in particular phenoxy-C _] _-C4 alkyl, especially 1-phenoxy-ethyl, or

wherein n = 0 ,

^R 5 = Ci-C4-alkylthio.

In a preferred embodiment, the compound is a compound of formula (IV)

wherein R5 = H or halogen,

R7 = H, C]_-C4-alkyl, C]_-C4-alkoxy, C1-C4- alkylcarbonyl, halo-Cχ-C4-alkyl or halogen,

Rg = H, C]_-C4~alkyl or halogen,

R9 = H, Cχ-C4-alkyl, C;]_-C4-alkoxy or halo- C]_-C4-alkyl,

R^o = H or halogen, and

Y, U and D are as defined above.

In more preferred compounds of formula (IV)

Rg = H or Br,

R7 = H, Cl, CH3, OCH3, CO-CH ₃ , CF3 _r

Rg = H, CH2CH3, CH ₃ , Cl,

R ₉ = H, CH ₃ , OCH ₃ , CF ₃ , _and

R ₁₀ = H, Br.

In even more preferred compounds of formula

(IV)

R ₆ = H,

R ₇ = H, CH3, OCH3, CO-CH ₃ , CF ₃ ,

R ₈ = H, Cl,

R ₉ = H, CH ₃ , OCH ₃ , CF ₃ , and

R ₁₀ = H.

In preferred embodiments of formula (III) and especially formula (IV) , n is 0 and

xs

I

wherein R4 is CH ₃ , CH (CH ₃ ) 2, and R]_χ is CH ₃ , CH ₂ CH ₃ , CH ₂ -C ₆ H ₅ ,

wherein Rj_ ₂ ^{is CH} 3 ^or CH2CH3, in particular

CH2CH3

or

wherein R5 is - (CH ₂ ) _m (CR]_3) (OAr), wherein R]_3 is H, C]_-C4~alkyl, in particular methyl, Ar is phenyl or heteroaryl, in particular phenyl, and m = 0, 1 or 2.

In another embodiment of the present invention the β-secretase inhibitor is a compound of formula (V)

wherein R is as defined above, U = -CH ₂ -, C=O, - (CH ₂ ) _n S-, -(CH ₂ ) _H O-, - (CH ₂ ) _n NH-, or

-(CH ₂ ) _I T J- (CH ₂ ) _n -, wherein the heterocycle is op-

tionally substituted, in particular by one or two C1-C4- alkyl groups or such that a bicycle is formed, and n independently from each other is 0, 1 or 2, Bl, B3, B5 are independently selected from N or C-R' ,

B2 and B4 are independently selected from C-

R' , and

R' is independently from each other selected from the group comprising hydrogen, halogen, in particular F, a C _] _-Cg-alkyl group, in particular a C]_-C4~ alkyl group, an -NR2R3 group wherein R2 and R3 are independently from each other H, linear or branched Cl- C6-alkyl, in particular linear or branched Cl-C4-alkyl, or R2 and R3 form together with the nitrogen to which they are bound an aliphatic or aromatic 5- or 6-membered one or more heteroatoms comprising heterocycle, e.g. a piperidino, a piperazino or a morpholino group, an amido group, an ester group, a

group

or two adjacent R' form a group

and n independently from each other is 0, 1 or 2. In a preferred embodiment R' is independently from each other selected from the group comprising hydrogen, halogen, in particular F, a C _] _-Cg-alkyl group, in particular a C]_-C4~alkyl group, an amido group, an ester group, a

group

or two adjacent R' form a group

and n independently from each other is 0, 1 or 2

Preferably, a compound of formula (VI]

wherein

R7 is selected from H, CH3, 00.3, COCH3

Bl is selected from

B2 is selected from

B3 is selected from N, CF, C-SO ₂ -N

B4 is selected from CH, C-N(CHs) ₂ , C-NHC (CH ₃ ) 3 B5 is selected from N or CH, or B4 and B5 are both CR ^Λ wherein said two R' form together a group of type

and [U] _n is selected from -(CH2)2NH-

^■ The compounds disclosed above are either commercially available or can be produced according to well known methods (see e.g. US 5,814,646).

It has been found that compounds of the formulas indicated above and their pharmaceutically acceptable salts that are also included in the present invention are efficient in inhibiting β-secretase activity. Thus, such compounds and their pharmaceutically acceptable salts are suitable in the treatment and prophylaxis of neurodegenerative diseases, in particular diseases related to β-secretase activity such as

Alzheimer's disease, Down's syndrome, and advanced aging of brain. β-secretase activity or β-secretase inhibition, respectively, as used in the context of the present invention can be determined by means of Aβl-40 (Sw) bioassay and/or SEAP bioassay and/or FRET assay.

The Aβl-40 (Sw) bioassay measures the amount of the amyoid peptide Aβl-40 in the supernatant of Swedish APP695 transgenic HEK293 cells in the presence (or absence) of the various BACE inhibitors via ELISA (enzyme-linked immunosorbent assay) .

The SEAP bioassay measures the amount of the secreted reporter enzyme SEAP (secreted alkaline phosphatase) in the supernatant of transiently transfected HEK293 cells. In these cells, a SEAP-

APP (Sw) 695 fusion protein is transiently expressed in the presence (or absence) of the various BACE inhibitors.

Secretion of the SEAP moiety upon cleavage at the APP β- site is quantitated via a luminescence readout.

The FRET assay measures the activity of recombinant BACE enzyme in the presence (or absence) of BACE inhibitors via a FRET (fluorescence resonance energy transfer) -based readout.

A β-secretase inhibitor of the present invention in general shows an inhibitory effect in at least one of the above mentioned tests, preferably in at least two of said tests, much preferred in all of said tests .

As already mentioned above, the compounds of the present invention can be administered for prophylactic and/or therapeutic treatment of diseases related to the deposition of amyloid β-protein, such as Alzheimer's disease, Down's syndrome, and advanced aging of the brain. In therapeutic applications, the compounds are administered to a host already suffering from the disease. The compounds will be administered in an amount sufficient to inhibit further deposition of plaques . The specific dose of compound (s) administered according to this invention to obtain therapeutic and/or prophylactic effects will, of course, be determined by the particular circumstances, such as the specific compound administered, the condition being treated, etc. A daily dose will contain a dosage level of from about 0.01 mg/kg to about 50 mg/kg of body weight of an active compound, preferably from about 0.05 mg/kg to about 20 mg/kg, for example from about 0.1 mg/kg to about 120 mg/kg. The compound can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal either as such, but preferable in a formulation comprising carriers adjuvants etc. Suitable pharmaceutically acceptable solid and liquid carriers and/or pharmaceutically acceptable adjuvants, such as

stabilizing agents, emulsifyers, etc. are known in the art.

For example, a typical pharmaceutical composition for intramuscular injection would contain about one μg to one mg of the compound in from one to four milliliters of sterile buffered water. The typical pharmaceutical composition for intravenous infusion would contain about one to one hundred milligrams of the compound in from one hundred to five hundred milliliters of sterile Ringer's solution.

The pharmaceutical formulations are prepared by known procedures using known and readily available ingredients .

It shall be understood that all above mentioned preferred and much preferred characteristics can independently be combined with each other.

Short Description of the Drawings Figure 1 shows the structure formulas of compounds Al to A14 of Tables 1 and 4

Figure 2 shows the structure formulas of compounds A15 to A60 of Table 5.

Figure 3 shows the structure formulas of compounds Bl to B4 of Tables 2 and 6

Figure 4 shows the structure formula of compounds Cl to Cβ of Tables 3 and 7.

Please note that "lacking" valences in the chemical structures signify the presence of a hydrogen.

Modes for Carrying Out the Invention Specific compounds and their β-secretase inhibiting effects are further described below.

Tables 1 to 3 make a relation between compound designation and structure.

Table 1:

Table 2:

5

Table 3

10

These compounds have been tested for their 5 performance as β-secretase inhibitors.

The results of three different tests performed are listed in Tables 4 to 6 below.

The tests performed were a) Aβl-40 (Sw) bioassay, which measures the 0 amount of the amyoid peptide Aβl-40 in the supernatant of Swedish APP695 transgenic HEK293 cells in the presence of the various BACE inhibitors via ELISA (enzyme-linked immunosorbent assay) . In the table, the inhibitory

concentration that reduces Aβl-40 secretion to 50 % is indicated (IC50) , or the % reduction of Aβl-40 secretion at the indicated concentration. b) SEAP bioassay, which measures the amount of the secreted reporter enzyme SEAP (secreted alkaline phosphatase) in the supernatant of transiently transfected HEK293 cells. A SEAP-APP (Sw) 695 fusion protein is transiently expressed in HEK293 cells in the presence of the various BACE inhibitors. Secretion of the SEAP moiety upon cleavage at the APP β-site is quantitated via a luminescence readout. In the table, the inhibitory concentration that reduces secreted SEAP activity to 50 % is indicated (IC50) , or the % reduction of secreted SEAP activity at the indicated concentration. c) FRET assay, which measures the activity of recombinant BACE enzyme in the presence of the various BACE inhibitors via a FRET (fluorescence resonance energy transfer) -based readout. In the table, the inhibitory concentration that reduces the activity of BACE to 50 % is indicated (IC50), or the % reduction of the activity of BACE at the indicated concentration. d) An additional in silico test was performed for the compounds listed in Tables 1 to 3. The compounds were docked with the FFLD approach (Budin et al . , Biol. Chem. 382, 1365-1372, 2001) and their binding energy was evaluated with the LIECE method (Huang and Caflisch, J. Med. Chem. 47, 5791-5797, 2004) . The affinity evaluated with LIECE is in the low micromolar range for most of these compounds .

Table 4

Compo Aβl-40 (Sw) SEAP bioassay FRET assay LIECE und bioassay (cell-based) (in vitro) Ri [μM] (cell-based)

Al IC50 3.0 μM IC50 3.5 μM IC50 58 μM 8.11

A2 IC50 3.2 μM 27 (3 μM) IC50 284 μM 9.35

A3 IC50 2.6 μM 23 (3 μM) IC50 97 μM 9.81

A4 IC50 7.5 μM 21 (6 μM) 33 (100 μM) 10.26

A5 IC50 14. 3 μM 0 (6 μM) 16 (100 μM) 15.99

Aβ IC50 23 μM 19 (12.5 μM) 21 (100 μM) 17.56

A7 IC50 12. 9 μM 0 (12.5 μM) 0 (100 μM) 18.64

A8 IC50 5.6 μM 14 (3 μM) 35 (100 μM) 32.34

A9 IC50 5.9 μM 17 (3 μM) IC50 46 μM 32.56

AlO IC50 3.1 μM 10 (1.6 μM) IC50 64 μM 34.71

All IC50 5.2 μM 0 (6 μM) 20 (200 μM) 38.37

A12 IC50 4.2 μM 14 (1.6 μM) IC50 131 μM 39.41

A13 IC50 3.8 μM 25 (3 μM) 37 (100 μM) 50.09

Al4 IC50 7.8 μM 0 6 μM) 0 (100 μM) 66.41

Table 5:

Aβl-40 FRET

ComLIECE assay

STRUCTURE pound Ki (Sw)

No. [μM] bioassay IC50

[μM]

A49 131

Table 6:

Table 7

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and prac- ticed within the scope of the following claims.