ES2870225T3 - Dipeptide analogs for treating conditions associated with the formation of amyloid fibrils - Google Patents

Abstract

A compound comprising a tryptophan (Trp) moiety coupled to a beta-lamellar breaking moiety, the compound being represented by a chemical structure selected from the group consisting of ** (See formula) ** to be used in the inhibition of the formation of amyloid fibrils or in the treatment of an amyloid-associated disease or disorder.

Description

DESCRIPTION

Dipeptide analogs for treating conditions associated with the formation of amyloid fibrils

Field and background of the invention

The present invention, in some of its embodiments, relates to new therapeutic agents and, more particularly, to dipeptides and their analogues, which prevent the formation of amyloid fibrils and, therefore, can be used in the treatment of diseases associated with amyloid. such as type II diabetes mellitus, dementia or Alzheimer's diseases, systemic and localized amyloidosis, eye diseases or disorders (eg, glaucoma), and prion-related encephalopathies.

The deposition of amyloid material (also called amyloid plaque formation) is a central feature of a variety of unrelated disease states including Alzheimer's disease, prion-related encephalopathies, type II diabetes mellitus, familial amyloidosis, and light chain amyloidosis. .

Amyloid material is composed of a dense network of unbranched proteinaceous fibrils of indefinite length that have a diameter of approximately 80 to 100 A. Amyloid fibrils contain a nuclear structure of polypeptide chains arranged in antiparallel or parallel p-pleated sheets that are with their longitudinal axes perpendicular to the longitudinal axis of the fibril [Both et al. (1997) Nature 385: 787-93; Glenner (1980) N. Eng. J. Med. 302: 1283-92; Balbach et al. (2002) Biophys J. 83: 1205-16].

Approximately twenty amyloid fibril proteins have been identified in vivo and correlated with specific diseases. These proteins share little or no amino acid sequence homology, yet the nuclear structure of the amyloid fibrils is essentially the same. This common nuclear structure of amyloid fibrils, and the presence of common substances in amyloid deposits, suggest that data characterizing a particular form of amyloid material may also be relevant for other forms of amyloid material and can therefore be put in practice in a template design for the development of drugs against diseases associated with amyloid, such as type II diabetes mellitus, dementia or Alzheimer's diseases, diseases and disorders of the eye and encephalopathies related to prions.

Furthermore, amyloid deposits do not appear to be inert in vivo, but instead are in a dynamic state of motion and may even regress if fibril formation is stopped [Gillmore et al. (1997) Br. J. Haematol. 99: 245-56].

Thus, therapies designed to inhibit amyloid polypeptide production or inhibit amyloidosis may be useful for testing amyloid-associated diseases.

One of the therapeutic proposals currently investigated to prevent the formation of amyloid fibrils involves small molecules that can enter the CNS (central nervous system) and interrupt the polymerization of p-amyloid peptides. Examples of these compounds that have been described in the art as effective animal models include cyclohexanehexol [McLaurin et al., Nature Medicine 12 (7), 2006, pp. 801-808], which include AZD-103; curcumin (Yang et al., J. Biol. Chem., 208 (7) 2005, 5892-5901; and hydroxycholesterol derivatives, described and claimed in WO 03/077869.

Some of the present inventors have previously described that the aggregation of peptides in the form of amyloid fibrils is driven by aromatic interactions. Based on these findings, a series of short peptides comprising aromatic amino acids and a lamellar beta-cleavage amino acid were prepared and found to be effective in inhibiting the formation of amyloid fibrils. US Patent No. 7,781,396 describes and claims these dipeptides. One potential peptide described therein is the D-Trp-Aib dipeptide. Derivatives of this dipeptide are also described in said document as derivatives of this dipeptide.

This peptide is also described by Frydman-Marom et al. (Angew. Chem. Int. Ed. 48, 11, pp. 1981-1986).

Bharat K Trivedi et al JOURNAL OF MEDICINAL CHEMISTRY, vol. 41, January 1, 1998 (1998-01-01), pages 38-45, describe similar compounds comprising a - (CO) O-adamantyl group.

Additional prior art includes US Patent No. 7,732,479 and Yescovi et al. [Org. Biomol. Chem., 2003, 1.2983-2997].

Summary of the invention

In a search for new small molecules with improved performance to inhibit the formation of amyloid fibrils, the present inventors have successfully designed and prepared and practiced modified dipeptides based on the previously described dTrp-Aib. These new dipeptides therefore include a tryptophan moiety coupled to a beta-lamellar cleavage moiety, each of which is selected in order to confer steric and / or lipophilic characteristics to the dipeptide compound to achieve the desired performance.

The present invention therefore provides a compound comprising a tryptophan (Trp) moiety coupled to a lamellar beta cleavage moiety, provided that said Trp moiety is not Trp said lamellar beta cleavage moiety is not acidic to - aminoisobutyric acid (Aib) nor an α-aminoisobutyric acid ester (Aib), the compound being represented by a chemical structure selected from the group consisting of:

for use in the inhibition of the formation of amyloid fibrils or in the treatment of an amyloid-associated disease or disorder.

In one embodiment, the compound is represented by the chemical structure:

In one embodiment, the amyloid-associated disease or disorder is one of: type II diabetes mellitus, Alzheimer's disease (AD); early-onset Alzheimer's disease; late-onset Alzheimer's disease, pre-symptomatic Alzheimer's disease; Parkinson's disease; SAA amyloidosis; hereditary Icelandic syndrome; multiple myeloma; medullary carcinoma; Aortic medical amyloid, insulin injection amyloidosis; systemic prion amyloidosis; amyloidosis with chronic inflammation, Huntington's disease; senile systemic amyloidosis; amyloidosis of the pituitary gland; hereditary renal amyloidosis; familial British dementia; Finnish hereditary amyloidosis; Familial non-neuropathic amyloidosis; an eye disease or disorder related to amyloidosis or prion disease.

In one embodiment, the amyloid-related eye disease or disorder is glaucoma or age-related macular degeneration.

In one embodiment, the compound is formulated for drop administration.

In one embodiment, the compound is a formulation for administration by injection, including intraocular injection.

Unless otherwise defined, all technical and / or scientific terms used in the present specification have the same meaning commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods or materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the invention, examples of methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will prevail.

Brief description of the drawings

Some embodiments of the invention are described herein, by way of example only, with reference to the accompanying drawings.

In the drawings;

Figs. 1A-C * show the chemical structures of exemplary beta lamellar cleavage moieties according to some embodiments of the invention, wherein the moiety is coupled to a Trp moiety through the alpha-amine;

Figs. 2A-J show the chemical structures and examples of Trp according to some embodiments of the invention, in which the moiety is coupled to a lamellar beta cleavage moiety through alpha-carboxylate;

Figs. 3A-Y show the chemical structures of exemplary dipeptide analogs according to some embodiments of the invention;

Fig. 4 is a schematic illustration of a synthetic pathway for preparing an exemplary dipeptide analog according to some embodiments of the invention;

Figs. 5A-B present Western blot analysis (Fig. 5A) and data obtained using a staining with 15% tris-tricine and Imperial protein (Fig. 5B) demonstrating the effect of compound 2, H2N-a-Me-Trp- Aib-OMe on globulomer inhibition at various inhibitor mole ratios; Ap peptide, obtained using the Hillen protocol;

Fig. 6 presents data demonstrating the effect of compounds 1 and 7 to inhibit Ap ^1-42 fibril formation at various inhibitor: Ap peptide mole ratios, using the Hillen protocol;

Fig. 7 presents data demonstrating the effect of compounds 3 and 5 to inhibit Ap ^1-42 fibril formation at various inhibitor: Ap peptide mole ratios, using the Hillen protocol; and

Fig. 8 presents comparative data demonstrating the effect of D-Trp-Aib (EG30) to inhibit Ap ^1-42 fibril formation at various inhibitor: Ap peptide mole ratios, using the Hillen protocol.

Description of specific embodiments of the invention

The present description relates to new therapeutic agents and, more particularly, to dipeptides and their analogues, for use in the inhibition of the formation of amyloid fibrils or in the treatment of an associated amyloid disease or disorder. The agents inhibit the formation of amyloid fibrils and, therefore, can be used in the treatment of amyloid associated diseases such as type II diabetes mellitus, dementia or Alzheimer's diseases, systemic and localized amyloidosis, eye diseases, and prion-related disorders and encephalopathies.

The dipeptides described in the present specification are based on a previously described D-Trp-Aib dipeptide (also mentioned in the present specification and in the state of the art as EG030), which incorporates a coupled aromatic amino acid residue (Trp) to a beta-laminar breaking moiety (the unnatural amino acid Aib) and which has been defined as a highly effective inhibitor of amyloid fibril formation. The dipeptides described in the present specification, therefore, are small molecules that are considered as analogs of the D-Trp-Aib dipeptide, in which one or more of the aromatic amino acid residues (Trp) of the beta-lamellar breaker residue they are modified in order to give the analog an improved performance compared to D-Trp-Aib.

In a search for new therapeutic agents to prevent amyloid plaque formation, the present inventors have successfully designed and prepared and practiced with dipeptides with improved performance. More specifically, the new dipeptides were designed to exhibit improved pharmacokinetic characteristics such as, for example, improved BBB permeability and improved inhibitory activity of amyloid plaque formation, and optionally improved biostability (for example, increased half-life), solubility improved and the like.

Although the present invention has been reduced in practice, the present inventors have identified some structural characteristics that confer a desired pharmacokinetic profile to the dipeptide analog. These include incorporation of a beta-lamellar switch moiety with increased steric hindrance and / or increased lipophilicity (compared to Aib); an aromatic residue incorporating a beta-laminar switch residue and an aromatic residue with increased lipophilicity and / or with functionalities that increase the half-life of the molecule.

Therefore, a dipeptide analog comprising a tryptophan (Trp) residue coupled to a beta-lamellar switch residue is described herein.

As used herein, the term "tryptophan residue" describes a chemical residue that is derived from the amino acid tryptophan. The term "tryptophan moiety" encompasses tryptophan itself, as well as derivatives and structural analogs thereof.

The term "derivative", as used herein in the context of any of the described moieties, describes a moiety that has been modified to include a new chemical functionality (namely, a functionality not present in the rest by itself). Derivation of a chemical moiety includes the substitution of one functional group for another, the addition of a functional group, or the omission of a functional group. By "functional group" is meant, for example, a substitution other than hydrogen.

A "tryptophan derivative" therefore includes, according to some aspects of the description, a tryptophan derived to include one or more indole ring substituents, a substituent on the indole nitrogen, a substituent on the alpha carbon atom and one or more substituents on the alpha nitrogen atom. Other derivatives are also contemplated.

The term "analog" as used herein in the context of any of the described moieties, describes chemical moieties that have structural characteristics similar to, but not identical to, the original moiety. Thus, an analog may differ from the original residue by a degree of saturation, a number of atoms in a ring, a type of hetero atom, a length of an alkylene chain, a configuration of a double bond, a configuration of one or more asymmetric carbon atoms and the like.

A "tryptophan analog" can therefore include an amino acid in which an indole residue is attached directly to the alpha carbon atom, through an ethylene chain, or through a double bond, a tryptophan modified to include a heteroaromatic residue other than indole and a tryptophan modified to include a heterocyclic residue, as defined herein, other than indole, as non-limiting examples.

As used herein, the term "beta-lamellar switch moiety" includes residues that are derived from natural and unnatural amino acids, and that are characterized by an angle fi of about -60 to 25 rather than the angle fi beta. -typical laminar about -120 to -140 degrees, thus disrupting the beta-laminar structure of amyloid fibril.

An example of a naturally occurring amino acid known as a beta-lamellar switch is proline. Other beta-laminar switch amino acids include, but are not limited to, aspartic acid, glutamic acid, glycine, lysine, and serine (from Chou and Fasman (1978) Annu. Rev. Biochem. 47, 258).

Compounds in which the beta-lamellar switch moiety is a synthetic amino acid such as a Camethylated amino acid, whose conformational limitations are restricted are described herein [Balaram, (1999) J. Pept. Res. 54, 195-199]. Unlike natural amino acids, which have a hydrogen atom attached to Ca, Ca-methylated amino acids have a methyl group attached to Ca, which greatly affects their steric properties relative to the ^ and ^ angles of the amino acid. Thus, although alanine has a wide range of allowed ^ and ^ conformations, α-aminoisobutyric acid (Aib, see Table 2 above) has limited ^ and ^ conformations.

In some embodiments, the beta-lamellar switch moiety is derived from alpha-aminoisobutyric acid (Aib), such that it is Aib itself or a derivative or analog of Aib, as defined herein.

A derivative of Aib includes, according to some embodiments, Aib derivative to substitute one or more of the methyl substituents in Calpha, to substitute the alpha-carboxylic acid, for example, with an ester, an amide, an acyl chloride, a thiocarboxylate, etc. And to include a substituent on the alpha nitrogen. Other derivatives are also contemplated.

In some embodiments, the beta-lamellar switch moiety is an alpha-methylated amino acid such as, for example, alpha-methyl-lysine, alpha-methyl-valine, and alpha-methylphenylalanine.

It should be understood that when the indicated moieties are cited in the context of the dipeptide analogs described, reference is made to a portion of each moiety formed upon coupling to the other moiety. In this context, the term "residue" is equivalent to the term "residue", as used in the context of amino acids in a peptide, so that it represents the part of an amino acid or an analog or derivative thereof that is present in the dipeptide, after being coupled to another amino acid (or a derivative or analog thereof) through a peptide bond or an analogous bond.

Consequently, a "dipeptide analog", as used herein, describes a peptide composed of residues of a Trp residue and a Beta-lamellar switch residue, as defined herein, and encompasses any of the tryptophan residues described herein, covalently linked to any of the beta-laminar switch moieties as described herein, through the formation of a peptide bond between an amino group of a residue and a group carboxylic from another moiety. The "dipeptide analog" can therefore also be referred to as a chemical conjugate comprising a tryptophan residue covalently linked to an Aib residue. The dipeptide analog is also referred to herein as a compound or a molecule.

Trp-Aib dipeptide and an ester derivative thereof are excluded from the scope of embodiments of the present invention. Embodiments of the invention encompass, however, dipeptide analogs in which one of the Trp or Aib residues (including an ester thereof), provided that the other residue is not Aib (or an ester thereof or Trp, respectively.

Particular dipeptide analogs to be used as defined in the invention are defined in the claims. Other dipeptide analogs are also described herein. The dipeptide analogs described herein can be collectively represented by the general formula I:

Formula I

in which:

dashed line indicates an optional double bond;

* indicates an (R) configuration or an (S) configuration (relevant for the carbon atom substituted with NR6R7 in the case of a single bond, in which the dashed line is absent;

R1 is selected from the group consisting of hydrogen, alkyl, aryl, hydroxy, alkoxy, aryloxy, thiol, thioalkoxy, thioaryloxy, halo, and amino;

R2 and R3 are each independently selected from the group consisting of alkyl, cycloalkyl, aryl, halo, haloalkyl, and benzyl or, alternatively, R2 and R3 together form a 3-8 membered saturated or unsaturated ring;

R4 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, thiocarbonyl, carboxylate, and thiocarboxylate or, alternatively, R4 and R3 together form a 4-8 membered saturated or unsaturated ring;

R5 is selected from the group consisting of hydrogen and alkyl, or is absent in the case where the dashed line indicates a double bond;

R ⁶ and R7 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, thiocarbonyl, carboxylate and thiocarboxylate, or, alternatively, R ⁶ and R7 together form a saturated or unsaturated ring of 4-8 members;

R ⁸ is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, thiocarbonyl, carboxylate, and thiocarboxylate; and

R9-R12 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, alkoxy, thioalkoxy, aryloxy, thioaryloxy, halo, hydroxythiol, carbonyl, carboxylate, and carbamate;

with the proviso that when R2 is alkyl and R3 is methyl, at least of R4, R5 and R8-R12 is not hydrogen.

In general formula I, the fragment - (R ⁴ ) -NC (R ² ) (R ³ ) -C (= O) -R ¹ is considered to be derived from the residue of Aib.

In some embodiments, R4 is hydrogen, R2 and R3 are each methyl, and R1 is hydroxy, such that the dipeptide analog comprises aminoisobutyric acid (Aib) as the beta-lamellar switch moiety.

In some embodiments, the Aib residue is derivatized to include other substituents on the alpha carbon atom.

Thus, in some embodiments, at least of R2 and R3 is higher alkyl than methyl (ie, having more than one carbon atom).

In some embodiments, one or both of R2 and R3 is butylalkyl such as, but not limited to isopropyl, isobutyl, tert-butyl, and the like.

As used herein and in the art, the term "bulky" in reference to a substituent or a certain group describes a chemical moiety that occupies a large volume. The bulkiness of a group is determined by the number and size of the atoms that comprise the group or by their ordering and by the interactions between the atoms (eg, bond lengths or repulsion interactions). In the context of the present embodiments, bulky groups are groups that comprise three or more carbon atoms. Furthermore, in the context of the present embodiments, bulky alkyl groups encompass branched alkyls or substituted alkyls.

In some embodiments, one or both of R2 and R3 is alkyl substituted with an aryl or cycloalkyl, in order to form bulky substituent (s). The alkyl can be substituted with other bulking groups. In exemplary embodiments one or both of R2 and R3 is benzyl in which the phenyl is substituted or unsubstituted.

In some embodiments, one of R2 and R3 is methyl and the other is higher alkyl or substituted alkyl, as described herein. Examples of these beta-laminar switch moieties include, but are not limited to, alpha-methylated amino acids such as a-Me-valine, a-Me-leucine, and a-Me-phenylalanine.

In some embodiments, R2 and R3 form a 3-8 membered saturated or unsaturated ring.

The ring can be alicyclic (cycloalkyl), heterocyclic, aromatic, or heteroaromatic.

In some embodiments, R2 and R3 together form a saturated alicyclic three-membered ring (cyclopropane), a 4-membered ring (cyclobutane), a 5-membered ring (cyclopentane), or a 6-membered ring (cyclohexane).

Alternatively, R2 and R3 together form an unsaturated non-aromatic ring (eg, cyclopentene or cyclohexene).

Additionally alternatively, R2 and R3 together form a heterocyclic ring, as defined herein.

In some embodiments, R2 and R3 together form an oxetane, a tetrahydrofuran, a tetrahydropyran, a dihydrofuran, or a dihydropyran.

In some embodiments, R2 and R3 together form an oxetane.

The ring formed by R2 and R3 can be substituted or unsubstituted, as described herein.

As demonstrated in the examples section that follows, it has been shown that dipeptide analogs in which R2 and R3 represent groups with greater bulk compared to the two corresponding methyl groups in Aib (i.e., dipeptide analogs in the that R2 and R3 together form a ring), exhibit superior inhibitory activity compared to D-Trp_Aib dipeptide. In some embodiments, R4 is hydrogen, as in Aib.

In some embodiments, R4 is other than hydrogen.

Thus, in some embodiments, R4 is alkyl (eg, methyl).

In some embodiments, R4 and R3 together form a ring, as described herein for R2 and R3. This ring can be a substituted or unsubstituted heteroalicyclic or heteroaromatic ring, as described herein.

In exemplary embodiments, when R4 and R3 together form said ring, R2 is an alkyl.

In some embodiments, R4 and R3 together form a saturated five-membered ring, such that the beta-laminar switch moiety is a Me-proline.

In some embodiments, the beta-lamellar switch moiety comprises one or more halo groups (eg, chlorine, fluorine, or bromine).

Although no particular theoretical linkages are desired, it is suggested that the presence of a halo group increases the lipophilicity and / or increases the half-life of the dipeptide analog and thus confers improved pharmacokinetic characteristics to the dipeptide analog.

A halo group can be introduced into the dipeptide analog as one of R2 and R3, as a substituent on an alkyl, cycloalkyl, or an aryl that forms one or more R2, R3, or R4, or as a substituent on a ring formed by R2. and R3 and / or R3 and R4.

In some embodiments, one or more of R2 and R3 is haloalkyl such as trihaloalkyl (eg, trifluoromethyl). In an exemplary embodiment, the beta-lamellar switch moiety is an amino-trifluoroisobutyric acid.

In some embodiments, one or more of R2 and R3 is halogenated benzyl. In an exemplary embodiment, the beta-lamellar switch moiety is a halogenated α-Me-phenylalanine.

Each of the beta-lamellar switch moieties described herein may end with a carboxylic acid group, such that R1 in formula I is hydroxy.

However, the present inventors have shown that derivatization of the carboxylic group to include an ester or an amide results in improved inhibitory activity and can also confer on the dipeptide analog improved pharmacokinetic characteristics such as improved lipophilicity and BBB permeability. Thus, in some embodiments, for any of the beta-lamellar switch moieties described herein, R1 is an alkoxy or amino.

In some embodiments, the alkoxy is a bulky group, as defined herein, such that the alkoxy is an O-alkyl group where the alkyl is a bulky alkyl, as defined herein. Representative examples include, but are not limited to, t-butoxy, isopropoxy, and the like.

In some embodiments when R1 in formula I is amino, the amine can be a primary amine (-NH2), a secondary amine, (-NR ') a tertiary amine (NR'R "), where R' and R" can each independently being alkyl, cycloalkyl or aryl, preferably each independently being an alkyl.

In some embodiments, R1 is a secondary amine.

Referring now to the Trp residue, presented in formula I by the fragment not included in the fragment - (R4) -N-C (R2) (R3) -C (= O) -Ri

In some embodiments, the Trp residue in the disclosed dipeptide analog includes a beta-laminar switch moiety, for example, in the form of a methyl substituent on the alpha carbon atom, such that the Trp residue is Me-tryptophan.

Consequently, in some embodiments R5 is alkyl.

In some embodiments, R5 is methyl.

In some embodiments, the α-amine of the Trp residue is derived such that at least one of R ⁶ and R7 is other than hydrogen and the α-amine is a secondary amine or a tertiary amine.

A secondary or tertiary amine imparts to the dipeptide analog improved lipophilicity and permeability. In some embodiments, the Trp residue alpha-amine is substituted with a carboxylate, carbonyl (including aldehyde) thiocarbonyl, and / or thiocarboxylate, in order to form an amide or a carbamate.

In some embodiments, the alpha-amine of the Trp residue is substituted with t-Boc.

In some embodiments R ⁶ and R7 together form a heterocyclic or heteroaromatic ring, as described earlier herein for R4 and R3.

In some embodiments, the Trp residue is such that the indole residue is derivatized to include one or more substituents. In some embodiments, the N-indole is substituted such that R ⁸ is other than hydrogen. In some embodiments R ⁸ is an alkyl (eg, methyl).

In some embodiments, R ⁸ is bulky alkyl (eg, isopropyl, isobutyl, or t-butyl).

In some embodiments, R ⁸ is t-butyl.

In some embodiments, the indole group is substituted with one or more substituents, denoted R9-R12, as detailed above.

In some embodiments, one or more of R9-R12 is halo, as defined herein. Although no particular theoretical linkages are desired, it is suggested that the introduction of one or more halo groups into the indole moiety provides improved pharmacokinetic characteristics as a consequence of improved lipolyphilicity and / or prolonged half-life.

As one of the main obstacles to using short peptide fragments in therapy is their proteolytic degradation by stereospecific cellular proteases, in some embodiments one or both of the optional asymmetric carbon atoms (marked by * in formula I) are derivatives of D-isomers. of the indicated amino acid residues and consequently have an (R) configuration.

In some embodiments, the alpha carbon of the Trp moiety is an asymmetric carbon atom having an (R) configuration.

In some embodiments, an indole group, as described herein, is attached to the alpha carbon atom through a double bond.

It should be understood that the number and nature of the modifications made to the dipeptide analog are governed by steric and electronic considerations that can affect the stability of the product obtained and the feasibility of its synthesis.

Consequently, in some embodiments, the dipeptide analogs are such that they do not contain two or more bulky groups in close proximity to each other or two or more electronegative groups in close proximity to each other.

As used herein, the term "amine" describes a group -R'R "and a group -NR'- in which R 'and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, as shown define these terms in the present specification.

The term "amine" is used to describe a group -NR'R "in cases where the amine is a terminal group, as defined below and is used to describe a group -NR'-in cases where the amine is a linking group.

Throughout this specification, the term "end group" describes a group (a substituent) that is attached to another moiety in the compound through an atom thereof.

The term "connecting group" describes a group (a substituent) that is attached to another moiety in the compound through two or more atoms thereof.

The amino group, therefore, can be a primary amine, where both R 'and R "are hydrogen, a secondary amine, where R' is hydrogen and R" is alkyl, cycloalkyl, or aryl, or a tertiary amine. , wherein R 'and R "are independently alkyl, cycloalkyl, or aryl.

Alternatively R 'and R "may each independently be hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, cyanoaryloxy , nitro, azo, sulfonamido, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amido, N-amido, guanyl, guanidino and hydrazino .

The term "alkyl" describes a saturated aliphatic hydrocarbon that includes straight chain and branched chain groups. Preferably, the alkyl group has one to 20 carbon atoms. Whenever a numerical range is stated in the present specification, for example "1-20", it implies that the group, in this case the alkyl group, can contain one carbon atom, 2 carbon atoms, 3 carbon atoms, etc. And that includes up to 20 carbon atoms. In some embodiments, the alkyl is a medium-sized alkyl having 1 to 10 carbon atoms. In some embodiments, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 6 or 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. The substituted alkyl can have one or more substituents, where each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, trihydroxy, trialkoxy, triaryloxy, cyano, nitro, azo, sulfonamido, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amido, N-amido, guanyl, gunidino and hydrazino.

The alkyl group can be a terminal group, as this term is defined above, which is attached to a single contiguous atom, or a connecting group, as this term is defined above, that connects two or more moieties through al minus two carbon atoms in its chain.

The term "cycloalkyl" describes a monocyclic or fused ring with all carbon atoms (that is, rings that share a pair of contiguous carbon atoms) group in which one or more of the rings does not have a completely pi electron system conjugate. The cycloalkyl group can be substituted or unsubstituted. The substituted cycloalkyl may have one or more substituents, where each substituent group may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamido, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amido, N-amido, guanyl, guanidino and hydrazino. The cycloalkyl group can be a terminal group, as this term is defined above, in which it is attached to a single contiguous atom, or a connecting group as this term is defined above, that connects two or more moieties at two or more positions of the same.

The term "aryl" describes monocyclic or polycyclic groups of fused rings with all carbon atoms (ie, rings that share pairs of contiguous carbon atoms) that have a fully conjugated pi electron system. The aryl group can be substituted or unsubstituted. The substituted aryl may have one or more substituents, where each substituent group may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamido, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amido, N-amido, guanyl, guanidino and hydrazino. The aryl group can be a terminal group, as this term is defined above, which is attached to a single contiguous atom, or a connecting group, as this term is defined above, which connects two or more moieties at two or more positions. of the same.

The term "heteroaryl" describes a group of monocyclic or fused rings (that is, rings that share a pair of contiguous atoms) that has one or more atoms in the ring (s), such as nitrogen, oxygen and sulfur and also has a fully conjugated pi electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. The heteroaryl group can be substituted or unsubstituted. The substituted heteroaryl may have one or more substituents, where each substituent group may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, trioaryloxy, cyano, nitro, azo, sulfonamido, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-cabamate, N-carbamate, C-amido, N-amido, guanyl, guanidino and hydrazino. The heteroaryl group can be a terminal group, as this term is defined above, in which it is attached to a single contiguous atom or a connecting group, as this term is defined above, that connects two or more moieties to two or more positions of the same. Representative examples are pyridine, pyrrole, oxazole, indole, purine, and the like.

The term "heterocyclic" describes a group of monocyclic or fused rings having in the ring (s) one or more atoms such as nitrogen, oxygen and sulfur. The rings can have one or more double bonds. However, the rings do not have a fully conjugated pi electron system. The heteroalicyclic can be substituted or unsubstituted. The substituted heteroalicyclic may have one or more substituents, where each substituent group may independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amino, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, trihydroxy, trialkoxy, triaryloxy, cyano, nitro, azo, sulfonamido, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-cabamate, N-carbamate, C-amido, N-amido, guanyl, guanidino and hydrazino. The heteroalicyclic group can be a terminal group, as this term is defined above in which it is attached to a single contiguous atom, or a connecting group, as this expression is defined above that connects two or more residues to two or more positions of the same. Representative examples are piperidino, piperazine, oxetane, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

The term "halo" is also referred to herein as "halide" and describes fluorine, chlorine, bromine or iodine.

The term "haloalkyl" describes an alkyl group as defined above, further substituted with one or more halides.

A "trihaloalkyl" describes, for example, a trihaloalkyl (eg, -CX3, where X is halide).

The term "carbonyl" as used herein describes a terminal group -C (= O) -R 'or a connecting group -C (= O) -, as these terms are defined above, where R' it is as defined herein.

The term "thiocarbonyl" as used herein, describes a terminal group -C (= S) R 'or a linking group -C (= S) -, as these expressions are defined above where R' is as defined herein.

The term "hydroxyl" or "hydroxy" describes an -OH group.

The term "alkoxy" describes an -O-alkyl and -O-cycloalkyl group, as defined herein.

The term "aryloxy" describes an -O-aryl group and an -O-heteroaryl group, as defined herein.

The term "thiohydroxy" or "thiol" describes a -SH group.

The term "thioalkoxy" describes an -S-alkyl group and an S-cycloalkyl group, as defined herein.

The term "thioaryloxy" describes a group -S-aryl and -S-heteroaryl, as defined herein.

The term "cyano" describes a group -C = N.

The term "isocyanate" describes a group -N = C = O.

The term "nitro" describes a -NO2 group.

The term "acyl halide" describes a group -C (= O) R "" where R "" is halide, as defined above.

The term "carboxylate" encompasses C-carboxylate, O-carboxylate, C-thiocarboxylate, and O-thiocarboxylate.

The term "C-Carboxylate describes a terminal group -C (= O) -R 'or a connecting group -C (= O) -O-, as these expressions are defined above, where R' is as defined in the present specification.

The term "O-carboxylate, describes a terminal group -OC (= O) R 'or a linking group -OC (= O) -, as these expressions are defined above, where R' is as defined herein. descriptive memory. The term "C-thiocarboxylate" describes a terminal group -C (S) -OR 'or a connecting group -C (= S) -O-, as these expressions are defined above, where R' is as defined in the present specification.

The term "O-thiocarboxylate" describes a terminal group, -OC (= S) R 'or a linking group -OC (= S) - as these expressions are defined above, where R' is as defined herein descriptive memory. The term "amido" encompasses C-amido and N-amido.

The term "C-amido" describes a terminal group -C (= O) -NR'R "or a connecting group -C (= O) -NR ', as these expressions are defined above, where R' and R "Are as defined herein.

The term "N-amido" describes a terminal group R'C (= O) -NR "- or a connecting group R'C (= O) -N-, as these expressions are defined above, where R 'and R "are as defined herein. The term "N-carbamate" describes a terminal group R "OC (= O) -NR '" or a connecting group -OC (= O) -NR' ", as these expressions are defined above where R 'and R "Are as defined herein.

The term "O-carbamate" describes a terminal group -OC (= O) -NR'R "or a connecting group -OC (= O) -NR'- as these expressions are defined above, where R 'and R "Are as defined herein.

The term "O-thiocarbamate" describes a terminating group -OC (= S) -NR'R "or a connecting group -OC (= S) -NR'-, as these expressions are defined above, where R 'and R "are as defined herein.

The term "N-thiocarbamate" describes a terminal group R "OC (= S) NR" or a connecting group -OC (S) NR'-, as these expressions are defined above, where R 'and R "are as are defined herein.

The term "S-dithiocarbamate" describes a terminal group -SC (= S) -NR'R "or a connecting group -SC (= S) NR'-, as these expressions are defined above, where R 'and R "Are as defined herein.

The term "N-dithiocarbamate" describes a terminal group R "SC (= S) NR'- or a connecting group -SC (= S) NR'-, as these expressions are defined above, where R 'and R" they are as defined herein.

The term "azo" or "diazo" describes a terminal group -N = NR 'or a connecting group -N = N-, as these expressions are defined above, where R' is as defined herein.

The term "sulfate" describes a terminal group -OS (= O) 2-OR ', as this term is defined above, or a connecting group -OS (= O) 2-O-, as these expressions are defined above. , where R 'is as defined above.

The term "thiosulfate" describes a terminal group -OS (= S) (= O) -OR 'or a connecting group -OS (= S) (= O) -O-, as these expressions are defined above, in which R 'is as defined above.

The term "sulfite" describes a terminal group -O-S (= O) -O-R 'or a connecting group -O-S (= O) -O- as these expressions are defined above where R' is as defined above.

The term "thiosulfite describes a terminal group -O-S (= S) -O-R 'or a connecting group -O-S (= S) -O-, as these expressions are defined above, where R' is as defined above.

The term "sulphinate" describes a terminal group -S (= O) -OR 'or a linking group -S (= O) -O-, as these expressions are defined above, where R' is as defined above .

The term "sulfoxide" or "sulfinyl" describes a terminal group -S (= O) R 'or a linking group -S (= O) -, as define these expressions above, where R 'is as defined above.

The term "sulfonate describes a terminal group -S (= O) 2-R 'or a connecting group -S (= O) 2-, as these expressions are defined above, where R' is as defined herein. descriptive memory.

The term "S-sulfonamido" describes a terminal group -S (= O) 2-NR'R "or a connecting group -S (= O) 2-NR'- as these expressions are defined above, where R ' and R "are as defined herein.

The term "N-sulfonamido" describes a terminal group R'S (= O) 2-NR "- or a connecting group -S (= O) 2-NR'-, as these expressions are defined above, where R 'and R "are as defined herein.

The term "disulfide" refers to an end group -S-SR 'or a linking group -S-S-, as these terms are defined above, where R' is as defined herein.

The term "phosphonate" describes a terminal group -P (= O) (OR ') (OR ") or a connecting group -P (O) (OR') (O) -, as these expressions are defined above, in that R 'and R "are as defined herein.

The term "thiophosphonate" describes a terminal group -P (= S) (OR ') (OR ") or a connecting group -P (= S) (OR') (O) -, as these expressions are defined above, where R 'and R "are as defined herein.

The term "phosphinyl" describes a terminal group -PR'R "or a linking group -PR'-, as these terms are defined above, where R 'and R" are as defined herein.

The term "phosphine oxide" describes a terminal group -P (= O) (R ') (R ") or a connecting group -P (= O) (R') - as defined in these expressions above, being R 'and R "as defined herein.

The expression "phosphine sulfide" describes a terminal group -P (= S) (R ') (R ") or a connecting group -P (= S) (R') -, as these expressions are defined above, being R 'and R "as defined herein.

The term "phosphite" describes a terminal group -O-PR '(= O) (OR ") or a connecting group -O-PH (= O) (O) -, as these expressions are defined above, where R' and R "as defined herein. The term "urea" which is also referred to herein as "ureido" describes a terminal group -NR'C (= O) -NR'R "'or a connecting group -NR'C (= O) -NR "- as these expressions are defined above, where R 'and R" are as defined herein and R' "is as defined herein for R 'and R".

The term "thiourea", which is also referred to herein as "thioureido" describes a terminal group -NR'-C (= S) -NR "R" 'or a connecting group -NR'-C (= S ) -NR "-, where R ', R" and R' "are as defined herein.

The term "guanyl" describes a terminal group R'R "NC (= N) - or a connecting group -R'NC (= N) -, as these expressions are defined above, where R 'and R" are as are defined herein. The term "guanidino" describes a terminal group -R'NC (= N) -NR "R" 'or a connecting group -R'NC (= N) -NR "- as these expressions are defined above, where R ', R "and R'" are as defined herein.

The term "hydrazino" describes a terminal group -NR'-NR "R '" or a connecting group -NR'-NR "-, as these expressions are defined above, where R', R" and R '"are as defined herein.

As used herein, the term "hydrazido" describes a terminal group -C (= O) NR'-NR "R '" or a connecting group -C (= O) -NR'-NR "-, as these terms are defined above, where R ', R "and R'" are as defined herein.

As used herein, the term "thiohydrazido" describes a terminal group -C (= S) -NR'-NR "R" 'or a connecting group -C (= S) -NR'-NR "- , as these expressions are defined above, where R ', R "and R" are as defined herein.

A list of beta-laminar switch moieties that are suitable for use in the context of the description is presented in Table 3 (see Example 1 below) and in Figure 1.

A list of examples of Trp moieties that are suitable for use in the context of the description is presented in Table 2 (see Example 1 below) and in Figure 2.

It should be appreciated that embodiments of the present disclosure encompass any combination of one of the Trp moieties described herein and one of the beta-laminar switch moieties disclosed herein.

An example list of dipeptide analogs according to some embodiments of the disclosure is presented in Table 1 (see Example 1 below) and in Figure 3.

As indicated above and as further demonstrated in the examples section that follows, the present inventors have disclosed that modifications of some structural and functional characteristics of a Trp residue and / or an Aib residue in a dipeptide composed of these Two residues give rise to dipeptide analogs of the previously described D-Trp-Aibs that are characterized by improved performance and, therefore, can be effectively used as therapeutic agents to inhibit the formation of amyloid fibrils.

Consequently, in some embodiments, a dipeptide analog as described herein is characterized by improved performance compared to D-Trp-Aib and previously described derivatives thereof.

In some embodiments, a dipeptide analog as described herein is characterized by an inhibitory activity of amyloid fibril formation that is greater than an inhibitory activity of D-Trp-Aib. In some embodiments, an increased inhibitory activity of the dipeptide analogs described herein is measured by the minimum amyloid-beta protein: inhibitor ratio required to inhibit the formation of amyloid-beta protein globulomers. In some embodiments, the dipeptide analogs described herein exhibit substantially complete inhibition of amyloid-beta protein globulomer formation at a 1:20 amyloid-beta protein: inhibitor mole ratio. This ratio twice less than the corresponding minimum amyloid-beta protein: inhibitor ratio required to inhibit the formation of D-Trp-Aib amyloid-beta protein globulomers and indicates substantially improved inhibitory activity of the dipeptide analogs described in the present specification.

In some embodiments, the dipeptide analogs described herein exhibit substantially complete inhibition of amyloid-beta protein globulomer formation at a 1:10 amyloid-beta protein: inhibitor mole ratio. In some embodiments, the dipeptide analogs described herein exhibit substantially complete inhibition of amyloid-beta protein globulomer formation at a 1: 1 amyloid-beta protein: inhibited mole ratio and even lower ratios. .

In some embodiments, a dipeptide analog as described herein is characterized by a BBB permeability that is greater than the BBB permeability of D-Trp-Aib. In some embodiments, a dipeptide analog as described herein is characterized by a lipophilicity that is greater than the lipophilicity of D-Trp-Aib.

In some embodiments, a dipeptide analog as described herein is characterized by a half-life that is greater than the half-life of D-Trp-Aib.

Collectively, a dipeptide analog as described herein is characterized by an improved therapeutic effect compared to previously described D-Trp-Aib and its ester derivatives. This enhanced effect can be evidenced by a reduced amount of the dipeptide analog required to treat an indicated condition, as detailed below, by a reduced number of administrations of the dipeptide analog, compared to D-Trp-Aib and / or with reduced side effects.

Consequently, each of the dipeptide analogs described herein can be independently characterized as an inhibitor of amyloid fibril formation.

According to some embodiments, each of the dipeptide analogs described herein can be independently identified for use in treating an amyloid-associated disease or disorder.

The present invention therefore provides a compound comprising a tryptophan (Trp) residue coupled to a beta-lamellar switch residue, provided that said Trp is not Trp or said beta-lamellar switch residue is not α-aminoisobutyric acid. (Aib) nor an α-aminoisobutyric acid ester (Aib), the compound being represented by a chemical structure selected from the group consisting of

for use in the inhibition of the formation of amyloid fibrils or in the treatment of an amyloid-associated disease or disorder in a subject.

In one embodiment, the compound is represented by the chemical structure:

Preferred individual subjects are mammals such as canines, felines, sheep, pigs, horses, bovines, humans, and the like.

As used throughout this specification, the terms "inhibition (or prevention) of amyloid fibril formation", "inhibition (or prevention) of amyloid plaque formation", "disintegration of amyloid fibrils", "inhibition ( or prevention) of amyloid-beta globulomerization ”, as well as grammatical variants and combinations thereof, are used interchangeably and generally refer to an interference with biological procedures that results in an aggregation of amyloid beta peptides in the form of oligomers and polymers, thus forming an amyloid plaque. Thus, these expressions describe an activity of the described peptide analogs that results in a reduction or prevention of amyloid plaque formation, or a substantial decrease in the appearance of plaque in the affected tissue.

The term "amyloid plaque" refers to a fibrillar amyloid as well as aggregated but non-fibrillar amyloid, hereinafter "protofibrillar amyloid", which can also be pathogenic.

It will be appreciated that when used for the treatment of amyloid diseases, the dipeptide analogs described herein are capable of preventing fibril formation, reducing fibril formation, or disintegrating the aggregates formed by competitive destabilization of the previously formed aggregate. . Alternatively, the dipeptide analogs described may act through self-aggregation and formation of heteromolecular complexes that are not arranged as homomolecular structures formed by amyloid fragments.

The term "amyloid-associated disease or disorder" as used herein describes a medical condition with a pathology involving the formation of amyloid plaque. This medical condition may involve other pathologies, however, it is treatable, at least to some extent, by reducing, preventing or inhibiting the formation of amyloid plaque.

Examples of amyloid-associated diseases treatable according to embodiments of the present invention include, but are not limited to, type II diabetes mellitus, Alzheimer's disease (AD), early-onset Alzheimer's disease, late-onset Alzheimer's disease, Alzheimer's disease Presymptomatic, Parkinson's disease, SAA amyloidosis, Hereditary Icelandic syndrome, Multiple myeloma, Medullary carcinoma, Medical aortic amyloid, Insulin injection amyloidosis, Prion systemic amyloidosis, Chronically inflamed amyloidosis, Huntington's disease, Senile systemic amyloidosis, Pituitary gland amyloidosis , hereditary renal amyloidosis, British familial dementia, Finnish hereditary amyloidosis, familial non-neuropathic amyloidosis [Gazit (2002) Curr. Med. Chem. 9: 1667-1675], amyloid-related eye diseases and disorders such as glaucoma [Guo et al., PNAS (2007), 104 (33), pp. 13444-13449] and age-related macular degeneration (AMD) and prion diseases including scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172: 21-38] and human prion diseases including (i) kuro, (ii) Creutzfeldt-Jakob disease (CJD), (iii) Gerstmann-Streussler-Sheinker disease (GSS), and (iv) fatal familial insomnia (FFI) [Gajdusek ( 1977) Science 197: 943-960; Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449].

In any of the methods and uses described herein, a therapeutically effective amount, as defined herein, of the dipeptide analog is provided to the subject. The dipeptide analog can be provided using one of a variety of delivery methods. Methods of delivery and suitable formulations are described below with respect to pharmaceutical compositions.

Suitable routes of administration may include, for example, oral, sublingual, inhalation, rectal, transmucosal, transdermal, intracavesmosal, topical, intestinal, or parenteral, including intramuscular, subcutaneous, and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal injections. , intranasal or intraocular.

The dipeptide analogs described herein can be provided to an individual subject on their own, or as part of a pharmaceutical composition in which they are mixed with a pharmaceutically acceptable carrier.

As used herein, a "pharmaceutical composition" refers to a preparation of one or more active ingredients described herein with other chemical components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

In the present specification, "active ingredient" refers to the dipeptide analog, which can be taken into account for the biological effect.

In the following, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" that can be used interchangeably refer to a carrier or diluent that does not cause significant irritation to an organism and does not nullify biological activity and properties. of the administered compound. An adjuvant is included in these expressions. One of the ingredients included in the pharmaceutically acceptable carrier can be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).

In the present specification, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Alternatively, a preparation can be administered in a local rather than systemic manner, for example, through an injection of the preparation directly to a specific area of a patient's body.

The pharmaceutical compositions of the present invention can be manufactured by procedures well known in the art, for example, by conventional mixing, dissolution, granulation, preparation of dragees, preparation of fine powders, emulsification, encapsulation, inclusion or lyophilization.

The pharmaceutical compositions to be used according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliary materials that facilitate the treatment of the active ingredients in the form of preparations that can be used for pharmaceutically . The appropriate formulation will depend on the chosen route of administration.

Formulations for topical administration include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays, and powders. Conventional, aqueous, powdered or oily carriers, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavors, dispersing aids, emulsifiers, or binders may be desirable.

Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents, or other suitable additives. Slow release compositions are intended for the treatment.

Compositions suitable for use in the context of the present embodiments include compositions in which the active ingredients are contained in an effective amount to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredients effective to prevent, alleviate or ameliorate the symptoms of a disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is within the abilities of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro tests. For example, a dose can be formulated in animal models and this information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture analyzes and animal studies can be used to formulate a range of dosages for use in humans. The dosage may vary depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage can be chosen by the individual practitioner considering the condition of the patient. [See, for example, the publication of Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 page 1].

Depending on the severity and the response of the condition to be treated, the dosage may be one or a plurality of administrations, with a course of treatment lasting from several days to several weeks or until healing is achieved or is effected. a decrease in the disease state.

The amount of a composition to be administered will naturally depend on the subject being treated, the severity of the condition, the manner of administration, the judgment of the attending physician, etc.

Compositions including the dipeptide analog as described herein, formulated in a compatible pharmaceutical carrier, can be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as described herein. descriptive.

If desired, the compositions can be presented in a container or delivery device, such as an FDA-approved test kit, which can be contain the active ingredient. The container may comprise, for example, a metal or plastic foil such as, but not limited to, a blister case or a pressurized (for inhalation) container. The container or delivery device may be accompanied by instructions for administration. The container or delivery device may also be accompanied by an indication associated with the container in a form prescribed for a state agency that regulates the manufacture, use, or distribution of pharmaceutical products, where the indication reflects the agency's approval of the form of delivery. compositions for human or veterinary administration. This indication, for example, may be a label approved by the US Food and Drug Administration for drug prescriptions or an insert of approved products.

In some embodiments, the pharmaceutical composition is packaged in a packaging material and is identified in print, in or on the packaging material, for use in the treatment of an amyloid-associated disease or disorder, as described herein. descriptive memory.

It will be appreciated that the treatment of amyloid-associated diseases according to the present invention can be combined with other methods of treatment known in the art (ie, combination therapy). Thus, the compounds according to embodiments of the present invention can be co-administered (simultaneously or separately) with additional anti-amyloid drugs. Examples of these anti-amyloid drugs include, but are not limited to, amyloid destabilizing antibodies, amyloid destabilizing peptides, and anti-amyloid small molecules (additional details of these drugs are provided in the background section above). Compounds according to embodiments of the present invention can be co-administered (simultaneously or separately) with additional drugs to treat an indicated disease or disorder.

Also described is a process for preparing dipeptide analogs described herein, which is performed by coupling a tryptophan moiety and a beta-lamellar breaker moiety.

In some embodiments, the procedure is carried out in the presence of a peptide coupling agent. Any suitable coupling agents to be used to couple amino acids are contemplated.

In some embodiments, the method further comprises, prior to coupling, protecting one or more functional groups on the tryptophan moiety and the beta-lamellar breaker moiety. Any suitable protecting group is contemplated. Suitable protecting groups are normally selected to be suitable for the functional group to be protected and to be easily removed under conditions that do not affect other functionalities of the dipeptide product or any of its intermediates.

In the case that the Trp moiety and / or the protected beta-lamellar breaking group are used, the process further comprises, subsequent to the coupling, removing the protecting group (s). In the event that more than one protecting group is present on the coupled dipeptide, the removal of the protecting groups can be performed simultaneously or sequentially, depending on the protecting groups used.

Selection and practice of the described procedure, with suitable protecting groups, may be well recognized by those skilled in the art.

In some embodiments, the process further comprises either before or after coupling, preparing the tryptophan moiety and / or the beta-lamellar breaker moiety of the dipeptide analog.

Consequently, in some embodiments, Trp is coupled to a beta-lamellar breaking moiety as described herein, to thereby provide a dipeptide analog that comprises Trp as the Trp moiety and subsequently Trp in this analog. The dipeptide is derivatized in order to produce a dipeptide analog with a Trp residue, as described herein for dipeptide analogs comprising a Trp residue other than Trp.

In some embodiments, a Trp moiety, as described herein, already modified as described herein is first prepared and then coupled to the beta-laminar breaker.

Similarly, in some embodiments, the Aib is coupled to a Trp moiety, to thereby provide a dipeptide analog with Aib as the beta-lamellar breaker moiety, and subsequently the Aib in this dipeptide analog is derivatized in order to produce a dipeptide analog with a beta-lamellar breaking moiety, as described herein for dipeptide analogs comprising a beta-lamellar breaking moiety other than Aib or an ester thereof.

An example of a general procedure for preparing the described dipeptide analogs is presented in the examples section that follows.

As used herein, the term "about" refers to ± 10%.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of" means "including and limited to".

The expression "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the composition. , method or structure claimed.

The term "example of" is used herein, to indicate "serving as an example, case, or illustration." Any embodiment described as an "example of" is not necessarily intended to be preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

The term "optionally" is used herein to indicate "is provided in some embodiments and not provided in other embodiments." A particular embodiment of the invention may include a plurality of "optional" features unless these features are in conflict.

As used herein, the singular form of the articles "an", "an", "the", "the" includes plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

Throughout this specification, various embodiments of the invention may be presented in a slot format. It should be understood that the description in the interval format is merely for the sake of convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Consequently, the description of an interval should be considered to have specifically described all possible subintervals, as well as the individual numerical values in that interval. For example, when describing an interval as 1 to 6, it should be considered that it has specifically described subintervals such as 1 to 3, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. , as well as individual numbers in that interval, for example 1,2, 3, 4, 5, and 6. This applies regardless of the width of the interval.

Whenever a numerical range is indicated in the present specification, it is intended to include any quoted numerical value (fractional or integer) in the indicated range. The terms "varying / varying between" a first indicated number and a second indicated number and "varying / varying from" a first indicated number "to" a second indicated number are used herein interchangeably and are intended that include the first and second numbers listed and all fractional and whole numbers in between.

As used herein, the term "method" refers to ways, means, techniques, and procedures for carrying out a given task including, but not limited to, known or already developed ways, means, techniques, and procedures. from manners, means, techniques and procedures known to users of chemical, pharmacological, biological, biochemical and medical techniques.

As used herein, the term "treating" includes suppressing, substantially inhibiting, slowing down or reversing the progress of a condition, substantially improving the clinical or aesthetic symptoms of a condition, or substantially preventing the appearance of clinical or aesthetic symptoms of a condition.

Various embodiments and aspects of the present invention, as outlined above and claimed and in the claims section below, find experimental support in the following examples.

Examples

Reference is now made to the following examples, which together with the descriptions above illustrate some embodiments of the invention.

Example 1

Chemical synthesis

Materials and methods:

Chemical reagents were purchased from Sigma-Aldrich, unless otherwise noted.

Solvents were purchased from Bio-Lab, unless otherwise indicated.

NMR measurements were performed on a Bruker Avance-200 MHz NMR device, using SiMe4 as a standard.

Electrospray-type mass spectroscopy (ESI-MS) measurements were performed on a Waters Micromass SYNAPT HDMS Mass spectrometer.

General procedure:

The dipeptide compounds described herein were prepared using a standard procedure to couple two amino acids as follows:

D-Trp or an analog thereof (eg, D / L-a-methyl-Trp) is coupled to a beta-lamellar breaking moiety derived from the unnatural amino acid 2-amino-isobutyric acid (Aib).

Protection of carboxylic function: amino acids are protected in their carboxylic function through a methyl or tert-butyl ester using thionyl chloride as reagent and methanol, ethanol or tert-butanol as solvent, based on the procedure described in the Bioorganic publication & Medicinal Chemistry 15 (14): 4903-9, 2007, with the following changes: thionyl chloride was not distilled before the reaction and the intermediate product was not purified via recrystallization but instead a column was used silica gel 60, with ethyl acetate in hexane as eluent. Intermediates are verified by 1H-200 MHz NMR using CDCb as solvent.

N-Boc protection: N-Boc protection of the tryptophan residue (for example, D-Trp-methyl ester or D / L-alpha-methyl-Trp-methyl ester) is carried out according to the procedure described in publication J Org. Chem. 71, 7106 9, 2006. The intermediate is purified using a silica gel 60 column using 20-50% ethyl acetate in hexane as eluent. The product is verified by 1H-200 MHz NMR using CDCb as solvent.

Deesterification of Boc-protected amino acid: Deesterification of the Boc-protected Trp residue e.g. D-Trp-methyl ester or D / L-alpha-methyl-Trp-methyl ester) is carried out using lithium hydroxide as follows: The protected amino acid is dissolved in two volumes of methanol and cooled in an ice bath. Three equivalents of lithium hydroxide are dissolved in a volume of water and added to the solution and the mixture is stirred at room temperature for a period of time ranging from two hours to overnight, while monitoring by TLC. The obtained product is purified using a silica gel 60 column using 1% acetic acid in ethyl acetate as eluent. The product is verified at through 200 MHz 1H-NMR using CDCI3 as solvent.

Coupling: Coupling of the Boc-protected Trp moiety (e.g. D-Trp or D / L-alpha-methyl- ^{Trp-methyl ester) is performed using HBTU as the coupling reagent,} DieA ^{as the catalyst, and DMF as the} solvent, depending on the procedure described in the publication J. Med. Chem. 48 (22), 6908-6917, 2005. Coupling can also be effected using other coupling agents. The product is purified through a silica gel column using 20% -50% ethyl acetate in hexane as eluent. The structure of the product is verified by 1H-200 MHz NMR using CDCb or DMSO-d6 as solvent.

Boc deprotection: Boc deprotection is carried out using 50% TFA in methylene chloride for 6 minutes, followed by evaporation under reduced pressure. In order to remove the TFA residues, an addition of benzene and an evaporation under reduced pressure are carried out, respectively, 2-3 times. Subsequently, water is added and the pH of the solution is neutralized. The water is then removed by evaporation under reduced pressure followed by lyophilization overnight. The product is verified by 200 MHz 1H-NMR using d6-DMSO as the solvent.

An optional additional deesterification is then performed, if desired, using the procedure described above. The final product is purified on a silica gel column, using 1% acetic acid in ethanol as eluent. The product is verified by 1H-NMR at 200 MHz using d6-DMSO as the solvent.

An example of a synthetic scheme, exposing the synthesis of H2N-Trp-Aib-OMe, for illustrative purposes, is presented in Figure 4.

Using the general procedure described above, the following examples of dipeptide compounds were synthesized, as follows:

Preparation of tert-butyl H2N-Trp-Aib-OtBu ((R) - (2- (2-amino-3- (1H-indol-3-yl) propanamido) -2-methylpropanoate; compound 1):

Compound 1 was prepared by coupling D-Trp and Aib-OtBu as described above.

1H-NMR (200 MHz; DMSO-d6): 6 = 1.22 (s broad, 9H, - (CH3) 3) 1.58 (s broad, 6H, (CH3) 2), 3.09-3, 45 (m, 1H, CH & 2H, CH2), 7.01-7.14 (m, 3H, Ar-CH), 7.37 (d, 1H, Ar-CH), 7.71 (d, 1H , Ar-CH), 8.07 (s broad, 2H, amido-NH), 10.97 (s broad, 1H, indole-NH).

MS (ESI): m / z = 344.2 (M + -H +), 10%.

Preparation of methyl (D / L) -H2N-a-Me-Trp-Aib-OMe (2- (2-amino-3- (1H-indol-3-yl) -2-methylpropanamido) -2-methylpropanoate; compound 2):

Compound 2 was prepared by coupling L / D-a-Me-Trp (racemic mixture) and Aib-OMe as described above.

1H-NMR (200 MHz; DMSO-d6): 6 = 1.381.48 (m, 9H, CHa & CHâ), 3.05-3.39 (m, 2H, CH2), 3.66 (s, 3H, CH3) , 3.85 (m, 1H, CH), 7.08-7.77 (m, 5H, Ar-CH), 11.13 (s broad, 1H, indole-NH);

MS (ESI): m / z = 337.2 (M ++ Li +) 100%.

Preparation of methyl (D / L) -H2N-a-Me-Trp-cyclopentane-OMe (1- (2-amino-3- (1H-indol-3-yl) -2-methylpropanamido) cyclopentanecarboxylate; compound 3) :

Compound 3 was prepared by coupling L / D-a-Me-Trp and methyl 1-aminocyclopentanecarboxylate as described above.

1H-NMR (200 MHz; CDCls): 6 = 0.83-1.79 (m, 11H, CH3 & 4 CH2), 3.18-3.58 (m, 2H, CH2), 3.89 (s, 3H, CH3), 4.3 (s broad, 2H, NH2), 6.91-7.57 (m, 5H, Ar-CH), 7.67 (s broad, 1H, amido-NH);

MS (ESI): m / z = 345.0 (M ++ 2H +) 10%.

Preparation of H2N-Trp-cyclopentane-OH ((R) -1- (2-amino-3- (1H-indol-3-yl) propanamido) cyclopentanecarboxylic acid; compound 4):

Compound 4 was prepared by coupling D-Trp and 1-aminocyclopentanecarboxylic acid as previously described.

1H-NMR (200 MHz; CDCls): 5 = 1.51-1.58 (m, 4H, CH2), 1.90-2.25 (m, 4H, 2CH2), 3.39-3.6 (m, 2H, CH2), 4.12 (m 1H, CH), 7.24-7.75 (m, 5H, Ar-CH), 8.1 (s broad, 1H, amido-NH);

MS (ESI): m / z = 346.2 (M + NaCl) 100%.

Preparation of H2N-Trp-cyclopropane-OH ((R) - (1- (2-amino-3- (1H-indol-3-yl) propanamido) cyclopropanecarboxylic acid; compound 5):

Compound 5 was prepared by coupling D-Trp and 1-aminocyclopropanecarboxylic acid as previously described.

1H-NMR (200 MHz; DMSO-d6): 5 = 1.33-1.90 (m, 4H, CH2), 3.25-3.62 (m, 2H, CH2), 4.22 (m, 1H, CH), 7.07 8.17 (m, 6H, Ar-CH & 1H, amide NH);

MS (ESI): m / z = 346.2 (M ++ NaCl) 100%.

Preparation of H2N-Trp-cyclopropane-OEt ((1- (2-amino-3- (1H-indol-3-yl) propanamido) cyclopropanecarboxylate ethyl); compound 6):

Compound 6 was prepared by coupling D-Trp and ethyl 1-aminocyclopropanecarboxylate as described above.

1H-NMR (200 MHz; CDCls): 5 = 1.15-1.52 (m, 7H, 2CH2 & Et-CHa), 2.96-3.52 (m, 2H, CH2), 4.24 (q, 2H), 4.56 4.66 (m, 1H, CH), 6.96-7.59 (m, 5H, Ar-CH), 7.72-8.0 (s broad, 1H, NH amide);

MS, ESI, MS (ESI): m / z = 351.1 (M ++ K +) 100%.

Preparation of H2N-Trp-cyclohexane-OH (1- (2-amino-3- (1H-indol-3-yl) propanamido) cyclohexanecarboxylic acid; compound 7):

Compound 7 was prepared by coupling D-Trp and 1-aminocyclohexanecarboxylic acid as previously described.

1H-NMR (200 MHz; CDCls): 5 = 0.76-0.92 (m, 6H, 3 CH2), 1.29-1.56 (m, 4H, 2 CH2), 3.03-3, 55 (m, 2H, CH2), 3.89 (m, 1H, CH), 7.02-7.62 (5H, Ar-CH), 7.88 (s broad, 1H, NH amide);

MS (ESI): m / z = 346.2 (M ++ H2O) 100%.

Preparation of ethyl H2N-Trp-cyclohexane-OEt ((1- (2-amino-3- (1H-indol-3-yl) propanamido) cyclohexanecarboxylate; compound 8):

Compound 8 was prepared by coupling D-Trp and ethyl 1-aminocyclohexanecarboxylate as described above.

¹ H-NMR (200 MHz; DMSO-da): 5 = 0.90-2.01 (m, 13H, Et-CH ³ & 5CH ² ), 3.04-3.43 (m, 2H, CH2), 4.01 (q, 2H), 6.99-8.10 (m, 5H, Ar-CH), 10.94 (s broad, 1H, indole-NH);

MS (ESI): m / z = 413.3 (M ++ NaCl) 100%.

Preparation of methyl H2N-Trp-a-Me-Proline-OMe (1- (2-amino-3- (1 H-indol-3-yl) propanoyl) -2-methylpyrrolidine-2-carboxylate; compound 9):

Compound 9 was prepared by coupling D-Trp and α-Me-Proline-OMe as described above.

¹ H-NMR (200 MHz; CDCb): 5 = 1.27-1.70 (m, 7H, CH3 & 2 CH2), 3.22 (m, 3H, CH2), 3.51-3.81 (m, 2H, CH2), 4.7 (m, 1H, CH), 7.23-7.70 (m, 5H, Ar-CH);

MS (ESI): m / z = 395.1 (M ++ 2Na + & + Cl-) 100%.

Preparation H2N-Trp-oxetane-OH ((R) -3- (2-amino-3- (1H-indol-3-yl) propanamido) oxetane-3-carboxylic acid; compound 10):

Compound 10 was prepared by coupling D-Trp and 1-aminooxethanecarboxylic acid as previously described.

Preparation of H2N-Trp-oxetane-NH2 ((R) -3- (2-amino-3- (1 H-indol-3-yl) propanamido) oxetane-3-carboxamide; compound 11):

Compound 11 was prepared by coupling D-Trp and 1-aminooxethanecarboxamide as described above.

The chemical structures of the conjugates are presented in Table 1 below.

Table 1

It should be appreciated that although in Table 1 the chemical structures are presented for D-Trp residues, conjugates comprising a corresponding L-Trp residue are also contemplated.

Using the general procedure described above, compounds comprising a Trp residue were prepared as set forth in Table 2: 5

Table 2

Each of the Trp moieties listed in Table 2 is coupled to one of the beta-lamellar breaker moieties listed in Table 3 below.

Table 3

Using further the general procedure described hereinabove, the following compounds were prepared as follows:

Preparation of compound A (Fig. 3):

Compound A was prepared as set forth in Scheme 1 below.

E s q u e m a 1

Preparation of compound A2: To a stirred solution of A (5.10 grams, 16.7 mmol) and HCl salt of phenylmethyl-2-amino-2-methylpropanoate (3.60 grams, 18.4 mmol) in Dm F (100 ml) Ho BT (3.40 grams, 5.1 mmol), DIEA (8.9 ml, 50.1 mmol and EDCI (4.50 grams, 25.1 mmol) were added at room temperature. Stir for 16 hours at room temperature under N2 atmosphere, the mixture was poured into ice / water (100 ml) and extracted with EtOAc (100 ml x 2). The organic phase was washed with aqueous 1N HCl solution (80 ml x 2), saturated NaHCO3 solution (80 ml x 2), brine (80 ml) and water over Na2SO4. The EtOAc solvent was then removed in vacuo. The residue was purified by flash chromatography on silica gel (Pe / EtOAc = 2: 1) to provide compound A2 (6.00 grams, 75%) as a white solid.

Preparation of compound A3: The solution of compound A2 (6.00 grams, 12.4 mmol) in Et2O / HCl (30 ml, 2.5 M) was stirred for two hours at room temperature under N2 atmosphere. The reaction mixture was concentrated, then NaHCO3 (sat.) Was added to bring the pH to 7, and then it was extracted with DCM (80 ml) and washed with brine (80 ml). The organic phase was dried over Na2SO4 and concentrated in vacuo to provide compound A3 (4.50 grams, 96%) as a white solid.

1HRMN (400 MHz, DMSO-d6): or = 1.50 (s, 6H), 2.86-2.91 (m, 1H), 3.35-3.40 (m, 1H), 3.67 -3.71 (m, 1H), 5.21 (s, 2H), 7.56 (d, J = 1.6 Hz, 1H), 7.12-7.25 (m, 2H), 7, 33-7.41 (m, 5H), 7.66 (d, J = 12Hz, 1H), 7.81 (s, 1H), 8.39 (broad, 1H).

Preparation of compound A4: To a stirred solution of compound A3 (731 mg, 1.73 mmol) in THF (20 ml) was added Ac2O (4.0 ml). After stirring for two hours at room temperature under N2 atmosphere, the reaction mixture was concentrated in vacuo. To this residue was added DCM (30 ml) and washed with saturated NhHCO3 (30 ml x 2) and brine (30 ml). The organic phase was dried over Na2SO4 and concentrated in vacuo to provide compound A4 (780 mg, 96%) as a white solid.

1HRMN (400 MHz, DMSO-d6): or = 1.39 (s, 6H), 1.94 (s, 3H), 3.05-3.11 (m, 1H), 3.27-3.32 (m, 1H), 4.73-4.76 (m, 1H), 5.09-5.18 (m, 2H), 6.32 (s, 1H), 6.40 (d, J = 7 , 6 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H), 7.13-7.24 (m, 2H), 7.32-7.40 (m, 6H), 7 , 65 (d, J = 8.0Hz, 1H), 8.24 (s, 1H).

Preparation of compound A: To a stirred solution of compound A4 (780 mg, 1.85 mmol) in THF (10 ml) was added 10% Pd / C (330 mg) and stirred for two hours under H2 (1 atm ) at room temperature. The reaction mixture was filtered, the filtrate was concentrated to provide compound A (321 mg, 52%) as a white solid.

1HRMN (400 MHz, DMSO-d6): or = 1.21 (s, 3H), 1.26 (s, 3H), 1.71 (s, 3H), 2.83-2.89 (m, 1H ), 3.04-3.09 (m, 1H), 4.54-4.60 (m, 1H), 6.96-7.08 (m, 2H), 7.13 (d, J = 13.2 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.6Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 10.0 Hz, 1H), 10.80 (s, 1H), 12.22 (s, 1H).

LC-MS (mobile phase: from 90% water and 10% H3CN to 5% water and 95% CH3CN in six minutes, finally under these conditions for 0.5 minutes): The purity is 97.9% , retention time = 2.336 minutes.

MS: Calc .: 331.1; found: 330.0 (M-H) -.

Preparation of compound C (Fig. 3):

The synthesis of compound C is set forth in Scheme 2 below.

Scheme 2

Preparation of compound C ': A mixture of HCOOH (1.5 ml, 30.4 mmol) and Ac2O (2.2 ml, 24.0 mmol) was stirred for 2 hours at 60 ° C and then cooled to room temperature . To this reaction mixture was added a solution of compound A3 (2.88 grams, 7.60 mmol, described above) in THF (20 ml). The mixture was stirred for two hours at room temperature under N2 atmosphere, concentrated in vacuo and the residue was diluted with DCM (10 ml), then washed with saturated NaHCO3 (100 ml x 2) and brine (100 ml) and dried over Na2SO4. The organic phase was concentrated in vacuo to provide compound C '(780 mg, 96%) as a white solid.

1HRMN (400 MHz, DMSO-d6): 5 = 1.39 (s, 6H), 3.05-3.11 (m, 1H), 3.32-3.37 (m, 1H), 4.78 -4.80 (m, 1H), 5.10 5.19 (m, 2H), 6.09 (s, 1H), 6.40 (d, J = 7.2 Hz, 1H), 7.04 (d, J = 1.5 Hz, 1H), 7.16-7.27 (m, 3H), 7.34-7.42 (m, 5H), 8.06 (s broad, 1H), 8 , 16 (s, 1H).

Preparation of compound C: A mixture of compound C '(350 mg, 0.86 mmol) and Pd / C (10%, 250 mg) in THF (10 ml) was stirred for 2 hours under H2 (1 atm) at temperature environment under N2 atmosphere. The reaction mixture was filtered, the filtrate was concentrated in vacuo to provide compound C 200 mg, 73%) as a white solid.

1HRMN (400 MHz, DMSO-d6): 5 = 1.35 (s, 3H), 1.1.38 (s, 3H), 2.86-2.92 (m, 1H), 3.07-3 , 12 (m, 1H), 4.64-4.70 (m, 1H), 6.96-7.08 (m, 2H), 7.14 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.92 (m, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.29 (s, 1H), 10.83 (s, 1H), 12.26 (s, 1H).

LC-MS (mobile phase: from 95% water and 5% CH3CN to 5% water and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes): purity is 98.6% , retention time = 2.309 minutes.

MS: calc .: 317.1; found: 316.0 (M-H) -.

Preparation of compound B (Fig. 3):

The synthesis of compound B is set forth in Scheme 3 below.

E s q u e m a 3

Preparation of compound B1: To a stirred solution of compound C '(780 mg, 1.85 mmol, described above) in anhydrous THF (20 ml) was added BH3. SMe2 (1.6 ml, 16 mmol, 10 M in THF) at 0 ° C. After stirring for 3 hours at room temperature under N2 atmosphere, the reaction mixture was quenched with HCl (conc.) At 0 ° C, then NhHCO3 (sat.) Was added to bring the pH to 8 and extracted with EtOAc (40 ml x 2). The combined organic phase was washed with brine (40 ml), dried over Na2SO4 and purified by flash chromatography on silica gel (EtOAc) to provide compound B1 (400 mg, 17%) as a white solid.

Preparation of compound B2: To a stirred solution of compound B1 (400 mg, 1.0 mmol) in THF (20 ml) was added Ac2Ü (2 ml) at room temperature. After stirring for two hours under a N2 atmosphere, the reaction mixture was diluted with DCM (30 ml), washed with saturated NaHCO3 (30 ml x 2) and brine (30 ml) and the organic phase was dried over Na2SO4, It was concentrated in vacuo and purified by flash chromatography on silica gel (PE / EtÜAc = 1: 1-1: 4) to provide compound B2 (300 mg, 68%) as a white solid.

1HRMN (400 MHz, DMSÜ-d6): 5 = 1.39 (s, 3H), 1.53 (s, 3H), 1.98 (s, 3H), 2.81 (s, 3H), 3, 08-3.14 (m, 1H), 3.31 3.36 (m, 1H), 5.21 (s, 2H), 5.40-5.44 (m, 1H), 6.71 (s , 1H), 6.97 (d, J = 2.0 Hz, 1H), 7.12-7.23 (m, 2H), 7.34-7.41 (m, 6H), 7.58- 7.64 (m, 1H), 8.1 (s, 1H).

Preparation of compound B: To a stirred solution of compound B2 (300 mg, 0.690 mmol) in THF (10 ml) was added 10% Pd / C (200 mg) and the mixture was stirred for 2 hours under H2 (1 atm ) at room temperature and then filtered. The filtrate was concentrated in vacuo to provide compound B (200 mg, 84%) as a white solid.

1HRMN (400 MHz, DMSÜ-d6): 5 = 1.31-1.39 (m, 6H), 1.65 (s, 1.5H), 1.92 (s 1.5H), 2.78 ( s, 1.5H), 2.91 (s, 1.5H), 2.96-3.06 (m, 1H), 3.17-3.21 (m, 1H), 4.55-4, 57 (m, 0.5H), 5.31-5.34 (m, 0.5H), 6.98-7.12 (m, 3H), 7.31-7.36 (m, 1H), 6.61-7.66 (m, 1H), 8.05 (s, 0.5H), 8.33 (s, 0.5H), 10.79 (s, 0.5H), 10.89 ( s, 0.5H), 12.24 (s, 1H).

LC-MS (mobile phase: from 95% water and 5% CH3CN to 5% water and 95% H3CN in 6 minutes, finally under these conditions for 0.5 minutes): the purity is 97.2% , retention time = 2.415 minutes.

MS: calc .: 345.1; found: 346.1 (M H) +.

Preparation of compounds U and T (Fig. 3):

The 'repair of compounds U and T is set forth in Scheme 4 below.

E s q u e m a 4

Preparation of compound U9: To a stirred solution of compound U8 (5.00 grams, 34.5 mmol) in DCM (50 ml) was added DMAP (2.10 grams, 17.3 mmol) and DIEA (9.0 ml , 51.7 mmol) and Boc2O (11.3 grams, 51.7 mmol). The mixture was stirred for 2 hours at room temperature under N2 atmosphere, washed with 1N HCl (100 ml x 2), saturated NaHCO3 solution (100 ml x 2) and brine (80 ml), the organic phase was dried Na2SO4, concentrated and purified by flash chromatography on silica gel (PE / EtOAc = 30: 1-10: 1-4: 1) to provide compound U9 (8.10 grams, 96%) as a white solid.

1HRMN (400 MHz, CDCla): or = 1.74 (s, 9H), 7.40-7.45 (m, 2H), 8.17-8.19 (m, 1H), 8.27 (s , 1H), 8.31-8.33 (m, 1H), 10.13 (s, 1H).

Preparation of compound U11: To a stirred solution of compound U10 (1.60 grams, 5.39 mmol) in DCM (15 ml) was added d Bu (753 mg, 4.95 mmol). The mixture was stirred for 10 minutes at room temperature, then a solution of compound U9 (1.10 grams, 4.49 mmol, in 10 ml of DCM) was added dropwise. After stirring for 3 hours at room temperature under N2 atmosphere, the reaction mixture was washed with 5% citric acid (20 ml x 2) and brine (80 ml), the organic phase was dried over Na2SO4, concentrated and Purified by flash chromatography on silica gel (PE / EtOAc = 25: 1-4: 1) to provide compound U11 (1.70 grams, 89%) as a yellow solid.

1HRMN (400 MHz, CDCls): or = 1.48 (s, 9H), 1.71 (s, 9H), 3.90 (s, 3H), 6.24 (broad, s, 1H), 7, 29-7.40 (m, 2H), 7.62 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H), 8.18 (d , J = 8.0 Hz, 1H).

Preparation of compound U12: To a stirred solution of compound U11 1.70 grams, 4.10 mmol) in THF / MeOH 1: 1, 20 ml) was added a solution of KOH (1.80 grams, 32.0 mmol in 10 ml of H2O). After stirring for 2 hours at 60 ° C under N2 atmosphere, HCl (conc.) Was added to the mixture to bring the pH = 3.4 under ice-cooling, diluted with H2O (80 ml), extracted with EtOAc (100 ml x 2), the combined organic phase was washed with brine (80 ml x 2), dried over Na2SO4 and concentrated in vacuo to provide compound U12 (900 mg, 75%) as a yellow solid .

1HRMN (400 MHz, DMSO-d6): or = 1.38 (broad, 9H), 7.11-7.21 (m, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.64 (s broad, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.81 (s, 1H), 8.27 (broad, 1H), 11.71 (broad, 1H), 12.24 (broad, 1H).

Preparation of compound U13: To a stirred solution of compound U12 (660 mg, 2.19 mmol) and methyl 2-amino-2-methylpropanoate (HCl) in DMF (20 ml) was added HATU (1.70 grams, 4 , 40 mmol) and DIEA (1.2 ml, 6.60 mmol) at room temperature and the mixture was stirred for 16 hours at room temperature under N2 atmosphere, then poured into ice / water (50 ml), extracted with EtOAc (50 ml x 2) and the combined organic phase was washed with 1N HCl (80 ml x 2), saturated NaHCO3 (50 ml x 2) and brine (50 ml), dried over Na2CO4, the solvent was concentrated in vacuo and the residue was purified by flash chromatography on silica gel (PE / EtOAc = 2: 1) to provide compound U13 (450 mg, 51%) as a yellow solid.

1HRMN (400 MHz, DMSO-d6): or = 1.47 (s, 6H), 1.67 (s, 9H), 3.82 (s, 3H), 6.94 (s, 1H), 7, 21-7.30 (m, 3H), 7.43 7.45 (m, 1H), 7.61 (s, 1H), 7.76-7.80 (m, 2H), 8.84 (s , 1 HOUR).

Preparation of compound U: To a stirred solution of compound U13 (250 mg, 0.63 mmol) in THF / MeOH (1: 1, 20 ml) was added a solution of LiOH (120 mg, 2.7 mmol, in 3 ml H2O) and the reaction mixture was stirred for 2 hours at 60 ° C under N2 atmosphere, then HCl (concentrated) was added to bring the pH to 3 under ice-cold water. The mixture was then diluted with 30 ml of H2O and extracted with EtOAc (50 ml x 2), the combined organic phase was washed with brine (50 ml x 2), dried over Na2SO4 and concentrated in vacuo to provide the compound U (200mg, 83%) as a red solid.

1HRMN (400 MHz, DMSO-d6): or = 1.40 (s, 6H), 1.48 (s, 9H), 7.11-7.13 (m, 2H), 7.43-7.45 (m, 2H), 7.69-7.75 (m, 4H), 8.6 (s, 1H), 11.64 (s, 1H).

13C NMR (100 MHz, MeOH-d4): α = 13.11, 19.52, 23.48, 23.73, 27.08, 27.29, 56.18, 60.19, 80.29, 109 , 74, 111.31, 111.43, 117.92, 120.11, 120.23, 122.18, 122.76, 124.54, 126.08, 126.63, 127.48, 135.98 , 166.54, 171.66, 176.82.

LCMS (mobile phase: from 95% water and 5% CH3CN to 5% water and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes): purity is 97.5%, time retention = 3.415 minutes.

MS: calc .: 387.1; found: 388.1 (M + H) +.

Preparation of compound T: To a stirred solution of compound U (150 mg, 0.39 mmol) and NH4Cl (70.0 mg, 1.30 mmol) in DMF (10 ml) was added HATU (240 mg, 0.630 mmol) and DIEA (0.5 ml, 2.80 mmol). After stirring for 16 hours at room temperature under N2 atmosphere, the reaction mixture was poured into ice water (30 ml) and extracted with EtOAc (50 ml x 2). The combined organic phase was washed with 1N HCl (50 mL x 2), saturated NaHCO3 (50 mL x 2), and brine (50 mL) and dried over Na2SO4, concentrated in vacuo, and purified by flash gel chromatography. silica (PE / EtOAc = 2: 1) to provide compound T (120 mg, 80%) as a yellow solid.

1HRMN (400 MHz, DMSO-d6): or = 1.30-1.51 (m, 15H), 7.10-7.20 (m, 4H), 7.41-7.46 (m, 2H) , 7.70-7.73 (m, 2H), 7.87 (s, 1H), 8.55 (s, 1H), 11.60 (s, 1H).

13C NMR (100 MHz, MeOH-d4): or = 24.42, 27.33, 56.69, 80.46, 111.29, 117.97, 120.03, 122.16, 123.41, 126 , 41, 136.03, 155.51, 166.70.

LCMS (mobile phase: from 95% water and 5% CH3CN to 5% water and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes): purity is 97.3%, time retention = 2,782 minutes.

MS: calc .: 386.2; found: 387.1 (M + H) +.

Preparation of compounds Q and W (Fig. 3):

Compounds U and W were prepared as set forth in Scheme 5 below.

E s q u e m a 5

Preparation of compound 12 (Scheme 5):

Compound 18 was prepared as set forth in Scheme 6 below.

Scheme 6

Preparation of compound 15: A solution of compound 14 (10.0 grams, 97.0 mmol) in dioxane (150 ml) and NaOH (5%, 150 ml) was cooled to 0 ° C, then Boc2Ü was added dropwise . The mixture was stirred for 16 hours at room temperature under N2 atmosphere, then HCl (concentrated) was added to bring the pH to 3 and the mixture was extracted with EtOAc (100 ml x 3), washed with brine, 50 ml) , dried over Na2SO4, concentrated in vacuo, and purified on a silica gel column (PE / EtOAc = 5: 1) to provide the product as a white solid (12.1 grams, 61% yield) .

Preparation of compound 16: To a stirred mixture of compound 15 (8.60 grams, 42.3 mmol) and K2CO3 (8.80 grams, 63.5 mmol) in DMF (150 ml) was added BnBr (6.20 ml , 51.0 mmol) dropwise at 0 ° C. After stirring for 15 hours at room temperature under N2 atmosphere, the reaction mixture was poured into water (200 ml) and extracted with EtOAc (200 ml x 3 ). The organic phase was washed with brine (100 ml), dried over Na2SO4 and concentrated in vacuo. Hexane (50 ml) was added to this residue and the mixture was stirred for 30 minutes, filtered to provide compound 16 (8.50 grams, 69%) as a white solid.

1HRMN (400 MHz, CDCb): 5 = 1.43 (s, 9H), 1.54 (s, 6H), 5.04 (broad, s, 1H), 5.19 (s, 2H), 7, 37-7.38 (m, 5H). Preparation of compound 17: To a stirred solution of compound 16 (5.00 grams, 17 mmol) in THF (100 ml) was added NaH (5.40 grams, 60%, 136 mmol, washed with hexane) cooled with ice and The mixture of The reaction was stirred for two hours in an ice / water bath, then Mel (4.50 mL, 85.0 mmol) was added dropwise. The mixture was stirred for 15 hours at 30 ° C under N2 atmosphere and then slowly poured into ice / water (100 ml) and extracted with EtOAc (100 ml x 3). The combined organic phase was washed with brine (100 mL x 2), dried over Na2SO4, and concentrated in vacuo to provide compound 17 (4.00 grams, 77%) as a yellow oil.

1HRMN (400 MHz, CDCla): or = 1.45 (s, 9H), 1.47 (s, 6H), 2.93 (s, 3H), 5.16 (s, 2H), 7.33- 7.38 (m, 5H).

Preparation of compound 18: The solution of compound 17 (4.0 grams, 13.0 mmol) in Et2O / HCl (30 ml, 2.5 M) was stirred for 2 hours at room temperature under N2 atmosphere. The solvent was then evaporated and hexane (40 ml) was added to the residue and the mixture was stirred for 30 minutes and filtered to provide the HCl salt of compound 18 (2.3 grams, 74%) as a white solid .

1HRMN (400MHz, CD3OD): or = 1.61 (s, 6H), 2.70 (s, 3H), 5.33 (s, 2H), 7.70-7.50 (m, 5H).

Preparation of compound 19: To a stirred solution of compound A1 (3.60 grams, 11.8 mmol) and salt of compound 18 (HCl) in DMF (100 ml) was added HATU (5.60 grams, 14.8 mmol ) and DMAP (3.60 grams, 29.4 mmol) at room temperature and the mixture was stirred for 16 hours at room temperature under N2 atmosphere, then poured into water (100 ml) and extracted with EtOAc (150 ml x 2). The combined organic phase was washed with 1N HCl (aqueous) (100 mL x 2), saturated NaHCO3 (10 mL x 2), and brine (100 mL), then dried over Na2SO4, concentrated in vacuo, and purified by chromatography. Flash on silica gel (Pe / EtOAc = 22: 1) to provide compound 19 (2.70 grams, 56%) as a yellow solid.

1HRMN (400 MHz, DMSO-d6): or = 1.32 (d, J = 8.8 Hz, 6H), 1.49 (s, 9H), 2.58 (s, 3H), 3.04- 3.11 (m, 2H), 4.94 4.99 (m, 1H), 5.10-5.21 (m, 2H), 5.41-5.43 (m, 1H), 6.95 (s, 1H), 7.12-7.21 (m, 2H), 7.29-7.39 (m, 6H), 7.72 (d, J = 7.6 Hz, 1H), 8, 15 (s, 1H).

Preparation of compound 20: a solution of compound 19 (500 mg, 1.01 mmol) in Et2O / HCl (2.5 M, 20 ml) was stirred for 2 hours at room temperature under N2 atmosphere and subsequently the reaction mixture concentrated under vacuum to provide compound 20 (450 mg, crude).

Preparation of compound 21: A solution of compound 20 (450 mg, crude) and Ac2O (2.0 ml) and DIEA (1.0 ml) in THF (20 ml) was stirred for 2 hours at room temperature under an atmosphere of N2 and subsequently the reaction mixture was concentrated in vacuo and diluted with EtOAc (30 ml), the organic phase was washed with 1N HCl (aqueous) (20 ml x 2), saturated NaHCO3 (20 ml x 2) and brine (20 ml), dried over Na2SO4 and the solvent was removed in vacuo to provide compound 21 (400 mg, 91%) as a yellow solid.

Preparation of compound Q: The mixture of compound 21 (500 mg, crude) and 10% Pd / C (250 mg) in THF (20 ml) was stirred for 2 hours at room temperature under H2 (1 atm), then It was filtered and the filtrate was concentrated to provide compound Q (250mg, 88%) as a white solid.

1HRMN (400 MHz, DMSO-d6): or = 1.25-1.31 (s, 6H), 1.79 (s, 3H), 2.83 (s, 3H), 2.85-2.88 (m, 1H), 3.00-3.05 (m, 1H), 4.94-5.00 (m, 1H), 6.98-7.09 (m, 2H), 7.14 (d , J = 2.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 8.8 Hz, 1H), 10.83 (s, 1H), 11.91 (s broad, 1H).

LC-MS (mobile phase: from 95 water and 5% CH3CN to 40% water and 60% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes): purity is> 95%, time retention = 1.958 minutes.

MS calc .: 345.2; found: 346.1 (M H) +.

Preparation of compound V: To a stirred solution of compound V (230 mg, 0.67 mmol) and MeNH2.HCl (222 mg, 3.33 mmol) in Dm F (20 ml) was added HOBT (1.35 mg, 1 mmol) and DIEA (0.8 ml, 4.70 mmol), then EDCI (192 mg, 1.0 mmol) was added at room temperature and the reaction mixture was stirred for 16 hours at room temperature under N2 atmosphere , concentrated in vacuo and purified by preparative HPLC to provide compound V (30 mg, 17%) as a white solid.

1HRMN (400 MHz, CDCI3): 8 = 1.35 (d, J = 5.2 Hz, 6H), 1.99 (s, 3H), 2.65 (d, J = 4.8 Hz, 3H) , 2.74 (s, 3H), 3.21 3.24 (m, 2H), 5.16-5.18 (m, 1H), 5.61 (s broad, 1H), 6.48 (s broad, 1H), 7.16-7.29 (m, 3H), 7.40 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 8.20 (s, 1H).

LC-MS [mobile phase: from 90% water (0.02% NH4Ac) and 10% CH3CN to 5% water (0.02% NH4Ac) and 95% CH3CN in 6 minutes, finely under these conditions for 0.5 minutes]: purity is> 95%, retention time = 2,090 minutes.

MS calc .: 327.2; found: 328.1 (M ++ H).

Preparation of compound R (Fig. 3): Compound R was prepared as set forth in Scheme 7 below. Scheme 7

The mixture of compound 19 (1.50 grams, 3.8 mmol) and Pd / C (10%, 400 mg) in THF (40) ml) was stirred for 15 hours at room temperature under H2 (1 atm), then It was filtered and the filtrate was concentrated to provide compound R (1.20 grams, 80%) as a red solid.

1HRMN (400 MHz, DMSO-d6): 8 = 1.20-1.32 (m, 15H), 2.87-2.30 (m, 5H), 4.60-4.65 (m, 1H) , 6.83-7.16 (m, 4H), 7.33-7.57 (m, 2H), 10.84 (s, 1H), 11.88 (s, 1H).

LC-MS (mobile phase: from 95% water and 5% CH3CN to 5% water and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes): purity is 98.6% , retention time = 3.685 minutes.

MS calc .: 403.2: found: 404.1 (M ++ H).

Preparation of compounds D, E and F (Fig. 3):

Compounds D, E and F were prepared as set forth in Scheme 8 below.

E s q u e m a 8

Compound D: R = NH2;

Compound E: R = NHMe;

Compound F: R = NMe2

Preparation of compound 40: To a stirred solution of compound A1 (2.0 grams, 6.6 mmol) in DMF (10 ml) was added amine (1.1 gram, 7.0 mmol), HOBt (945 mg, 7 mmol) EDC (1.9 gram, 10 mmol) and DIEA (2.6 grams, 20 mmol). After stirring for 16 hours at room temperature under N2 atmosphere, the reaction mixture was poured into ice / water (20 ml) and extracted with EtOAc (20 ml x 3) and the combined organic phase was washed with brine (50 ml), dried over Na2SO4 and concentrated in vacuo. The residue was purified on a silica gel column (PE: EtOAc = 8: 1) to provide compound 40 (3.2 grams, 81% yield).

Preparation of compound 41: To a solution of compound 40 (4.8 grams, 12 mmol) in MeOH (50 ml) and H2O (10 ml) was added LOH.H2O (608 mg, 15 mmol). After stirring for 4 hours at room temperature, HCl (concentrated) was added to the reaction mixture to bring the pH to 5, water (40 ml) was added and the mixture was extracted with EtOAc (40 ml x 3). The combined organic phase was washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo to provide compound 41 as a white solid (4.2 grams, 90% yield).

Preparation of compounds 42, 43 and 44:

To a solution of compound 41 (300 mg, 0.77 mmol) in DMF (10 ml) was added amine salt (2.3 mmol, selected according to the desired product), HOBt (157 mg, 1.2 mmol), EDC (2.23 grams, 1.2 mmol) and DIEA (298 mg, 2.3 mmol). After stirring for 16 hours at room temperature under N2 atmosphere, the mixture was poured into H2O (50 ml) and extracted with EtOAc (50 ml x 3). The combined organic phase was washed with brine (50 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified on a silica gel column (PE: EtOAc = 8: 1) to provide the product (42, 43 or 44).

Preparation of compounds D, E and F:

A solution of compound 42 (or 43 or 44) in EtOAc / HCl (4N, 10 mL) was stirred for 2 hours at room temperature, then concentrated in vacuo and purified by preparative HPLC to provide the corresponding product as a white solid.

Compound D: 90mg, 51% yield;

1H NMR (400 MHz, DMSO-da): 6 = 1.28 (s, 6H), 3.30-3.03 (m, 2H), 4.03 (t, J = 7.0 Hz, 1H) , 7.20-6.90 (m, 5H), 7.37 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 8.25- 8.00 (s broad, 3H), 8.54 (s, 1H), 11.04 (s, 1H).

LC-MS [mobile phase: from 95% water (0.05% TFA) and 5% CH3CN to 5% water (0.05% TFA) and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes]: purity is> 95%, retention time = 2.308 minutes.

MS: calc .: 288.1; found: 289.1 (M ++ H).

Compound E: 75mg, 57% yield;

1H NMR (400 MHz, DMSO-d6): 6 = 1.27 (s, 6H), 2.52 (s, 3H), 3.30-3.02 (m, 2H), 4.02 (t, J = 7.2 Hz, 1H), 7.20 7.00 (m, 3H), 7.38 (d, J = 7.6 Hz, 1H), 7.53 (d, J = 7.6 Hz , 1H), 7.70 (d, J = 8.0 Hz, 1H), 8.35-7.90 (s broad, 3H), 8.59 (s, 1H), 11.03 (s, 1H ).

LC-MS [mobile phase: from 95% water (0.05% TFA) and 5% CH3CN to 5% water (0.05% TFA) and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes]: purity is> 95%, retention time = 2.391 minutes.

MS: calc .: 302.1; found: 303.1 (M ++ H).

Compound F: 50mg, 47% yield;

1H NMR (400 MHz, DMSO-d6): 6 = 1.32 (s, 3H), 1.34 (s, 3H), 3.10-2.65 (m, 8H), 3.47 (dd, J = 7, 6, 5, 4 Hz, 1H), 7.15-7.00 (m, 3H), 7.34 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 10.90 (s, 1H).

LC-MS [mobile phase: from 95% water and 5% CH3CN to 5% water and 95% CH3CN in 6 minutes, finally under these conditions for 0.5 minutes]: purity is 98.3% , retention time = 2,568 minutes.

MS: calc .: 316.1; found: 317.2 (M ++ H).

Preparation of compound W (Fig. 3):

Compound W was prepared as set forth in Scheme 9 below, using benzyloxycarbonyl (Z) as the N-terminal protecting group of the dipeptide analog and phenylacyl ester (2-phenyl-2-oxoethyl ester (Pac) as the protecting group for C terminal of the dipeptide analog, thus generating ZD-Trp-Aib-OPac The two protecting groups Z and Pac are separated by catalytic hydrogenation.

E s q u e m a 9

Preparation of Z-D-Trp-Aib-OMe (Compound 51)

33.84 grams (100 mmol) of Z-D-Trp-OH were dissolved in 150 ml of EtOAc. 30.44 grams (110 mmol) of N- (4,6-dimethoxy-1,3,5-triazin-2-yl) -N-methylmorpholinium chloride were added at 0-5 ° C. The H-Aib- OMe was supplied from 28.56 grams (186 mmol) of hydrochloride salt and was added to the reaction mixture. 20 ml (150 mmol, 1.5 equivalents) of triethylamine was also added and the reaction mixture was stirred at room temperature for one day. The reaction mixture was subsequently treated by adding 150 ml of water, separating the phases, washing the organic phase with 150 ml of saturated NaHCO3 and 150 ml of saturated NaCl, drying in Na2SO4 and evaporating the solvent. The crude compound 51 was obtained as an orange oil (45.0 grams, 103 mmol, 103%) and was purified by column chromatography on silica (using toluene: MeOH 9: 1 as eluent) in order to produce compound 51 as an orange oil (40.0 grams, 91.4 mmol, 91.4% yield).

Preparation of Z-D-Trp-Aib-OH (Compound 52):

A solution of 20.0 grams (46 mmol) of ZD-Trp-Aib-OMe in a mixture of 100 ml of methanol, 100 ml of water and 2.2 grams (55 mmol) of NaOH was stirred at room temperature for a day. The reaction mixture was subsequently subjected to treatment by evaporating the solvent, adding 200 ml of water to the residue obtained, extracting with 3 x 50 ml of cyclohexane, adjusting the pH of the aqueous phase to 3, with 10 ml of 85% H3PO4, filtering the formed precipitate while washing the precipitate with 400 ml of water and drying in vacuo at 40 ° C. Compound 52 was obtained as a white solid product (17.0 grams, 40 mmol, 87% yield).

Preparation of ZD-Trp-Aib-OPAc (compound 53):

To a solution of 4.23 grams (10 mmol) of ZD-Trp-Aib-OH (compound 52) and 199 grams (10 mmol) of 2'-bromo acetophenone in 20 ml of EtOAc, 1.4 ml ( 10 mmol) of triethylamine and the reaction mixture was stirred at room temperature overnight. The reaction mixture was subsequently worked up by adding 50 ml of EtOAc, washing the organic phase with 20 ml of KHSO41M, 20 ml of water, 20 ml of 1M NaHCO3 and 20 ml of water and evaporating the solvent. The obtained crude product was purified by column chromatography on silica (using hexane: EtOAc 1: 1 as eluent) to yield compound 53 as a clear oil (7.9 mmol, 79% yield).

Preparation of Z- (N-tBu) -D-Trp-Aib-OPac (compound 54):

4.3 grams (7.9 mmol) of Z-D-Trp-Aib-OPac (compound 53) was placed in a metal reactor along with 40 ml of dichloromethane and 0.24 ml of concentrated H2SO4 as a catalyst. Isobutylene was condensed in the reactor at -5 ° C (cooled in a salt-ice bath). The reaction mixture was stirred for one day at room temperature and 2.5 bar pressure and was subsequently treated by evaporating the isobutylene, washing the residual organic phase with 50 ml of 10% NaOH and evaporating the organic phase. The crude product thus obtained was purified by column chromatography on silica (using CHCb: MeOH 98: 2 as eluent) to provide compound 54 as a light yellow solid product (2.85 grams, 4.8 mmol, 60 % yield).

Preparation of H- (N-tBu) -D-Trp-Aib-OH (compound W):

1.2 grams (2 mmol) of Z- (N-tBu) -D-Trp-Aib-OPac (compound 54 were dissolved in 100 ml of MeOH, the solution was placed in a reactor and 0.12 grams were added to it of SelCat Q6 catalyst (Pd / C type) The reaction mixture was stirred for one day at room temperature and 1.5 bar of H2 pressure and subsequently subjected to treatment by filtration, washing the catalyst with 2 x 50 ml of hot methanol, combining the organic phases and evaporating the solvent The solid residue was purified by column chromatography on silica (using toluene: methanol 1: 1 as eluent) to provide compound W as a light yellow solid (126 mg, 0.36 mmol, 18% yield).

1H-NMR (recorded at 11.7 Tesla on a Bruker Avance-500 MHz spectrometer (two channels) at 300 Ken DMSO): or = 1.35 (s), 1.64 (s), 2.80, 3, 07, 3.45 (t), 6.98 (t), 7.07 (t), 7.58 (d), 7.63 (d), 8.37, 8.52.

Example 2

Activity tests

Peptide solutions:

The D-Trp-Aib analogs described herein, as well as other peptides tested for comparison purposes, were dissolved in DMSO at a concentration of 500 mM and 100 mM, sonicated for 20 seconds on ice, and then diluted with DMSO to their final concentrations.

Oligomer formation (Hillen's protocol):

Intermediates AP1-42 and globulomers were produced according to Barghorn et al. [J. Neurochem. 2005 Nov; 95 (3): 834-47] .. Synthetic lyophilized p-amyloid polypeptides (Ap 1-42> 98% pure) were purchased from Bachem (Bubendorf, Switzerland). To avoid preaggregation, synthetic lyophilisate AP1-42 was pretreated with HFIP. AP1-42 was dissolved in 100% HFIP, sonicated for 20 seconds and incubated for 2 hours at 37 ° C under shaking at 100 rpm. After evaporation in a speedVac device, the AP1-42 was resuspended in DMSO, with or without the peptide tested, at 5 mM and diluted with NaH2PO420 mM, NaCl 140 mM, pH 5.4, to a concentration 400 pM final and 1/10 volume 2% DSDS (0.2% final concentration). Ap globulomers were generated by further dilution with two volumes of H2O and incubation for 18 hours or more. The Ap aggregation products were then separated using 15% tristricin gel and stained using imperial protein dye.

Western blot analysis:

To evaluate the effect of D-Trp-Aib analogs on the transformation of Ap1-42 into toxic structures, the tested peptides were incubated with Ap1-42 in increasing mole ratios and the reaction mixtures were resolved on SDS-PAGE followed by Western blot analysis using a specific anti-AP1-42 antibody (6E10) (SIGNET).

Thioflavin T Fluorescence Assay: AP1-42 polypeptide fibrilization was verified using a thioflavin T (ThT) dye binding assay. A solution of Ap1-4210 pM was prepared as described above and mixed immediately with the stock solutions of analogs tested (100 pM), in order to achieve a final concentration of 5 pM for Ap and various concentrations of the analog tested. For each measurement, ThT was added to 0.1 ml of sample to provide a final concentration of ThT 0.3 pM and Ap O, 4 pM. The samples were incubated at 37 ° C. Fluorescence measurements, made after addition of the ThT solution to each sample at 37 ° C, were carried out using a Jobin Yvon FluroMax-3 spectrometer (excitation 450 nm, slot of 2.5 nm; emission 480 nm, slot 5 nm, integration time 1 second). The background was subtracted from each sample. Each experiment was repeated in quadruplicate.

Results:

All dipeptide analogs described herein tested using Hillen's protocol for the generation of AP1-42 globulomers were found to exhibit inhibition of the generation of AP1-42 globulomers to a greater extent measured for D-Trp- Aib (MRZ99030) at a 1: 1 mole ratio (inhibitor: Ap peptide) and even at lower concentrations. All of the analogs tested showed complete inhibition at a 20: 1 mole ratio (inhibitor: Ap peptide).

Compound 2, HhN-a-Me-Trp-Aib-OMe (see Table 1 above), showed complete globulomer inhibition at a mole ratio of 1: 1 (inhibitor: Ap peptide), using the Hillen protocol (See Fig. 5A for data obtained using Western blot analysis and Fig. 5B for data obtained using 15% Tris-tricine gel and Imperial protein stain).

Compounds 1 and 7 (see Table 1 above) were shown using the Hillen protocol to inhibit Ap1-42 fibril formation at a mole ratio of 10: 1 and 20: 1 (inhibitor: Ap peptide) (see Fig. 6).

Compounds 3 and 5 (see Table 1 above) were shown in the Hillen protocol, for inhibiting Ap1-42 fibril formation at a 10: 1 mole ratio (inhibitor: Ap peptide) (see Fig. 7).

For comparison purposes, data were obtained for inhibition of Ap 1-42 globulomer generation measured for D-Trp-Aib (EG30), using the Hillen protocol, which showed complete inhibition only at a mole ratio of 40: 1 (inhibitor: Ap peptide) (see Fig. 8).

For further comparison, it is appreciated that the following peptides tested did not show any activity to inhibit the generation of Ap1-42 globulomers: dF-dF-Aib; Y-Aib-Aib; Aib-Y-Y; dY-Aib; and N-Y-Y-P.

Example 3

Activity Assays (SPR Binding Assay)

Surface plasmon resonance (SPR) studies were performed using a Biacore X100 biosensor instrument (GE Lifesciences, Uppsala, Sweden), equipped with two flow cells on a detector chip. The Ap monomers were covalently coupled to a CM7 detector chip flow cell (GE Lifesciences, Uppsala, Sweden) through primary amines using the amine coupling test kit (GE Lifesciences, Uppsala, Sweden). As a control, ethanolamine was immobilized on the reference channel. 3 different types of chips were used and the immobilization levels for Ap were comparable (21605RU, 22180RU and 2192RU, respectively). One RU represents approximately 1pg / mm2 of the analytes on the surface matrix of the detector chip.

Compounds tested were dissolved in DMSO and further diluted in DMSO to provide 1000x concentrated stock solutions. Stock solutions were diluted 1: 1000 in HBS-EP buffer containing 0.01 M HEPES, pH 7.4, 0.5 M NaCl, 3 mM EDTA and 0.005% PB: 0 surfactant. HBS-EP 0.1% (v / v) DMSO was used as the assay performance buffer. The test solutions were injected onto the detector chip in concentrations ranging from 0.1 Nm to 300 Nm at a flow rate of 10 µl / min for 180 seconds at 25 ° C. The concentrations were tested in duplicate.

The RUs caused by the compound injected into the ethanolamine control flow cell was adjusted as the reference response and subtracted from the RUs caused by the same compound injected into the Ap saturated flow cell. The ratios between each RU obtained in the Steady state of binding (platform of the binding curve) and compound concentration layer were plotted.

After the analyte injection was stopped, HBS-EP buffer was flowed over the chip for 180 s to allow the bound analyte to dissociate from the immobilized Ap and dissociation curves were obtained. After the dissociation phase, the regeneration solution (1 M NaCl, 50 mM NaOH) was injected and flowed over the chip for 30 seconds to remove residual analytes from the immobilized Ap.

Biacore X100 Ver 1.1 software was used to record the binding curves and Biacore X100 Ver 1.1 evaluation software was used to analyze the curves (plot of each RU at steady state versus analyte concentration, plot fit, determination of Kd values ). The equilibrium dissociation constant Kd of the analyte with respect to the immobilized Ap was determined from the steady state levels estimating the maximum RU, Rmax and calculating the Kd as the concentration of the compound that caused half the Rmax. Table 4 below presents the IC-50 values obtained by performing repeated tests for compound examples, as described herein.

Table 4

Example 4

Pharmacological parameters

The aqueous solubility of the dipeptide analog examples described herein was tested at pH 7.4. The data obtained are presented in Table 5 below and indicate that all the tested compounds showed good solubility above 250.

Examples of dipeptide analogs described herein were tested for to membrane permeability in MDCKII cells transfected with human PGP, to assess intestinal absorption. The data obtained are presented in Table 5 below, in which AB is the apical-basolateral flow IAI is the asymmetry index.

The metabolic stability of the peptide analog examples described herein was tested in human and rat liver microsomes (h Lm and RLM, respectively). The data obtained are presented in Table 5 below, as% of compound remaining after 30 minutes. All peptides were found to be sufficiently stable.

Table 5

Although the invention has been described in conjunction with its specific embodiments, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art.

Claims (6)

1. A compound comprising a tryptophan (Trp) moiety coupled to a beta-lamellar breaking moiety, the compound being represented by a chemical structure selected from the group consisting of

for use in the inhibition of the formation of amyloid fibrils or in the treatment of an amyloid-associated disease or disorder. 2. The compound to be used of claim 1, represented by the chemical structure: The compound to be used of claims 1 or 2, wherein said amyloid-associated disease or disorder is one of: type II diabetes mellitus, Alzheimer's disease (AD); early-onset Alzheimer's disease; late-onset Alzheimer's disease, pre-symptomatic Alzheimer's disease; Parkinson's disease; SAA amyloidosis; hereditary Icelandic syndrome; multiple myeloma; medullary carcinoma; Aortic medical amyloid, insulin injection amyloidosis; systematic prion amyloidosis; amyloidosis with chronic inflammation, Huntington's disease; senile systemic amyloidosis; amyloidosis of the pituitary gland; hereditary renal amyloidosis; familial British dementia; Finnish hereditary amyloidosis; Familial non-neuropathic amyloidosis; an eye disease or disorder related to amyloidosis or prion disease. The compound to be used of claim 3, wherein said amyloid-related eye disease or disorder is glaucoma or age-related macular degeneration. The compound to be used of any of claims 1-4, wherein said compound is formulated for administration in the form of drops. The compound to be used of any of claims 1-4, wherein said compound is a formulation for administration by injection including intraocular injection.