Patent Application
20100267609
Alzheimer's disease is a progressive mental deterioration in a human resulting, inter alia, in loss of memory, confusion and disorientation. Alzheimer's disease accounts for the majority of senile dementias and is a leading cause of death in adults (Anderson, R. N., Natl. Vital Stat. Rep. 49:1-87 (2001), the teachings of which are incorporated herein in their entirety). Histologically, the brain of persons afflicted with Alzheimer's disease is characterized by a distortion of the intracellular neurofibrils and the presence of senile plaques composed of granular or filamentous argentophilic masses with an amyloid protein core, largely due to the accumulation of β-amyloid peptide (Aβ) in the brain. Aβ accumulation plays a role in the pathogenesis and progression of the disease (Selkoe, D. J., Nature 399: 23-31 (1999)) and is a proteolytic fragment of amyloid precursor protein (APP). APP is cleaved initially by β-secretase followed by γ-secretase to generate Aβ (Lin, X., et al., Proc. Natl. Acad. Sci. USA 97:1456-1460 (2000); De Stropper, B., et al., Nature 391:387-390 (1998)).
There is a need to develop effective compounds and methods for the treatment of Alzheimer's disease.
The present invention is directed to compounds and pharmaceutical compositions containing compounds represented by Structural Formula I:
In Formula I, Y is a carrier molecule; Z is a bond, —OP(O)−2O—, —C(O)OR33—, C(O)NHR33 or an amino acid sequence cleavable by a hydrolase; R33 is a bond or an alkylene; k is 0 or an integer from 1 to about 100; r is an integer from 1 to about 100; and A1, for each occurrence, is a compound represented by the following Formula II, or optical isomers, diastereomers, or pharmaceutically acceptable salts thereof:
In Formula II, X is C═O or S(O)n. n is 1 or 2. P1 is an aliphatic group, a hydroxyalkyl, an aryl, an aralkyl, a heterocycloalkyl, or an alkylsulfanylalkyl. P2, P1′, and P2′ are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted heterocycloalkyl. R is —H. R1 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted alkoxy, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocycle, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted heterocyclooxy, a substituted or unsubstituted heterocycloalkoxy, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted heteroaralkoxy, or —NR5R6. Alternatively, R1, together with X, is a peptide or Y—Z—. R4 is H; or R4 and P1′, together with the atoms connecting R4 and P1′, form a five or six membered heterocycle. R2 and R3 are each, independently, selected from the group consisting of H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocycle, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted heteroaryl, and a substituted or unsubstituted heteroaralkyl; or one of R2 and R3, together with the nitrogen to which they are attached, is a peptide or Y—Z—. Alternatively, R2 and R3 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocycle or a substituted or unsubstituted heteroaryl. R5 and R6 are each, independently, H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocycle, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heteroaralkyl. Alternatively, R and one of R5 or R6, together with X and the nitrogen atoms to which they are attached, form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle or substituted or unsubstituted heteroaryl ring. However, A1 does not include the following compounds:
In one embodiment, the invention is directed to compounds and pharmaceutical compositions containing compounds represented by Formula III:
In Formula III, Y, Z, k and r are defined as in Formula I, and A2, for each occurrence, is a compound represented by the following Formula IV, or optical isomers, diastereomers, or pharmaceutically acceptable salts thereof:
In Formula IV, X, P1, P2, P1′, P2′, R2, R3 and R4 are defined as in Formula II, and R19 an aliphatic group substituted with one or more substituents, wherein at least one substituent is a substituent selected from the group consisting of —NR15C(O)R16, —NR15C(O)2R16 and —NR15S(O)2R16. R15 and R16 are each, independently, H, an aliphatic group, an aryl, an aralkyl, a heterocycle, a heterocycloalkyl, a heteroaryl or a heteroaralkyl, wherein the aliphatic group, aryl, aralkyl, heterocycle, heterocyclalkyl, heteroaryl or heteroaralkyl are optionally substituted with one or more substituents selected from the group consisting of an aliphatic group, hydroxy, —OR9, a halogen, a cyano, a nitro, —NR9R10, guanidino, —OPO3−2, —S(O)pR9, —OC(O)R9, —C(O)R9, —C(O)2R9, —NR9C(O)R10, —C(O)NR9R10, —OC(O)NR9R10, —NR9C(O)2R10, an aryl, a heteroaryl, a heteroaralkyl, and a heterocycle. p is 0, 1, or 2. However, when R19 is substituted with —NR15C(O)R16 or —NR15C(O)2R16, —NR2R3 is not a group having the following structural formula:
In another embodiment, the invention is directed to compounds and pharmaceutical compositions containing compounds that selectively inhibit hydrolysis of a memapsin 2 β-secretase site relative to a memapsin 1 β-secretase site. Compounds of the invention that selectively inhibit hydrolysis of a memapsin 2 β-secretase site relative to a memapsin 1 β-secretase site are represented by Formula V:
In Formula V, Y, Z, k and r are defined as in Formula I, and A3, for each occurrence, is a compound represented by the following Formula II, or optical isomers, diastereomers, or pharmaceutically acceptable salts thereof:
In another embodiment, the invention is directed to compounds and pharmaceutical compositions containing compounds represented by Formula VI:
In Formula VI, Y, Z, k and r are defined as in Formula I, and A4, for each occurrence, is a compound represented by the following Formula VII, or optical isomers, diastereomers, or pharmaceutically acceptable salts thereof:
In Formula VII, X, P1, P2, P1′, P2′, R2, R3 and R4 are defined as in Formula II, are defined as in Formula II, and R18 is a substituted or unsubstituted heteroaralkoxy, a substituted or unsubstituted heteroaralkyl, or —NR20OR21. R20 and R21 are each, independently, —H or a substituted or unsubstituted heteroaralkyl. Alternatively, R and one of R20 or R21, together with X and the nitrogen atoms to which they are attached, form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle or substituted or unsubstituted heteroaryl ring.
In another embodiment, the invention is directed to compounds and pharmaceutical compositions containing compounds represented by Formula VIII:
In Formula VIII, A5, for each occurrence, in the compounds represented by Formula VIII is selected from the group of compounds in Table 1 or optical isomers, diastereomers, or pharmaceutically acceptable salts thereof.
In another embodiment, the present invention relates to a method of inhibiting hydrolysis of a β-secretase site of a β-amyloid precursor protein in an in vitro sample by administering to the in vitro sample a compound represented by Formula I, III, V, VI or VIII.
In another embodiment, the present invention relates to a method of decreasing β-amyloid protein (Walsh, D. M., et al., J. Biol. Chem. 274:25945-25952 (1999) and Liu, K., et al., Biochemistry 41:3128-3136 (2002)) in an in vitro sample by administering to the in vitro sample a compound represented by Formula I, III, V, VI or VIII.
In another embodiment, the present invention relates to a method of decreasing β-amyloid protein in a mammal by administering to the mammal a compound represented by Formula I, III, V, VI, or VIII.
In another embodiment, the present invention relates to a method of selectively inhibiting hydrolysis of a β-secretase site by memapsin 2 relative to memapsin 1 in an in vitro sample by administering to the in vitro sample a compound represented by Formula I, III, V, VI or VIII.
In another embodiment, the present invention relates to a method of selectively inhibiting hydrolysis of a β-secretase site by memapsin 2 relative to memapsin 1 in a mammal by administering to the mammal a compound represented by Formula I, III, V, VI or VIII.
In another embodiment, the present invention relates to a method of inhibiting hydrolysis of a β-secretase site of a β-amyloid precursor protein in a mammal by administering a compound represented by Formula I, III, V, VI or VIII.
In another embodiment, the present invention relates to a method of treating Alzheimer's disease in a mammal by administering to the mammal a compound represented by Formula I, III, V, VI, or VIII.
In another embodiment, the present invention relates to a crystallized protein selected from the group consisting of amino acid residues 1-456 of SEQ ID NO: 8, amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60), amino acid residues 27-456 of SEQ ID NO: 8 (SEQ ID NO: 61), amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) and amino acid residues 45-456 of SEQ ID NO: 8. (SEQ ID NO: 63); and a compound represented by Formula I, III, V, VI or VIII. The crystallized protein has an x-ray diffraction resolution limit not greater than about 4.0 Å.
In another embodiment, the present invention relates to a crystallized protein comprising a protein of SEQ ID NO: 6 and a compound represented by Formula I, III, V, VI or VIII. The crystallized protein has an x-ray diffraction resolution limit not greater than about 4.0 Å.
In another embodiment, the present invention relates to a crystallized protein comprising a protein encoded by SEQ ID NO: 5 and a compound is represented by Formula I, III, V, VI, or VIII. The crystallized protein has an x-ray diffraction resolution limit not greater than about 4.0 Å.
In another embodiment, the present invention relates to a crystallized complex comprising a protein selected from the group consisting of amino acid residues 1-456 SEQ ID NO: 8, amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60), amino acid residues 27-456 of SEQ ID NO: 8 (SEQ ID NO: 61), amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) and amino acid residues 45-456 of SEQ ID NO: 8 (SEQ ID NO: 63); and a compound in association with said protein, wherein said substrate is in association with said protein at an S3′ binding pocket, an S4′ binding pocket and an S4 binding pocket. Preferably, the compound is a compound of Formula I, III, V, VI, or VIII.
In another embodiment, the present invention relates to a crystallized complex comprising a protein selected from the group consisting of amino acid residues 1-456 SEQ ID NO: 8, amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60), amino acid residues 27-456 of SEQ ID NO: 8 (SEQ ID NO: 61), amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) and amino acid residues 45-456 of SEQ ID NO: 8 (SEQ ID NO: 63); and a compound in association with said protein, wherein said compound is in association with said protein at an S3 binding pocket. Preferably, the compound is a compound Formula V, VI, or VIII.
In another embodiment, the present invention relates to a crystallized complex comprising a protein selected from the group consisting of amino acid residues 1-456 SEQ ID NO: 8, amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60), amino acid residues 27-456 of SEQ ID NO: 8 (SEQ ID NO: 61), amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) and amino acid residues 45-456 of SEQ ID NO: 8 (SEQ ID NO: 63); and a compound represented by Formula V, VI, or VIII in association with said protein, wherein said compound is in association with said protein at an S3 binding pocket.
The invention described herein provides compounds for inhibiting the activity of memapsin 2 (β-secretase) and methods of using the compounds, for example, to inhibit the hydrolysis of a β-secretase site of a β-amyloid precursor protein, treat Alzheimer's disease and decrease β-amyloid protein. Advantages of the claimed invention include, for example, the selectivity of compounds for inhibiting memapsin 2 activity relative to the activity memapsin 1 activity, thereby providing a specific inhibitor for β-secretase and treatment of diseases or conditions associated with β-secretase activity. The claimed methods, by employing memapsin 2 inhibitors, provide methods to inhibit a biological reaction which is involved in the accumulation or production of β-amyloid protein, a phenomenon associated with Alzheimer's disease in humans.
Thus, the compounds of the invention can be employed in the treatment of diseases or conditions associated with β-secretase activity, which can halt, reverse or diminish the progression of the disease or condition, in particular Alzheimer's disease.
The features and other details of the invention, either as steps of the invention or as combinations of parts of the invention, will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention. The teachings of all of the references cited herein are incorporated by reference in their entirety.
The term “aliphatic” as used herein means straight-chain, branched C1-C12 or cyclic C3-C12 hydrocarbons which are completely saturated or which contain one or more units of unsaturation but which are not aromatic. For example, suitable aliphatic groups include substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. The terms “alkyl”, used alone or as part of a larger moiety, includes both straight, branched, or cyclic saturated hydrocarbon chains containing one to twelve carbon atoms. Preferably, alkyl groups are straight chain hydrocarbons having from one to about four carbons.
An alkylene, as used herein, is an alkyl group that has two points of attachment to another moiety, such as methylene.
A heteroalkyl, as used herein, is an alkyl group in which one or more carbon atoms is replaced by a heteroatom. A preferred heteroalkyl is methoxymethoxy.
A hydroxyalkyl, as used herein, is an alkyl group that is substituted with one or more hydroxy groups.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl” or “aralkoxy”, are carbocyclic aromatic ring systems (e.g. phenyl), fused polycyclic' aromatic ring systems (e.g., naphthyl and anthracenyl) and aromatic ring systems fused to carbocyclic non-aromatic ring systems (e.g., 1,2,3,4-tetrahydronaphthyl and indanyl) having five to about fourteen carbon atoms.
The term “heteroatom” refers to any atom other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur, and phosphorus and includes, for example, any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen.
The term “heterocycle”, as used herein includes non-aromatic ring systems having five to fourteen members, preferably five to ten, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom. Examples of heterocyclic rings include, tetrahydrofuranyl, tetrahydropyrimidin-2-one, pyrrolidin-2-one, hexahydro-cyclopenta[b]furanyl, hexahydrofuro[2,3-b]furanyl, tetrahydropyranyl, tetrahydropyranone, [1,3]-dioxanyl, [1,3]-dithianyl, tetrahydrothiophenyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, pyrrolidinone, piperazinyl, piperidinyl, and thiazolidinyl. Also included within the scope of the term “heterocycle”, as it is used herein, are groups in which a non-aromatic heteroatom-containing ring is fused to one or more aromatic or non-aromatic rings, such as in an indolinyl, chromanyl, phenantrhidinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring. Preferred heterocycles are tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, tetrahydropyrimidin-2-one, and pyrrolidin-2-one.
The term “heteroaryl”, used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy”, refers to aromatic ring system having five to fourteen members and having at least one heteroatom. Preferably a heteroaryl has from one to about four heteroatoms. Examples of heteroaryl rings include pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidinyl, purinyl, pyridazinyl, pyrazinyl, thiazolyl, thiadiazolyl, isothiazolyl, triazolyl, thienyl, 4,6-dihydro-thieno[3,4-c]pyrazolyl, 5,5-dioxide-4,6-dihydrothieno[3,4-c]pyrazolyl, thianaphthenyl, 1,4,5,6,-tetrahydrocyclopentapyrazolyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, azaindolyl, indazolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, and benzoisazolyl. Preferred heteroaryl groups are pyrazolyl, furanyl, pyridyl, quinolinyl, indolyl and imidazolyl.
A heteroazaaryl is a heteroaryl in which at least one of the heteroatoms is nitrogen. Preferred heteroazaaryl groups are pyrazolyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, benzimidazolyl, quinolinyl, benzotriazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, and benzoisazolyl. Pyrazolyl is a most preferred heteroazaaryl.
An aralkyl group, as used herein, is an aryl substituent that is linked to a compound by a straight chain or branched alkyl group having from one to twelve carbon atoms. Preferred aralkyl groups are benzyl and indanylmethyl.
An heterocycloalkyl group, as used herein, is a heterocycle substituent that is linked to a compound by a straight chain or branched alkyl group having from one to twelve carbon atoms. Preferred heterocycloalkyl groups are tetrahydrofuranylmethyl and pyrrolidinylmethyl.
An heteroaralkyl group, as used herein, is a heteroaryl substituent that is linked to a compound by a straight chain or branched alkyl group having from one to twelve carbon atoms. Preferred heteroaralkyl groups are pyrazolylmethyl, 2-pyrazolylethyl, 2-pyrazolyl-1-methylethyl, and 2-pyrazolyl-1-isopropylethyl.
An alkoxy group, as used herein, is a straight chain or branched or cyclic C1-C12 or a cyclic C3-C12 alkyl group that is connected to a compound via an oxygen atom. Examples of alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, isopropoxy, and t-butoxy.
A heterocyclooxy, as used herein, is a heterocyclic group that is attached to a molecule via an oxygen substituent.
A aralkoxy group, as used herein, is a aralkyl group that is attached to a compound via an oxygen substituent on the C1-C12 alkyl portion of the aralkyl. A preferred arylalkoxy is phenylmethoxy.
A heteroaralkoxy group, as used herein, is a heteroaralkyl group that is attached to a compound via an oxygen substituent on the C1-C12 alkyl portion of the heteroaralkyl. A preferred arylalkoxy are pyrazolylmethoxy and 2-pyrazolylethoxy.
A heterocycloalkoxy group, as used herein, is a heterocycloalkyl group that is attached to a compound via an oxygen substituent on the C1-C12 alkyl portion of the heteroaralkyl.
An alklysulfanylalkyl group, as used herein, is a sulfur atom that is linked to two C1-C12 alkyl groups, wherein one of the alkyl groups is also linked to a compound.
A halogen is a —F, —Cl, —Br, or —I.
A haloalkyl is an alkyl group that is substituted by one or more halogens.
A haloalkoxy is an alkoxy group that is substituted with one or more halogens.
An aryl (including aralkyl, aralkoxy and the like) or heteroaryl (including heteroaralkyl and heteroaralkoxy and the like) may contain one or more substituents. Examples of suitable substituents include aliphatic groups, aryl groups, haloalkoxy groups, heteroaryl groups, halo, hydroxy, OR24, COR24, COOR24, NHCOR24, OCOR24, benzyl, haloalkyl (e.g., trifluoromethyl and trichloromethyl), cyano, nitro, SO3−, SH, SR24, NH2, NHR24, NR24R25, NR24S(O)2—R25, and COOH, wherein R24 and R25 are each, independently, an aliphatic group, an aryl group, or an aralky group. Other substituents for an aryl or heteroaryl group include —R26, —OR26, —SR26, 1,2-methylene-dioxy, 1,2-ethylenedioxy, protected OH (such as acyloxy), phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH2(Ph), substituted —CH2CH2(Ph), substituted —CH2CH2(Ph), —NR26R27, —NR26CO2R27, —NR26NR27C(O)R28, —NR26R27C(O)NR28R29, —NR26NR27CO2R28, —C(O)C(O)R26, —C(O)CH2C(O)R26, —CO2R26, —C(O)R26, —C(O)NR26R27, —OC(O)NR16R27, —S(O)2R26, —SO2NR26R27, —S(O)R26, —NR26SO2NR26R27, —NR26SO2R27, —C(═S)NR26R27, —C(═NH)—NR26R27, —(CH2)yNHC(O)R26, wherein R26, R27 and R28 are each, independently, hydrogen, a substituted or unsubstituted heteroaryl or heterocycle, phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH2 (Ph), or substituted —CH2 (Ph); and y is 0-6. Examples of substituents on the aliphatic group or the phenyl group include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl. Preferred substitutents for a heteroaryl group such as a pyrazole group, are a substituted or unsubstituted aliphatic group, —OR9, —R23—O—R9, a halogen, a cyano, a nitro, NR9R10, guanidino, —OPO3−2, —PO3−2, —OSO3−, —S(O)pR9, —OC(O)R9, —C(O)R9, —C(O)2R9, —NR9C(O)R10, —C(O)NR9R10, —OC(O)NR9R10, —NR9C(O)2R10 a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted heterocycloalkyl, wherein R9 and R10 are each, independently, H, an aliphatic group, an aryl, an aralkyl, a heterocycle, a heterocycloalkyl, a heteroaryl or a heteroaralkyl, wherein the aliphatic group, aryl, aralkyl, heterocycle, heterocyclalkyl, heteroaryl or heteroaralkyl are optionally substituted with one or more aliphatic groups.
An aliphatic group, an alkylene, the carbon atoms of a heteroalkyl, and a heterocycle (including heterocycloalkyl, heterocyclooxy, and heterocycloalkoxy) may contain one or more substituents. Examples of suitable substituents on the saturated carbon of an aliphatic group of a heterocycle include those listed above for an aryl or heteroaryl group and the following: ═O, ═S, ═NNHR29, ═NNR29R30, ═NNHC(O)R29, ═NNHCO2(alkyl), ═NNHSO2(alkyl), or ═NR29, where each R29 and R30 are each, independently, selected from hydrogen, an unsubstituted aliphatic group or a substituted aliphatic group. Examples of substituents on the aliphatic group include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, thioalkyl, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl.
Suitable substitutents on the nitrogen of a non-aromatic heterocycle or on an unsaturated nitrogen of a heteroaryl include —R31, —NR31R32, —C(O)R31, —CO2R31, —C(O) C(O)R31, C(O)CH2C (O)R31, —SO2R31, —SO2NR31R32, —C(═S)NR31R32, —C(═NH)—NR31R32, and —NR31SO2R32; wherein R31 and R32 are each, independently, hydrogen, an aliphatic group, a substituted aliphatic group, phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH2(Ph), or a heteroaryl or heterocycle. Examples of substituents on the aliphatic group or the phenyl ring include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or haloalkyl.
A hydrophobic group is a group that does not reduce the solubility of a compound in octane or increases the solubility of a compound in octane. Examples of hydrophobic groups include aliphatic groups, aryl groups, and aralkyl groups.
As used herein, the term “natural amino acid” refers to the twenty-three natural amino acids known in the art, which are as follows (denoted by their three letter acronym): Ala, Arg, Asn, Asp, Cys, Cys-Cys, Glu, Gln, Gly, H is, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. The term “side-chain of an amino acid”, as used herein, is the substituent on the alpha-carbon of a natural amino acid.
The term “non-natural amino acid” refers to compounds of the formula NH2—C(R32)2—COOH, where R32 for each occurrence is, independently, any side chain moiety recognized by those skilled in the art; examples of non-natural amino acids include, but are not limited to: hydroxyproline, homoproline, 4-amino-phenylalanine, norleucine, cyclohexylalanine, α-aminoisobutyric acid, N-methyl-alanine, N-methyl-glycine, N-methyl-glutamic acid, tert-butylglycine, α-aminobutyric acid, tert-butylalanine, ornithine, α-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid, etc. and the derivatives thereof, especially where the amine nitrogen has been mono- or di-alkylated.
A peptide substituent is a sequence of natural or non-natural amino acids that are linked together via an amide bond which is formed by reaction of the α-amine of one amino acid with the a-carboxylic acid of an adjacent amino acid. Preferably, a peptide sequence includes only natural amino acids. In one embodiment, a peptide substituent is a sequence of about 6 natural amino acids. In another embodiment, a peptide substituent is a sequence of 2 natural amino acids. In yet another embodiment, a peptide substituent is 1 natural amino acid.
A “transition state isostere,” or “isostere,” as used herein, is a compound having a sequence of two or more natural or non-natural amino acids, wherein at least one amide linkage between two consecutive amino acids has been modified such that the —NH— group of the amide has been replaced with a —CH2— and the carbonyl of the amide group has been replaced with a —CH(OH)—. This isostere is also referred to herein as a “hydroxyethylene isostere” because the amide linkage between a pair of amino acids of a peptide is modified to form a hydroxyethylene linkage between the amino acids. A hydroxyethylene group is an isostere of the transition state of hydrolysis of an amide bond. Preferably, an isostere has only one modified amide linkage. The hydroxyethylene component of a peptide isostere is also referenced herein as “*” or “Ψ”. For example, the representation of the di-isostere Leucine*Alanine, Leu*Ala, L*A, or LΨA each refer to the following structure:
where the boxed portion of the molecule represents the hydroxyethylene component of the molecule.
“Binding pockets” or “binding subsites” or subsites” refer to locations in an enzyme or protease that interact with functional groups or side chains of a compound or substrate bond thereto. The subsites in memapsin 1 and memapsin 2 are labeled Sq and Sq′ and interact with or otherwise accommodate side chains Pq and Pq∝ of a peptide substrate or peptide isostere, such as the compounds of the invention, such that the Pq side chain of the peptide substrate or peptide inhibitor interact with amino acid residues in the Sq subsite of the enzyme. q is an integer that increases distally relative to the scissile bond of a peptide substrate that is cleaved by the enzyme or relative to the hydroxyethyl group of a hydroxyethyl isosteric inhibitor, such as the compounds of the invention, according to the nomenclature of Schecter and Berger (Schechter, I., Berger, A Biochem. Biophys. Res. Commun. (1967), 27:157-162). The composition of a subsite is a listing of the amino acids of the enzyme or protease which are within an interacting distance of the compound when the compound is bound to the subsite, or otherwise form a contiguous solvent accessible surface, indicated by their numbers in the amino acid sequence. Representative references to aspartic protease subsites include: Davies, D. R., Annu. Rev. Biophys. Biophys. Chem., 19:189-215 (1990) and Bailey, D. and Cooper, J. B., Protein Science, 3:2129-2143 (1994), the teachings of which are incorporated herein by reference in their entirety. More specifically, a subsite is defined by defining a group of atoms of the enzyme which represent a contiguous or noncontiguous surface that is accessible to a water molecule, with that surface having the potential for an interaction with a functional group or side chain of a peptide substrate or a peptide inhibitor, such as the compounds of the invention, when the peptide substrate or a peptide inhibitor is bound to the subsite.
An “interacting distance” is defined as a distance appropriate for van der Waals interactions, hydrogen bonding, or ionic interactions, as described in fundamental texts, such as Fersht, A., “Enzyme Structure and Mechanism,” (1985), W.H. Freeman and Company, New York. Generally, atoms within 4.5 Å of each other are considered to be within interacting distance of each other.
In many of the compounds of the invention, the amino acid residues whose side chains would be labeled P3, P4, etc. when using the above nomenclature have been replaced by a chemical group that is not an amino acid. Thus, in the compounds of formulas II, IV and VII, R1 together with X, R19 together with X, and R18 together with X, respectively, may include amino acid residues but also include other chemical groups as defined above (see definition of R1, R19 and R18). When R1 together with X, R19 together with X, or R18 together with X, in the compounds of the invention is a peptide group, the side chains of the peptide group are labeled P3, P4, etc. and bind in the enzyme subsites S3 and S4 as in the nomenclature described above. When R1 together with X, R19 together with X, or R18 together with X, in the compounds of the invention is a non-peptide moiety, these groups may also bind in the S3 and/or S4 subsite of the enzyme.
A “substrate” is a compound that may bind to the active site cleft of the enzyme according to the following scheme:
In the above reaction scheme, “E” is an enzyme, “S” is a substrate, and “E.S” is a complex of the enzyme bound to the substrate. Complexation of the enzyme and the substrate is a reversible reaction.
Compounds of Formulas II, IV, VII and the compounds in Table 1 may exist as salts with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates, (−)-tartrates or mixtures thereof including racemic mixtures, succinates, benzoates and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.
Certain compounds of Formulas II, IV, VII and the compounds in Table 1 which have acidic substituents may exist as salts with pharmaceutically acceptable bases. The present invention includes such salts. Example of such salts include sodium salts, potassium salts, lysine salts and arginine salts. These salts may be prepared by methods known to those skilled in the art.
Certain compounds of Formulas II, IV, VII and the compounds in Table 1 may contain one or more chiral centres, and exist in different optically active forms. When compounds of Formulas II, IV, VII or the compounds in Table 1 contain one chiral centre, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as racemic mixtures. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may be separated, for example, by crystallization; formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.
When a compound of Formulas II, IV, VII or a compound in Table 1 contain more than one chiral center, it may exist in diastereoisomeric forms. The diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. The present invention includes each diastereoisomer of compounds of Formula I and mixtures thereof.
Certain compounds of Formulas II, IV, VII and the compounds in Table 1 may exist in zwitterionic form and the present invention includes each zwitterionic form of compounds of Formula (I) and mixtures thereof.
In a preferred embodiment, the compounds of Formula II or IV, separately or with their respective pharmaceutical compositions, have an R1 or R19, respectively, group that together with X is an natural or non-natural amino acid derivative. The compounds of this embodiment are preferably represented by Formula IX:
In Formula IX, P1, P2, P1′, P2′, R2, R3 and R4 are defined as in Formula II. R16 is defined as in Formula IV, and R17 is a substituted or unsubstituted aliphatic group.
In another preferred embodiment, the compounds of Formula II or VII, separately or with their respective pharmaceutical compositions, have an R1 or R18 group, respectively, that is a substituted or unsubstituted heteroaralkoxy or a substituted or unsubstituted heteroaralkyl. The compounds of this embodiment are preferably represented by Formula X:
In Formula X, P1, P2, P1′, P2′, R2, R3 and R4 are defined as in Formula II. X1 is —O—, —NR22— or a covalent bond. R7 is a substituted or unsubstituted alkylene. m is 0, 1, 2, or 3. R8 is a substituted or unsubstituted aliphatic group, —OR9, —R23—O—R9, a halogen, a cyano, a nitro, NR9R10, guanidino, —OPO3−2, —PO3−2, —OSO3−, —S(O)pR9, —OC(O)R9, —C(O)R9, —C(O)2R9, —NR9C(O)R10, —C(O)NR9R10, —OC(O)NR9R10, —NR9C(O)2R10 a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heteroaralkyl, a substituted or unsubstituted heterocycle, or a substituted or unsubstituted heterocycloalkyl. p is 0, 1 or 2. R9 and R10 are defined as in Formula IV. R23 is a substituted or unsubstituted alkylene. R22 is —H. Alternatively, R and R22, together with X and the nitrogen atoms to which they are attached, form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle or substituted or unsubstituted heteroaryl ring.
In one preferred embodiment, R1 of Formula II is —OR15 or —NR15R16. R15 and R16 are defined as in Formula IV.
In another preferred embodiment, R1 of Formula II is a substituted aliphatic group. More preferably, R1 is an aliphatic group that is substituted with one or more substituents selected from the group consisting of —NR15C(O)2R16, —NR15C(O)R16, and —NR15S(O)2R16. R15 and R16 are defined as in Formula IV.
In another preferred embodiment, R1 of Formula II together with X is a peptide represented by Formula XI:
In Formula XI, P3 and P4 are each, independently, an amino acid side chain. P5 is an amino acid side chain selected from the group consisting of tryptophan side chain, methionine side chain, and leucine side chain. P6 is tryptophan side chain. P7 is an amino acid side chain selected from the group consisting of tryptophan side chain, tyrosine side chain; and glutamate side chain. P8 is an amino acid side chain selected from the group consisting of tryptophan side chain, tyrosine side chain; and glutamate side chain. More preferably, P5, P6, P7, and P8 are each a tryptophan side chain.
In another preferred embodiment, P1 of Formula II, IV, or VII is an aliphatic group. More preferably, P1 is selected from the group consisting of isobutyl, hydroxymethyl, cyclopropylmethyl, cyclobutylmethyl, phenylmethyl, cyclopentylmethyl, and heterocycloalkyl.
In another preferred embodiment, P2′ of Formula II, IV, or VII is a hydrophobic group. More preferably, P2′ is isopropyl or isobutyl.
In another preferred embodiment, P2 of Formula II, IV, or VII is a hydrophobic group. More preferably, P2 is —R11SR12, —R11S(O)R12, —R11S (O)2R12, —R11C(O)NR12R13, —R11OR12, —R11OR14OR13, or a heterocycloalkyl, wherein the heterocycloalkyl is optionally substituted with one or more alkyl groups. R11 and R14 are each, independently, an alkylene. R12 and R13 are each, independently, H, an aliphatic group, an aryl, an aralkyl, a heterocycle, a heterocyclalkyl, a heteroaryl, or a heteroaralkyl. Even more preferably, P2 is —CH2CH2SCH3, —CH2CH2S(O)CH3, —CH2CH2S(O)2CH3, —CH2C(O)NH2, —CH2C(O)NHCH2CH═CH2, tetrahydrofuran-2-yl, tetrahydrofuran-2-yl-methyl, tetrahydrofuran-3-yl, tetrahydrofuran-3-yl-methyl, pyrrolidin-2-yl-methyl, pyrrolidin-3-yl-methyl, or —CH2CH2OCH2OCH3.
In another preferred embodiment, R2 is H and R3 together with the nitrogen to which it is attached is a peptide in Formula II, IV or VII.
In another preferred embodiment, R2 is H and R3 is selected from the group consisting of 2-furanylmethyl, phenylmethyl, indan-2-yl, n-butyl, isopropyl, isobutyl, 1-fluoromethyl-2-fluoroethyl, indol-3-yl, and 3-pyridylmethyl in Formula II, IV or VII.
In another preferred embodiment, R2 and R3 in Formula II, IV or VII, together with the nitrogen to which they are attached, form morpholino, piperazinyl or piperidinyl, wherein the morpholino, piperazinyl and piperidinyl are optionally substituted with one or more aliphatic groups.
In another embodiment, R1 of formula II is a substituted or unsubstituted heteroaralkoxy or a substituted or unsubstituted heteroaralkyl.
In another preferred embodiment, R1 of Formula I or R18 or Formula VII is a substituted or unsubstituted heteroaralkoxy or a substituted or unsubstituted heteroaralkyl in which the heteroaryl group of the heteroaralkoxy or heteroaralkyl is selected from the group consisting of substituted or unsubstituted pyrazolyl, substituted or unsubstituted furanyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted thienyl, substituted or unsubstituted 4,6-dihydro-thieno[3,4-c]pyrazolyl, substituted or unsubstituted 5,5-dioxide-4,6-dihydrothieno[3,4-c]pyrazolyl, substituted or unsubstituted thianaphthenyl, substituted or unsubstituted carbazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted benzofuranyl, substituted or unsubstituted indolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzooxazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted isoindolyl, substituted or unsubstituted acridinyl, and substituted or unsubstituted benzoisazolyl. In a more preferred embodiment, R1 of Formula I or R18 or Formula VII is a substituted or unsubstituted heteroaralkoxy or a substituted or unsubstituted heteroaralkyl in which the heteroaryl group of the heteroaralkoxy or heteroaralkyl is a heteroazaaryl. In an even more preferred embodiment, the heteroazaaryl is selected from the group consisting of substituted or unsubstituted pyrazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzooxazolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoindolyl, and substituted or unsubstituted benzoisazolyl.
In another preferred embodiment, the compounds of the invention do not include a carrier molecule. In this embodiment, k is 0 and r is 1 in Formula I, III, V, or VIII.
In another preferred embodiment of the invention, k is 1 and r is 1 in Formula I, III, V, or VIII. In this embodiment, each isosteric inhibitor is attached to one carrier molecule.
The compounds of the invention (also referred to herein as “an inhibitor(s)” or “an inhibitor compound(s)”) are referenced by a number. The inhibitors are also referred to as “GT-1” followed by a numeric designation (e.g., GT-1138), “MM” followed by a numeric designation (e.g., MM 138), “MMI” followed by a numeric designation (e.g., MMI-138) or “OM” followed by a numeric designation (e.g., OM-138). The designations “GT-1,” “MM,” “MMI” and “OM,” as described herein, are equivalent. Likewise, use of the numerical value following the designation “GT-1,” “MM,” “MMI” and “OM” without “GT-1,” “MM,” “MMI” and “OM” refer to the same compound with the “GT-1,” “MM,” “MMI” and “OM” prefix. Thus, for example, “GT-1138,” “MM 138,” “MMI-138,” “OM-138” and “138” refer to the same inhibitor compound.
In another embodiment, the invention includes a method of selectively inhibiting memapsin 2 β-secretase activity relative to memapsin 1 β-secretase activity, comprising the step of administering the compounds of the invention. The selective inhibition of memapsin 2 β-secretase activity compared to memapsin 1 β-secretase activity can be in an in vitro sample or in a mammal.
“Selectively inhibiting” or “selective inhibition,” as used herein, refers to a greater ability of a compound of the invention to inhibit, prevent or diminish the β-secretase activity of memapsin 2 than the ability of the same compound to inhibit, prevent or diminish β-secretase activity of memapsin 1 under the same conditions, as measured by the percent inhibition (“% inh”) of each. “Percent inhibition” is calculated as follows: % inh=(1−Vi/Vo)×100. For example, as shown in
“Relative to memapsin 1,” as used herein, refers to the β-secretase activity of memapsin 2 compared to the β-secretase activity of memapsin 1. The ability of an inhibitor compound of the invention to inhibit β-secretase activity can be assessed by determining the extent to which a compound inhibits memapsin 2 cleaving of a β-secretase site of a β-amyloid precursor protein compared to the extent to which the same compound inhibits memapsin 1 cleaving of a β-secretase site of a β-amyloid precursor protein. These data can be expressed, for example, as Ki, Ki apparent, Vi/Vo, or percentage inhibition and depict the inhibition of a compound for memapsin 2 β-secretase activity relative to memapsin 1 β-secretase activity. For example, if the Ki of a reaction between an inhibitor compound of the invention and memapsin 1 is 1000 and the Ki of a reaction between an inhibitor compound of the invention and memapsin 2 is 100, the inhibitor compound inhibits the β-secretase activity of memapsin 2 ten (10) fold, relative to memapsin 1.
Ki is the inhibition equilibrium constant which indicates the ability of compounds to inhibit the β-secretase activity of memapsin 2 and memapsin 1. Numerically lower Ki values indicate a higher affinity of the compounds of the invention for memapsin 2 or memapsin 1. The K1 value is independent of the substrate, and converted from Ki apparent.
Ki apparent is determined in the presence of substrate according to established techniques (see, for example, Bieth, J., Bayer-Symposium V: Proteinase Inhibitors, pp. 463-469, Springer-Verlag, Berlin (1994)).
Vi/Vo depicts the ratio of initial cleavage velocities of the substrate FS-2 (Ermolieff, et al., Biochemistry 40:12450-12456 (2000)) by memapsin 1 or memapsin 2 in the absence (Vo) or presence (Vi) of a compound of the invention. A Vi/Vo value of 1.0 indicates that a compound of the invention does not inhibit the β-secretase activity of the enzyme memapsin 1 or memapsin 2. A Vi/Vo value less than 1.0 indicates that a compound of the invention inhibits β-secretase activity of the enzyme memapsin 1 or memapsin 2. The Vi/Vo values depicted in Table 1 were determined at conditions under which the enzyme and inhibitor concentrations were equal (e.g., about 80 nM, 100 nM).
Where stereochemistry is not shown, the compound is a mixture of isomers.
N.I., no inhibition.
The standard error for the Ki apparent is the error from the nonlinear regression of the Vi/Vo data measured at different concentrations of the compounds of the invention (e.g., between about 10 nM to about 1000 nM) employing well-known techniques (see, for example, Bieth, J., Bayer-Symposium V: Proteinase Inhibitors, pp. 463-469, Springer-Verlag, Berlin (1994)).
The Kiapp (apparent IQ values of inhibitors against memapsins 1 and 2 were determined employing previously described procedures (Ermolieff, J., et al., Biochemistry 39:12450-12456 (2000), the teachings of which are incorporated herein by reference in their entirety). The relationship of Ki (independent of substrate concentration) to Kiapp is a function of substrate concentration in the assay and the Km for cleavage of the substrate by either memapsin 1 or memapsin 2 by the relationship: Kiapp=Ki (1+[S]/Km).
“Memapsin 1” or “memapsin 1 protein,” as defined herein, refers to a protein that includes amino acids 58-461 of SEQ ID NO: 4 (SEQ ID NO: 64).
In one embodiment, memapsin 1 includes a transmembrane protein (SEQ ID NO: 2 (
Constructs encoding memapsin 1 can be expressed in host cells (e.g., mammalian host cells such as HeLa cells or 293 cells or E. coli host cells). The nucleic acid sequence encoding the promemapsin 1-T1 (SEQ ID NO: 3 (
A nucleic acid construct encoding promemapsin 1-T1 (SEQ ID NO: 4 (FIG. 7)) was expressed in E. coli, the protein purified from inclusion bodies and autocatalytically activated by incubation at pH 3-5 for 30 minutes (37° C.) to obtain memapsin 1 with an amino terminus of alanine (amino acid residue 58 of SEQ ID NO: 4 (FIG. 7)), which was employed in assays to assess the inhibition of memapsin 2 relative to memapsin 1 by compounds of the invention.
“Memapsin 2” or “memapsin 2 protein,” is any protein that includes an amino acid sequence identified herein that includes the root word “memapsin 2,” or any sequence of amino acids, regardless of whether it is identified with a SEQ ID NO, that is identified herein as having been derived from a protein that is labeled with a term that includes the root word memapsin 2 (e.g., amino acid residues 1-456 of SEQ ID NO: 8, amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60), amino acid residues 27-456 of SEQ ID NO: 8 (SEQ ID NO: 61), amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) and amino acid residues 45-456 of SEQ ID NO: 8 (SEQ ID NO: 63); and the various equivalents derived from SEQ ID NO: 9) and can hydrolyze a peptide bond. Generally, memapsin 2 is capable of cleaving a β-secretase site (e.g., the Swedish mutation of APP SEVNLDAEFR, SEQ ID NO: 11; the native APP SEVKMDAEFR, SEQ ID NO: 12). In one embodiment, memapsin 2 consists essentially of an amino acid sequence that results from activation, such as spontaneous activation, autocatalytic activation, or activation with a protease, such as clostripain, of a longer sequence. Embodiments of memapsin 2 that consist essentially of an amino acid fragment that results from such activation are those that have the ability to hydrolyze a peptide bond. Crystallized forms of memapsin 2 are considered to continue to be memapsin 2 despite any loss of β-secretase activity during crystallization. Embodiments of memapsin 2 are also referred to as BACE, ASP2 and β-secretase.
In one embodiment, memapsin 2 is a transmembrane protein (SEQ ID NO: 6 (
In another embodiment, memapsin 2 is promemapsin 2-T1 (nucleic acid sequence SEQ ID NO: 7 (
The nucleic acid construct of the resulting promemapsin 2-T1 SEQ ID NO: 7 (
It is also envisioned that promemapsin 2-T1 can be expressed in E. coli and autocatalytically activated to generate memapsin 2 which includes amino acid residues 16-456 of SEQ ID NO: 8 (SEQ ID NO: 60) (
A memapsin 2 including amino acid residues 60-456 of SEQ ID NO: 8 (SEQ ID NO: 67) (
Compounds that selectively inhibit memapsin 2 β-secretase activity relative to memapsin 1 β-secretase activity are useful to treat diseases or conditions or biological processes association with memapsin 2 β-secretase activity rather than diseases or conditions or biological processes associated with both memapsin 1 and memapsin 2 β-secretase activity. Since both memapsin 1 and memapsin 2 cleave amyloid precursor protein (APP) at a β-secretase site to form β-amyloid protein (also referred to herein as Aβ, Abeta or β-amyloid peptide), memapsin 1 and memapsin 2 have β-secretase activity (Hussain, I., et al., J Biol. Chem. 276:23322-23328 (2001), the teachings of which are incorporated herein in their entirety). However, the β-secretase activity of memapsin 1 is significantly less than the β-secretase activity of memapsin 2 (Hussain, I., et al., J. Biol. Chem. 276:23322-23328 (2001), the teachings of which are incorporated herein in their entirety). Memapsin 2 is localized in the brain, and pancreas, and other tissues (Lin, X., et al., Proc. Natl. Acad. Sci. USA 97:1456-1460 (2000), the teachings of which are incorporated herein in their entirety) and memapsin 1 is localized preferentially in placentae (Lin, X., et al., Proc. Natl. Acad. Sci. USA 97:1456-1460 (2000), the teachings of which are incorporated herein in their entirety). Alzheimer's disease is associated with the accumulation of Aβ in the brain as a result of cleaving of APP by β-secretase (also referred to herein as memapsin 2, ASP2 and BACE). Thus, methods employing the compounds which selectively inhibit memapsin 2 β-secretase activity relative to memapsin 1 β-secretase activity are important in the treatment of memapsin 2-related diseases, such as Alzheimer's disease. Selective inhibition of memapsin 2 β-secretase activity makes the compounds of the invention suitable drug candidates for use in the treatment of Alzheimer's disease.
In yet another embodiment, the invention is a method of treating Alzheimer's disease in a mammal (e.g., a human) comprising the step of administering to the mammal the compounds of the invention. The mammals treated with the compounds of the invention can be human primates, nonhuman primates and non-human mammals (e.g., rodents, canines). In one embodiment, the mammal is administered a compound that inhibits β-secretase (inhibits memapsin 1 and memapsin 2 β-secretase activity). In another embodiment, the mammal is administered a compound that selectively inhibits memapsin 2 β-secretase activity and has minimal or no effect on inhibiting memapsin 1 β-secretase activity.
In an additional embodiment, the invention is a method of inhibiting hydrolysis of a β-secretase site of a β-amyloid precursor protein in a mammal, comprising the step of administering to the mammal the compounds of the invention.
A “β-secretase site” is an amino acid sequence that is cleaved (i.e., hydrolyzed) by memapsin 1 or memapsin 2 (also referred to herein as β-secretase and ASP2). In a specific embodiment, a β-secretase site is an amino acid sequence cleaved by a protein having the sequence 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) (
In another embodiment, the compounds of the invention are administered to a mammal to inhibit the hydrolysis of a β-secretase site of a β-amyloid precursor protein. In another embodiment, the compounds are administered to an in vitro sample to inhibit the hydrolysis of a β-secretase site of a β-amyloid precursor protein.
“In vitro sample,” as used herein, refers to any sample that is not in the entire mammal. For example, an in vitro sample can be a test tube in vitro combination of memapsin 2 and an inhibitor compound of the invention; or can be an in vitro cell culture (e.g., Hela cells, 293 cells) to which the inhibitor compounds and/or memapsin proteins (memapsin 1 or 2) are added.
In a further embodiment, the invention is a method of decreasing the amount or production of β-amyloid protein in an in vitro sample or a mammal comprising the step of administering the compounds of the invention. The amount of β-amyloid protein or a decrease in the production of β-amyloid protein can be measured using standard techniques including western blotting and ELISA assays. A decrease in β-amyloid protein or a decrease in the production of β-amyloid protein can be measured, for example, in cell culture media in an in vitro sample or in a sample obtained from a mammal. The sample obtained from the mammal can be a fluid sample, such as a plasma or serum sample; or can be a tissue sample, such as a brain biopsy.
The compounds of the invention can be administered with or without a carrier molecule. “Carrier molecule,” as used herein, refers to a cluster of atoms held together by covalent bonds (the molecule) that are attached or conjugated to a compound or compounds of the invention. To penetrate the blood brain barrier (BBB), the carrier molecule must be relatively small (e.g., less than about 500 daltons) and relatively hydrophobic. The compounds of the invention may be attached or conjugated to the carrier molecule by covalent interactions (e.g., peptide bonds) or by non-covalent interactions (e.g., ionic bonds, hydrogen bonds, van der Waals attractions). In addition, carrier molecules may be attached to any functional group on a compound of the invention. For example, a carrier molecule may be attached to an amine group at the amine terminus of a peptide inhibitor of the invention. For example, R1 of Formula II may be a carrier molecule. A carrier molecule may be attached to a carboxylic acid group at the carboxylic acid terminus of a peptide inhibitor of the invention. For example, NR3R3 of Formula II may be a carrier molecule. Alternatively, the carrier molecule may be attached to a side chain (e.g., P1, P2, P3, P4, P5, P6, P7, P8, P1′, P2′, P3′, P4′, etc.) of an amino acid residue that is a component of the compounds of the invention.
The confocal microscopic images of cells incubated with CPI-1 revealed that inhibitors of the invention were not evenly distributed inside the cells. Some high fluorescence intensity was associated with intracellular vesicular structures including endosomes and lysosomes. These images indicated that the inhibitor was trapped inside of these subcellular compartments. This indicated that when CPI-1 enters lysosomes and endosomes, the carrier peptide moiety, in this case tat, was modified by proteases within lysosome or endosome resulting in an inhibitor that was unable to exit the lysosomal or endosomal compartment.
Lysosomes and endosomes contain many proteases, including hydrolase such as cathepsins A, B, C, D, H and L. Some of these are endopeptidase, such as cathepsins D and H. Others are exopeptidases, such as cathepsins A and C, with cathepsin B capable of both endo- and exopeptidase activity. The specificities of these proteases are sufficiently broad to hydrolyze a tat peptide away from the inhibitor compound, thus, hydrolyzing the carrier peptide away from the isosteric inhibitor.
These facts make it possible to use tat and other carrier peptides for specific delivery of pharmaceutical agents, such as the compound of Formula II, IV, VII, or a compound in Table 1 to lysosomes and endosomes. For example, a compound of Formula II, IV, VII or a compound in Table 1 to be delivered is chemically linked to a carrier peptide like tat to make a conjugated drug. When administered to a mammal by a mechanism such as injections, the conjugated compound will penetrate cells and permeate to the interior of lysosomes and endosomes. The proteases in lysosomes and endosomes will then hydrolyze tat. The conjugated compound will lose its ability to escape from lysosomes and endosomes.
The carrier peptide can be tat or other basic peptides, such as oligo-L-arginine, that are hydrolyzable by lysosomal and endosomal proteases. Specific peptide bonds susceptible for the cleavage of lysosomal or endosomal proteases may be installed in the linkage peptide region between a compound of Formula II, IV, VII or a compound in Table 1 and the carrier peptides. This will facilitate the removal of carrier peptide from the compound. For example, dipeptides Phe-Phe, Phe-Leu, Phe-Tyr and others are cleaved by cathepsin D.
Furthermore, the dissociable carrier molecule may be an oligosaccharide unit or other molecule linked to the compound by phosphoester or lipid-ester or other hydrolyzable bonds which are cleaved by glycosidases, phosphatases, esterases, lipases, or other hydrolases in the lysosomes and endosomes.
This type of drug delivery may be used to deliver the inhibitors of the invention to lysosomes and endosomes where memapsin 2 is found in high concentrations. This drug delivery system may also be used for the treatment of diseases such as lysosome storage diseases.
In one embodiment, the carrier molecule is a peptide, such as the tat-peptide Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 13) (Schwarze, S. R., et al., Science 285:1569-1572 (1999), the teachings of which are incorporated herein in their entirety) or nine arginine residues Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 14) (Wender, P. A., et al., Proc. Natl. Acad. Sci. USA 97:13003-13008 (2000), the teachings of which are incorporated herein in their entirety). In another embodiment, the carrier molecule includes cationic molecules (i.e., molecules that are ionized at physiologic pH) and preferably polycationic molecules. Preferred functional groups that form cations include guanidine, amino, or imidizole. Carrier molecules include saccharides or lipids that contain about 1-10 of the following functional groups: guanidine, amino, or imidizole. Carrier molecules also include peptides of length about 10 amino acids, consisting of a combination of about 1-10 lysine, 1-10 arginine, or 1-10 histidine residues, or 1-10 residues of amino acids that contain the following functional groups: guanidine, amino, or imidizole. Carrier molecules also include other constructions that are not peptides but contain the side chains of amino acids, consisting of a combination of about 1-10 lysine, 1-10 lysine, 1-10 arginine, or 1-10 histidine side chains, or 1-10 side chains that contain the following functional groups: guanidine, amino, or imidizole. When a compound of the invention is conjugated or attached to a carrier molecule, the resulting conjugate is referred to herein as a “Carrier Peptide-Inhibitor” conjugate or “CPI.” The CPI conjugate can be administered to an in vitro sample or to a mammal thereby serving as a transport vehicle for a compound or compounds of the invention into a cell in an in vitro sample or in a mammal. The carrier molecules and CPI conjugates result in an increase in the ability of the compounds of the invention to effectively penetrate cells and the blood brain barrier to inhibit memapsin 2 from cleaving APP to subsequently generate Aβ.
In another embodiment, the invention is a pharmaceutical composition of the compounds of the invention. The pharmaceutical composition of the compounds of the invention, with or without a carrier molecule, or the compounds of the invention, with or without a carrier molecule, can be administered to a mammal by enteral or parenteral means. Specifically, the route of administration is by intraperitoneal (i.p.) injection; oral ingestion (e.g., tablet, capsule form) or intramuscular injection. Other routes of administration as also encompassed by the present invention, including intravenous, intraarterial, or subcutaneous routes, and nasal administration. Suppositories or transdermal patches can also be employed.
The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Where the compounds are administered individually it is preferred that the mode of administration is conducted sufficiently close in time to each other (for example, administration of one compound close in time to administration of another compound) so that the effects on decreasing β-secretase activity or β-amyloid production are maximal. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer the compounds of the invention.
The compounds can be administered alone or as admixtures with a pharmaceutically suitable carrier. “Pharmaceutically suitable carrier,” as used herein refers to conventional excipients, for example, pharmaceutically, physiologically, acceptable organic, or inorganic carrier substances suitable for enteral or parenteral application which do not deleteriously react with the extract. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the compounds of the invention. The preparations can also be combined, when desired, with other active substances to reduce metabolic degradation. A preferred method of administration of the compounds is oral administration, such as a tablet or capsule. The compounds of the invention when administered alone, or when combined with an admixture, can be administered in a single or in more than one dose over a period of time to confer the desired effect (e.g., decreased β-amyloid protein).
When parenteral application is needed or desired, particularly suitable admixtures for the compounds of the invention are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. In particular, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene-block polymers, and the like. Ampules are convenient unit dosages. The compounds of the invention can also be incorporated into liposomes or administered via transdermal pumps or patches. Pharmaceutical admixtures suitable for use in the present invention are well-known to those of skill in the art and are described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309, the teachings of both of which are hereby incorporated by reference.
The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, including of a disease that results in increased activity of memapsin 2 or increased accumulation of β-amyloid protein, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., Alzheimer's disease), kind of concurrent treatment, complications from the disease being treated or other health-related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of Applicants' invention. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art.
In an additional embodiment, the invention is a crystallized protein comprising SEQ ID NO: 6 (
In yet another embodiment, the invention is a crystallized protein comprising a protein selected from the group consisting of amino acid residues 1-456 of SEQ ID NO: 8 (
The crystallized protein is formed employing techniques described herein (infra). Briefly, a nucleic acid construct encoding amino acids of SEQ ID NO: 6 (
In still another embodiment, the invention is a crystallized protein comprising a protein of SEQ ID NO: 6 (
In yet another embodiment, the invention is a crystallized protein comprising a protein encoded by SEQ ID NO: 5 (
An embodiment of the invention includes compounds that selectively inhibit memapsin 2 activity relative to memapsin 1. The compounds of the invention are employed in methods to decrease β-secretase activity, to decrease the accumulation of β-amyloid protein and in the treatment of diseases or conditions associated with β-secretase activity and β-amyloid protein accumulation. The compounds of the invention can be employed in methods to treat Alzheimer's disease in a mammal.
The present invention relates to the discovery of compounds that inhibit memapsin 2 (also referred to as BACE or ASP2). An embodiment of the invention includes compounds that selectively inhibit memapsin 2 activity relative to memapsin 1. The compounds of the invention can be employed in methods to decrease β-secretase activity, to decrease the accumulation of β-amyloid protein and in the treatment of diseases or conditions associated with β-secretase activity and β-amyloid protein accumulation. The compounds of the invention can be employed in methods to treat Alzheimer's disease in a mammal.
The present invention is further illustrated by the following examples, which are not intended to be limiting in any way.
Inhibitors were designed, constructed and evaluated for their ability to selectively inhibit memapsin 2 relative to memapsin 1.
The protease domain of memapsin 1 (amino acid residues 15-461 of SEQ ID NO: 4 (SEQ ID NO: 70) (
The E. coli produced promemapsin 1-T1 (amino acid residues 1-461 of SEQ ID NO: 4 (
Memapsin 2 (amino acid residues 1-456 of SEQ ID NO: 8 (
Memapsin 2 (
Peptide sequence EVNLAAEF (SEQ ID NO: 15), known to be a memapsin 2 substrate (Ghosh A. K., et ai., J. Am. Chem. Soc. 122:3522-3523 (2000), the teachings of which are incorporated herein by reference in their entirety) was used as a template structure to study residue preferences in substrate mixtures. For characterization of each of the eight subsites, separate substrate mixtures were obtained by addition of an equimolar mixture of 6 or 7 amino acid derivatives in the appropriate cycle of solid-state peptide synthesis (Research Genetics, Invitrogen, Huntsville, Ala.). The resulting mixture of 6 or 7 peptides differed only by 1 amino acid at a single position. At each position, 19 varied amino acids (less cysteine) were accommodated in three substrate mixtures, requiring 24 substrate mixtures to characterize eight positions. A substrate of known kcat/KM was also added to each mixture to serve as an internal standard. To facilitate the analysis in MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry), the template sequence was extended by 4 residues at the C-terminus (EVNLAAEFWHDR; SEQ ID NO: 16) for variations at Pi′, P2′, P3′, and P4′ and at the N-terminus (RWHHEVNLAAEF; SEQ ID NO: 17) to study positions P1, P2, P3, and P4. “Substrate mixtures,” as referred to herein, are mixtures of variants of SEQ ID NO: 16 and 17, as described above. An example of a substrate mixture is set forth below in Table 2.
a[mix] indicates an equimolar mixture of amino acid derivatives is incorporated at that position in the synthesis, resulting in a mixture of peptides which vary at that position by the amino acid in the mixture.
bMixture of amino acid derivatives added in the [mix] position.
Substrate mixtures were dissolved at 2 mg/ml in 10% glacial acetic acid and diluted into 0.009 M NaOH to obtain a mixture of substrates in the μM range at pH 4.1. After equilibration at 25° C., the reactions were initiated by the addition of an aliquot of memapsin 2. Aliquots were removed at time intervals, and combined with an equal volume of MALDI-TOF matrix (a -hydroxycinnamic acid in acetone, 20 mg/ml) and immediately spotted in duplicate onto a stainless-steel MALDI sample plate. MALDI-TOF mass spectrometry was performed on a PE Biosystems Voyager DE instrument at the Molecular Biology Resource Center on campus. The instrument was operated at 25,000 accelerating volts in positive mode with a 150 ns delay. Ions with a mass-to-charge ratio (m/z) were detected in the range of 650-2000 atomic mass units. Data were analyzed by the Voyager Data Explorer module to obtain ion intensity data for mass species of substrates and corresponding products in a given mixture. Relative product formation was calculated as the ratio of signal intensity of the product to the sum of signal intensities of both product and the corresponding substrate. The quantitative aspect of this analysis was established as follows. From a mixture consisting of seven substrate peptides, EVNLXAEFWHDR (SEQ ID NO: 18) (X=amino acids A, S, T, I, D, E, and F), their hydrolytic peptide products, XAEFWHDR (SEQ ID NO: 19), were prepared by complete hydrolysis. A series of mock partial digestions was prepared by combining known amounts of the substrate mixture with the hydrolysate, and each was subjected to MALDI-TOF/MS analysis. The ratios of product to sum of product and substrate peptide from observed intensity data correlated with the expected ratios for each pair of peptides in the mixture (average slope 1.04±0.01; average intercept 0.019±0.021; average correlation coefficient 0.987±0.006). Relative product formed per unit time was obtained from non-linear regression analysis of the data representing the initial 15% formation of product using the model
1−e−kT
where k is the relative hydrolytic rate constant and T is time in seconds. The initial relative hydrolytic rates of unknown substrates were converted to the relative kcat/KM by the equation
Relative kcat/KM=vx/vs
where vx and vs are the initial hydrolytic rates of a substrate x the reference substrates. For convenience of discussion, the relative kcat/Km value is also referred to as preference index.
The combinatorial inhibitor library was based on the sequence of OM99-2, EVNL*AAEF (SEQ ID NO: 20; “*” represents hydroxyethylene transition-state isostere and is equivalent to Ψ as used herein), with random amino acids (less cysteine) at 4 positions, P2, P3, P2′ and P3′. Di-isostere Leu*Ala was used in a single step of synthesis, thus fixed the structures at positions P1 and P1′. Peptides were synthesized by solid-state peptide synthesis method and left attached on the resin beads. By using the ‘split-synthesis’ procedure (Lam, K. S., et al. Nature 354:82-84 (1991)), each of the resin beads contained only one sequence while the sequence differed from bead to bead. The overall library sequence was
where Xxa residues (where a represents either 1, 2, 3, or 4) are randomized at each position with 19 amino acids. A shorter version of the peptides, starting at P2′ (sequence:Xx3-Xx-4-Phe-Arg-Met-Gly-Gly-(Resin bead) (SEQ ID NO: 22)), was also present in each bead with a ratio to the longer sequence at about 7:3. Without isostere, the short sequence would not bind memapsin 2 with significant strength but its presence was convenient for identifying the residues at P2′ and P3′ by automated Edman degradation. The residues were identified from the randomized positions as follows:
The assignment of Xx3 and Xx2 had no ambiguity since they are the only unknown residue at cycle 1 and 3, respectively. Amino acids Xx1 and Xx4 were assigned from their relative amounts. The presence of a methionine was designed to permit MS/MS identification of peptide fragments from released following CNBr cleavage.
About 130,000 individual beads, representing one copy of the library and estimated to be contained in 1.1 ml of settled beads, was hydrated in buffer A (50 mM Na acetate, 0.1% Triton X-100, 0.4 M urea, 0.02% Na azide, 1 mg/ml bovine serum albumin, pH 3.5; filtered with a 5 micron filter). The beads were soaked in 3% bovine serum albumin in buffer A for 1 h, to block the non-specific binding, and rinsed twice with the same buffer. Recombinant memapsin 2 was diluted into buffer A to 4 nM and incubated with the library for 1 hour. A single stringency wash was performed which included 6.7 μM transition-state isosteric inhibitor OM99-2 in buffer B (50 mM Na acetate, 0.1% Triton X-100, 0.02% sodium azide, 1 mg/ml BSA, pH 5.5; filtered with 5 micron filter), followed by two additional washes with buffer B without OM99-2. Affinity-purified IgG specific for recombinant memapsin 2 was diluted 100-fold in buffer B and incubated 30 minutes with the library. Following three washes with buffer B, affinity-purified anti-goat/alkaline phosphatase conjugate was diluted into buffer B (1:200) and incubated for 30 min, with three subsequent washes. A single tablet of alkaline phosphatase substrate (BCIP/NBT; Sigma) was dissolved in 10 ml water and 1 ml applied to the beads and incubated 1 hour. Beads were resuspended in 0.02% sodium azide in water and examined under a dissecting microscope. Darkly stained beads were graded by sight, individually isolated, stripped in 8 M urea for 24 h, and destained in dimethylformamide. The sequence determination of the beads were carried out in an Applied Biosystem Protein Sequencer at the Molecular Biology Resource Center on campus. The phenylthiohydantoin-amino acids were quantified using reversed-phase high-pressure liquid chromatography.
Inhibitor OM00-3 (ELDL*AVEF, SEQ ID NO: 23) was synthesized using the method as described by Ghosh, et al. (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000)).
The kinetic parameters, KM and kcat, using single peptide substrate, and Ki against free inhibitors, were determined as previously described (Ermolieff, J. et al., Biochemistry 39:12450-12456 (2000)).
Ki is the inhibition equilibrium constant which indicates the ability of compounds to inhibit the β-secretase activity of memapsin 2 and memapsin 1. Numerically lower Ki values indicate a higher affinity of the compounds of the invention for memapsin 2 or memapsin 1. The Ki value is independent of the substrate, and converted from Ki apparent.
Ki apparent is determined in the presence of substrate according to established techniques (see, for example, Bieth, J., Bayer-Symposium V: Proteinase Inhibitors, pp. 463-469, Springer-Verlag, Berlin (1994)).
Vi/Vo depicts the ratio of initial cleavage velocities of the substrate FS-2 (Ermolieff, et al., Biochemistry 40:12450-12456 (2000)) by memapsin 1 or memapsin 2 in the absence (Vo) or presence (Vi) of a compound of the invention. A Vi/Vo value of 1.0 indicates that a compound of the invention does not inhibit the β-secretase activity of the enzyme memapsin 1 or memapsin 2. A Vi/Vo value less than 1.0 indicates that a compound of the invention inhibits β-secretase activity of the enzyme memapsin 1 or memapsin 2. The Vi/Vo values depicted in Table 1 were determined at conditions under which the enzyme and inhibitor concentrations were equal (e.g., about 80 nM, 100 nM).
The standard error for the Ki apparent is the error from the nonlinear regression of the Vi/Vo data measured at different concentrations of the compounds of the invention (e.g., between about 10 nM to about 1000 nM) employing well-known techniques (see, for example, Bieth, J., Bayer-Symposium V: Proteinase Inhibitors, pp. 463-469, Springer-Verlag, Berlin (1994)).
The residue preferences at each subsite for different substrate side chains are defined by the relative kcat/KM values, which are related to the relative initial hydrolysis rates of these mixtures of competing substrates under the condition that the substrate concentration is lower than KM (Fersht, A., Enzyme Structure and Mechanism, 2nd edition, W. H. Freeman, New York (1985)). This method is a less laborious method to determine the residue preference by measuring the initial velocity of substrate mixtures and has been used to analyze the specificity of other aspartic proteases (Koelsch, G., et al., Biochim. Biophys. Acta 1480:117-131 (2000); Kassel, D. B., et al., Anal. Biochem. 228:259-266 (1995)). The rate determination was improved by the use of MALDI-TOF/MS ion intensities for quantitation of relative amounts of products and substrates.
The substrate side chain preference, reported as preference index in eight subsites of memapsin 2 is depicted in
In the familial Alzheimer's disease caused by the Swedish mutation of APP (SEVNLDAEFR; SEQ ID NO: 11), the change of P2−P1 from Lys-Met to Asn-Leu results in an increase of about 60 fold of the kcat/KM of memapsin 2 cleavage indicating that the greatest increase in catalytic efficiency is derived from the change in P2 (
Side Chain Preference Determined from a Combinatorial Inhibitor Library
The preference of memapsin 2 binding to side chains was also determined using a combinatorial library. The base-sequence of the library was derived from OM99-2: EVNL*AAEF (SEQ ID NO: 20) (“*” designates hydroxyethylene transition-state isostere), in which the P3, P2, P2′, and P3′ (boldface) were randomized with all amino acids except cysteine. After incubating the bead library with memapsin 2 and stringent selection of washing with OM99-2 solution, about 65 beads from nearly 130,000 beads were darkly stained, indicating strong memapsin 2 binding. The residues at the four randomized positions were determined for the ten most intensely stained beads. Table 3 shows that there is a clear consensus at these positions. This consensus is not present in the sequence of two negative controls (Table 3). To confirm this, a new inhibitor, OM00-3: ELDL*AVEF (SEQ ID NO: 23), was designed based on the consensus and synthesized. OM00-3 was found to inhibit memapsin 2 with Ki of 0.31 nM, nearly five-fold lower than the Ki of OM99-2. In addition, the residue preferences determined at P3, P2 and P2′ of the inhibitors agreed well with the results from substrate studies (
aLibrary template: Gly-Xx1-Xx2-Leu*Ala-Xx3-Xx4-Phe-Arg-Met-Gly-Gly-Resin (SEQ ID NO: 21), wherein Xx1 corresponds to an amino acid residue with side chain P3; Xx2 corresponds to an amino acid residue with side chain P2; Xx3 corresponds to an amino acid residue with side chain P2′; and Xx4 corresponds to an amino acid residue with side chanin P3′.
bNeg 1 and Neg 2 are two randomly selected beads with no memapsin 2 binding capacity.
The Determination of Relative kcat/km of Substrates in Substrate Mixtures
The relative initial hydrolysis rates of individual peptides in a mixture of substrates was determined. Since these relative rates are proportional to their kcat/Km values, they are taken as residue preferences when the substrates in the mixture differ only by one residues. The preference index was calculated from the relative initial hydrolitic rates of mixed substrates and is proportionate to the relative kcat/Km. The design of substrate mixtures and the condition of experiments are as described above.
Since memapsin 1 hydrolyzes some of the memapsin 2 cleavage sites (Farzan, M., et al., Proc. Natl. Acad. Sci., USA 97:9712-9717 (2000), the teachings of which are incorporated herein by reference in their entirety), the substrate mixture successfully used for studying subsite specificity of memapsin 2 (template sequence EVNLAAEF, SEQ ID NO: 15, was adopted for this study. Each substrate mixture contained six or seven peptides which differed only by one amino acid at a single position. At each position, each of the 19 natural amino acids (cysteine was not employed to prevent, for example, dimer formation by disulfide bonds) was accommodated in three substrate mixtures. A substrate of known kcat/Km value was also added to each set to serve as an internal standard for normalization of relative initial rates and the calculation of kcat/Km value of other substrates.
For four P′ side chain (P1′, P2′, P3′ and P4′), the template sequence was extended by four amino acid residues at the C-terminus (EVNLAAEFWHDR (SEQ ID NO: 16)) to facilitate detection in MALDI-TOF MS. Likewise, four additional amino acid residues were added to the N-terminus to characterize four P side chain (RWITHEVNLAAEF, SEQ ID NO: 17). The procedure and conditions for kinetic experiments were essentially as previously described for memapsin 2 (supra). The amount of substrate and hydrolytic products were quantitatively determined using MALDI-TOF mass spectrometry as described above. The relative keat/Km values are reported as preference index.
The combinatorial inhibitor library was based on the sequence of OM99-2: EVNLΨAAEF (SEQ ID NO: 20), where letters represent amino acids in single letter code and Ψ represents a hydroxyethylene transition-state isostere, as previously described (U.S. Application Nos. 60/141,363, filed Jun. 28, 1999; 60/168,060, filed Nov. 30, 1999; 60/177,836, filed Jan. 25, 2000; 60/178,368, filed Jan. 27, 2000; 60/210,292, filed Jun. 8, 2000; 09/603,713, filed Jun. 27, 2000; 09/604,608, filed Jun. 27, 2000; 60/258,705, filed Dec. 28, 2000; 60/275,756, filed Mar. 14, 2001; PCT/US00/17742, WO 01/00665, filed Jun. 27, 2000; PCT/US00/17661, WO 01/00663, filed Jun. 27, 2000; U.S. patent application entitled “Compounds which Inhibit Beta-Secretase Activity and Methods of Use Thereof,” filed Oct. 22, 2002 and having Attorney Docket No. 2932.1001-003; and Ghosh, et al. (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000), the teachings of all of which are hereby incorporated by reference in their entirety). Four positions, P2, P3, P2 and P3′, were filled with random amino acids residues (less cysteine). Positions P1 and P1′ were fixed due to the use of diisostere LeuΨAla in a single step of solid-state peptide synthesis of inhibitors (Ghosh, A. K., et al., J Am. Chem. Soc. 122:3522-3523 (2000), the teachings of which are incorporated herein by reference in their entirety). By using the split-synthesis procedure (Lam, K. S., et al., Nature 354:82-84 (1991), the teachings of which are incorporated herein by reference in their entirety), each of the resin beads contained only one sequence while the sequence differed among beads. The overall library sequence was: Gly-Xx1-Xx2-LeuΨAla-Xx3-Xx-4-Phe-Arg-Met-Gly-Gly- (Resin bead) (SEQ ID NO: 21).
Probing the binding of memapsin 1 to the combinatorial library and the sequence determination of the inhibitors was performed as described above. Affinity purified antibodies against memapsin 2 were used since the antibodies cross react with proteins memapsin 1 and memapsin 2.
Inhibitors of the invention are prepared by synthesis of the isostere portion of the inhibitor followed by coupling to a peptide having one or more an amino acids and/or modified amino acids.
A leucine-alanine isostere unit is included in inhibitors MMI-001-MMI-009; MMI-011-MMI-020; MMI-022-MMI-026; MMI-034-MMI-035; MMI-039; MMI-041-MMI-047; MMI-049-MMI-060; MMI-063-MMI-077; MMI-079-MMI-091; MMI-093-MMI-100; MMI-103-MMI-105; MMI-107-MMI-131; MMI-133-MMI-144; MMI-146-MMI-154; MMI-156-MMI-163; MMI-165-MMI-167;MMI-171; MMI-173-MMI-177; MMI-180; MMI-183-MMI-86; MMI-188-MMI-90; MMI-193-MMI-200; MMI-203-MMI-210; MMI-2,2-MMI-217; MMI-219-MMI-130. The leucine-alanine isostere was prepared using the method shown in Scheme 1.
To a stirred solution of N,O-dimethylhydroxyamine hydrochloride (5.52 g, 56.6 mmol) in dry dichloromethane (25 mL) under a N2 atmosphere at 0° C., was added N-methylpiperidine (6.9 mL, 56.6 mmol) dropwise. The resulting mixture was stirred at 0° C. for 30 minutes. In a separate flask, commercially available N-(t-butyloxycarbonyl)-L-leucine (11.9 g, 51.4 mmol) was dissolved in a mixture of tetrahydrofuran (THF) (45 mL) and dichloromethane (180 mL) under a N2 atmosphere. The resulting solution was cooled to −20° C. To this solution was added 1-methylpiperidine (6.9 mL, 56.6 mmol) followed by isobutyl chloroformate (7.3 mL, 56.6 mmol) dropwise. The resulting mixture was stirred for 5 minutes at −20° C. and the above solution of N,O-dimethyl-hydroxylamine was added dropwise. The reaction mixture was stirred at −20° C. for 30 minutes followed by warming to room temperature. The reaction was quenched with water and the layers were separated. The aqueous layer was extracted with CH2Cl2 (3 times). The combined organic layers were washed with 10% citric acid, saturated sodium bicarbonate, brine, dried over Na2SO4 and concentrated under reduced pressure. Flash column chromatography (25% ethyl acetate (EtOAc) in hexanes) yielded 1 (13.8 g, 97%). [α]D23-23 (c 1.5, MeOH); 1H-NMR (400 MHZ, CDCl3) δ 5.06 (d, 1H, J=9.1 Hz), 4.70 (m, 1H), 3.82 (s, 3H), 3.13 (s, 3H), 1.70 (m, 1H), 1.46-1.36 (m, 2H) 1.41 (s, 9H), 0.93 (dd, 6H, J=6.5, 14.2 Hz); 13C-NMR (100 MHZ, CDCl3) δ 173.9, 155.6, 79.4, 61.6, 48.9, 42.1, 32.1, 28.3, 24.7, 23.3, 21.5; HZ. (neat) 3326, 2959, 2937, 2871, 1710, 1666, 1502, 1366, 1251, 1046 cm−1; HRMS m/z (M+H)+ calc'd for C13H27N2O4 275.1971, found 275.1964.
To a stirred suspension of lithium aluminum hydride (LAH) (770 mg, 20.3 mmol) in diethyl ether (60 mL) at −40° C. under N2 atmosphere, was added dropwise a solution of 1 (5.05 g, 18.4 mmol) in diethyl ether (20-mL). The resulting reaction mixture was stirred for 30 minutes followed by quenching with 10% aqueous NaHSO4 (30 mL) and warming to room temperature for 30 minutes. This solution was filtered and the filter cake was washed with diethyl ether (two times). The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over MgSO4 and concentrated under reduced pressure to afford 2 (3.41 g) which was used immediately without further purification. Crude 1H-NMR (400 MHZ, CDCl3) δ 9.5 (s, 1H), 4.9 (s, 1H), 4.2 (m, 1H), 1.8-1.6 (m, 2H), 1.44 (s, 9H), 1.49-1.39 (m, 1H), 0.96 (dd, 6H, J=2.7, 6.5 Hz).
To a stirred solution of ethyl propiolate (801 mL) in THF (2 mL) at −78° C. was added a 1.0 M solution of lithium hexamethyldisilazide (7.9 mL) dropwise over a 5 minutes period. The mixture was stirred for 30 min, after which N-(tert-butoxycarbonyl)-L-leucinal 2 (or N-Boc-L-leucinal) (1.55 g, 7.2 mmol) in 8 mL of dry THF was added. The resulting mixture was stirred at −78° C. for 30 minutes. The reaction was quenched with saturated aqueous NH4Cl at −78° C. followed by warming to room temperature. Brine was added and the layers were separated. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. Flash column chromatography (15% EtOAc in hexanes) yielded a mixture of acetylenic alcohols 3 (68%). 1H-NMR (300 MHZ, CDCl3) δ 4.64 (d, 114, J=9.0 Hz), 4.44 (br s, 1H), 4.18 (m, 21-1), 3.76 (m, 1H), 1.63 (m, 1H), 1.43-1.31 (m, 2H), 1.39 (s, 9H), 1.29-1.18 (m, 3H), 0.89 (m, 6H); IR (neat) 3370, 2957, 2925, 2854, 1713, 1507, 1367, 1247, 1169, 1047 cm−1.
To a stirred solution of 3 (1.73 g, 5.5 mmol) in methanol (MeOH) (20 mL) was added 10% Pd/C (1.0 g). The resulting mixture was placed under a hydrogen balloon and stirred for 1 hour. After this period, the reaction was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The residue was dissolved in toluene (20 mL) and acetic acid (100 L). The resulting mixture was refluxed for 6 hours followed by cooling to room temperature and concentrating under reduced pressure. Flash column chromatography (40% diethyl ether in hexanes) yielded 4 (0.94 g, 62.8 mmol) and less than 5% of its diastereomer. Lactone 4: M.p. 74-75° C.; [α]D23-33.0 (c 1.0, MeOH); lit. (Fray, A. H., et al., J. Org. Chem. 51:4828-4833 (1986)) [α]D23-33.8 (c 1.0, MeOH); 1H-NMR (400 MHZ, CDCl3) δ 4.50-4.44 (m, 2H), 3.84-3.82 (m, 1H), 2.50 (t, 2H, J=7.8 Hz), 2.22-2.10 (m, 2H), 1.64-1.31 (m, 3H), 1.41 (s, 9H), 0.91 (dd, 6H, J=2.2, 6.7 Hz); 13C-NMR (75 MHZ, CDCl3) δ 177.2, 156.0, 82.5, 79.8, 51.0, 42.2, 28.6, 28.2, 24.7, 24.2, 23.0, 21.9; IR (neat) 2956, 2918, 2859, 1774, 1695, 1522, 1168 cm−1; mass (EI) m/z 294 (M++Na); HRMS: m/z (M+Na)+ calc'd for C14H25NO4Na, 294.1681, found 294.1690.
To a stirred solution of lactone 4 (451.8 mg, 1.67 mmol) in THF (8 mL) at −78° C. under a N2 atmosphere, was added dropwise lithium hexamethyldisilazide (3.67 mL, 1.0 M in THF, 3.67 mmol). The resulting mixture was stirred at −78° C. for 30 minutes. Methyl iodide (MeI) (228 mL) was added dropwise and the resulting mixture was stirred at −78° C. for 20 minutes. The reaction was quenched with saturated aqueous NH4Cl and allowed to warm to room temperature. The reaction mixture was concentrated under reduced pressure and the residue was extracted with EtOAc (three times). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. Flash column chromatography (15% EtOAc in hexanes) yielded 5 (0.36 g, 76%). The stereochemistry of C2-methyl group was assigned based upon NOESY and COSY experiments. Irradiation of the C2-methyl group exhibited 6% NOE with the C3 α-proton and 5% NOE with the C4-proton. The α- and β-protons of C3 were assigned by 2 D-NMR. [a]D23-19.3 (c 0.5, CHCl3); 1H-NMR (300 MHZ, CDCl3) δ 4.43 (t, 1H, J=6.3 Hz), 4.33 (d, 1H, J=9.6 Hz), 3.78 (m, 1H), 2.62 (m, 1H), 2.35 (m, 1H), 1.86 (m, 1H), 1.63-1.24 (m, 3H), 1.37 (s, 9H), 1.21 (d, 3H, J=7.5 Hz), 0.87 (dd, 6H, J=2.6, 6.7 Hz); 13C-NMR (75 MHZ, CDCl3) δ 180.4, 156.0, 80.3, 79.8, 51.6, 41.9, 34.3, 32.5, 28.3, 24.7, 23.0, 21.8, 16.6; IR (neat) 2962, 2868, 1764, 1687, 1519, 1272, 1212, 1008 cm−1; HRMS: m/z (M+Na)+ calc'd for C15H27NO4Na, 308.1838, found 308.1828.
To a stirred solution of lactone 5 (0.33 g, 1.17 mmol) in a mixture of THF and water (5:1; 6 mL) was added LiOH.H2O (0.073 g, 1.8 equiv). The resulting mixture was stirred at room temperature for 1 hour. The volatiles were removed under reduced pressure and the remaining solution was cooled to 0° C. and acidified with 25% aqueous citric acid to pH 3. The resulting acidic solution was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure to yield the corresponding hydroxy acid (330 mg) as a white foam. This hydroxy acid was used directly for the next reaction without further purification.
To the above hydroxy acid (330 mg, 1.1 mmol) in dimethylformamide (DMF) was added imidazole (1.59 g, 23.34 mmol) and tert-butyldimethylchlorosilane (1.76 g, 11.67 mmol). The resulting mixture was stirred at room temperature for 24 hours. MeOH (4 mL) was added and the mixture was stirred for an additional 1 hour. The mixture was acidified with 25% aqueous citric acid to pH 3 and was extracted with EtOAc three times. The combined extracts were washed with water, brine, dried over Na2SO4 and concentrated under reduced pressure. Flash column chromatography (35% EtOAc in hexanes) yielded 6 (0.44 g, 90%). M.p. 121-123° C.; [α]D23-40.0 (c 0.13, CHCl3); 1H-NMR (400 MHZ, DMSO-d6, 343 K) δ 6.20 (br s, 1H), 3.68 (m, 1H), 3.51 (br s, 1H), 2.49-2.42 (m, 1H), 1.83 (t, 1H, J=10.1 Hz), 1.56 (m, 1H), 1.37 (s, 9H), 1.28-1.12 (m, 3H), 1.08 (d, 3H, J=7.1 Hz), 0.87 (d, 3H, J=6.1 Hz) 0.86 (s, 9H), 0.82 (d, 3H, J=6.5 Hz), 0.084 (s, 3H), 0.052 (s, 3H); IR (neat) 3300-3000, 2955, 2932, 2859, 171.1 cm−1; HRMS: m/z (M+Na)+ calc'd for C21H43NO5NaSi, 440.2808, found 440.2830.
A. Isostere Used to Prepare MMI-133
The methyl diastereomers of the Leu-Ala isostere were synthesized using the minor product of the alkylation step (see Section I, step E).
Other isosteres with simple alkyl substituents in P1′ (MMI-010, MMI-021, MMI-027-MMI-033, MMI-036, MMI-202, MMI-211, MMI-218) were produced following the general procedure for preparing the leucine-alanine isostere as set forth above except that a different alkylating agent was used in Section I, step E for alkylating the lactone. For example:
B. Leucine-Allyl Isostere Used to Prepare MMI-010 and MMI-021
To a solution of 4 (2.41 g, 8.89 mmol) in THF (50 mL) was added lithium hexamethyldisilazane (1.0 Min THF, 19.56 mL, 19.56 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, allyl iodide (0.89 mL, 9.78 mmol) was added dropwise at −78° C. and the resulting mixture was stirred at −78° C. for 15 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (15% EtOAc in hexanes) to give 7 (1.94 g, 70%).
LiOH (66 mg, 1.58 mmol) was added to a solution of 7 (325 mg, 1.05 mmol) in dioxane/water (3:1, 4 mL) and stirred for 1 hour. The reaction mixture was acidified to pH 3 with 25% aqueous citric acid, extracted with EtOAc, dried over Na2SO4, and concentrated under reduced pressure to yield the corresponding hydroxyl acid (307 mg, 89%).
To a solution of the above hydroxyl acid (307 mg, 0.93 mmol) in DMF (8 mL) were added imidazole (1.07 g, 14.9 mmol) and TBSCI (1.12 g, 7.47 mmol). The reaction was stirred at room temperature for 15 hours. After this period, MeOH (4 mL) was added and the resulting mixture was stirred for 1 hour. The mixture was then diluted with 25% aqueous citric acid and extracted with EtOAc. The organic layer was washed with brine, dried with Na2SO4, and purified by column chromatography (10% EtOAc in hexanes) to yield 8 (383 mg, 93%).
C. Leucine-Homoserine Isostere Used to Prepare MMI-037:
The isostere portion of MMI-037 is produced by coupling the above Leucine-Allyl isostere 8 with Valine-N-benzyl amide under standard EDCI/HOBt conditions (Section IV) to provide 9.
Ozone was bubbled through a solution of compound 9 in CH2Cl2/MeOH (1:1, 6 mL) at −78° C. until the blue color persisted (ca. 10 minutes). Oxygen was bubbled through the mixture until the blue color dissipated after which nitrogen was bubbled through the mixture for 10 minutes. Triphenylphosphine (124 mg, 0.47 mmol) was added at −78° C. and the mixture stirred and allowed to warm to room, temperature over 1 hour. The solvent was removed under reduced pressure and the residue was purified by column chromatography (30% EtOAc in hexanes) to yield the corresponding aldehyde (86 mg, 56%).
NaBH4 (7.4 mg, 0.2 mmol) was added to a solution of the above aldehyde (86 mg, 0.13 mmol) in THF (3 mL) at 0° C. and stirred for 15 minutes. The reaction was quenched by addition of saturated aqueous NH4Cl, extracted with EtOAc, dried with Na2SO4, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (60% EtOAc in hexanes) to yield 10 (87%).
D. Leucine-Methionine Isostere Used to Prepare MMI-164:
The isostere portion of MMI-164 is produced by treatment of a solution of 10 (70 mg, 0.11 mmol) in CH2Cl2 (2 mL) with Et3N (0.03 mL, 0.22 mmol) and methane sulfonyl chloride (0.01 mL, 0.12 mmol) and stirred for 1 hour. The reaction mixture was diluted with CH2Cl2 and washed with saturated aqueous NH4Cl. The organic layer was dried with Na2SO4 and concentrated under reduced pressure to yield the corresponding mesylate (67 mg).
To the above mesylate in DMF (2 mL) was added NaSMe (15 mg, 0.22 mmol) followed by heating to 70° C. for 1 hour. The reaction was cooled to room temperature, diluted with EtOAc, and washed with water. The organic layer was dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (20% EtOAc in hexanes) to yield 11 (65% for 2 steps).
E. Leucine-Asparagine Isostere Used to Prepare MMI-038:
Pyridinium dichromate (302 mg, 0.81 mmol) was added to a solution of 10 (170 mg, 0.27 mmol) in DMF (2 mL) and stirred at room temperature for 12 hours. The reaction mixture was diluted with Et2O and filtered through Celite®. The filtrate was concentrated under reduced pressure and purified by column chromatography (10% MeOH in CHCl3) to afford 12 (130 mg, 77%).
HOBt (7.1 mg, 0.05 mmol) and EDCI (10 mg, 0.05 mmol) were added to 12 (30 mg, 0.04 mmol) in CH2Cl2 (2 mL). After stirring for 30 minutes at room temperature, the solution was added to liquid NH3 in CH2Cl2 at −78° C. After stirring at −78° C. for 30 minutes, the reaction mixture was warmed to room temperature, diluted with CH2Cl2, and washed with water. The organic layer was dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (60% EtOAc in hexanes) to yield 13 (19 mg, 63%).
F. Leucine-Serine Isostere Used to Prepare MMI-078 and MMI-132:
To a solution of known carboxylic acid 18 (Tetrahedron 1996, 8451) (1.05 g, 6.78 mmol) in THF (30 mL) at −20° C. was added Et3N (1.2 mL, 8.82 mmol) dropwise followed by pivaloyl chloride (1.08 mL, 8.82 mmol). The mixture was stirred for 30 minutes at −20° C. followed by cooling to −78° C.
In a separate flask, oxazolidinone 15 (1.56 g, 8.82 mmol) was dissolved in THF (25 mL), cooled to −78° C. and BuLi (5.5 mL, 1.6 M in hexanes, 8.82 mmol) was added dropwise. After stirring for 30 minutes, the solution was transferred via cannula into the first flask containing the mixed anhydride at −78° C. The resulting mixture was stirred for 30 minutes and quenched with NaHSO4 (5 g in 30 mL H2O) at −78° C. and warmed to room temperature. The organic layer was dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (20% EtOAc in hexanes) to yield 16 (2.37 g, 71%).
To a solution of 16 (47 mg, 0.15 mmol) in THF (1 mL) was added lithium hexamethyldisilazane (1.0 M in THF, 0.19 mL, 0.19 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, benzylchloromethyl ether (0.027 mL, 0.19 mmol) was added dropwise at −78° C. and the resulting mixture was stirred at −78° C. for 15 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (10% EtOAc in hexanes) to give 17 (68%).
To a solution of 17 (157 mg, 0.36 mol) in DME/H2O (3:1, 8 mL) was added NBS (70.8 mg, 0.4 mmol) at 0° C. After stirring for 45 minutes at 0° C., the reaction was quenched by the addition of H2O and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (10% EtOAc in hexanes) to give 18 (59%).
The reaction of 18 (73 mg, 0.21 mmol) with NaN3 (27 mg, 0.42 mmol) in DMPU (1 mL) at room temperature for 3 days yielded 19 (66%) after column chromatography (15% EtOAc in hexanes).
Completion of the isostere synthesis was accomplished following procedures previously described (Section I, Step F) to afford 20 followed by coupling of Valine-N-benzyl amide under standard EDCI/HOBt coupling conditions (Section IV) and hydrogenation of the azide and benzyl protecting group following standard hydrogenation conditions to provide the corresponding aminoalcohol.
Standard hydrogenation procedure: A mixture of the alkene, benzyl-protected alcohol, or azide (135 mg, 0.4 mmol) and Pd(OH)2/C (20%, 20 mg) in MeOH, EtOAc or a mixture thereof (5 mL) was stirred under an H2 atmosphere for 5 hours. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to yield the corresponding saturated compound, free alcohol, or free amine quantitatively.
G. Leucine-CH2 Isostere used to prepare MMI-145;
To a solution of 19 (35 mg, 0.11 mmol) in MeOH (2 mL) was added Boc2O (0.038 mL, 0.16 mmol) and Pd(OH)2/C (20% Pd, 5 mg). The mixture was placed under a hydrogen atmosphere and stirred for 12 hours at room temperature. The reaction was filtered through Celite®, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (50% EtOAc in hexanes) to yield 21 (44%).
To a solution of (diethylamino)sulfur trifluoride (0.0095 mL, 0.07 mmol) in CH2Cl2 (1 mL) at −78° C. was added dropwise a solution of 21 (20 mg, 0.06 mmol) in CH2Cl2 (1 mL). The reaction was warmed to room temperature and stirred for 12 hours. After this period, the reaction mixture was cooled to 0° C. and quenched with H2O. The organic layer was dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (25% EtOAc in hexanes) to yield 22 (61%).
To a solution of 22 (46.5 mg, 0.15 mmol) in DME:H2O (1:1, 3 mL) was added 1 N LiOH (0.46 mL) and stirred at room temperature for 2 hours followed by acidification with 1 N HCl to pH 3 and extraction with EtOAc. The organic layer was dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography to yield a mixture of products. The mixture (44 mg, 0.13 mmol) was dissolved in DMF (1 mL) and imidazole (207 mg, 3.04 mmol) and TBSCl (209 mg, 1.38 mmol) was added and stirred for 12 hours. The reaction was quenched MeOH (1 mL), stirred for 1 hour, acidified with 5% citric acid to pH 3, extracted with EtOAc, dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography to yield 23 (13.8 mg) and the isostere 24 (37.6 mg).
H. Leucine-Tyrosine Isostere Used to Prepare MMI-101 and MMI-102:
To a solution of 4 (220 mg, 0.81 mmol) in THF (50 mL) was added lithium hexamethyldisilazane (1.0 M in THF, 1.78 mL, 1.78 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, iodide 25 (22 mg, 0.89 mmol) was added dropwise at −78° C. and the resulting mixture was stirred at −78° C. for 15 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (10% EtOAc in hexanes) to give 26 (242 mg, 63%).
LiOH (32 mg, 0.77 mmol) was added to a solution of 26 (242 mg, 0.51 mmol) in dioxane/water (3:1, 4 mL) and stirred for 1 hour. The reaction mixture was acidified to pH 3 with 25% aqueous citric acid, extracted with EtOAc, dried over Na2SO4, and concentrated under reduced pressure to yield the corresponding hydroxy acid (240 mg, 96%).
To a solution of the above hydroxyl acid (240 mg, 0.49 mmol) in DMF (6 mL) were added imidazole (533 mg, 7.84 mmol) and TBSCl (588 mg, 3.92 mmol). The reaction was stirred at room temperature for 15 hours. After this period, MeOH (4 mL) was added and the resulting mixture was stirred for 1 hour. The mixture was then diluted with 25% aqueous citric acid and extracted with EtOAc. The organic layer was washed with brine, dried with Na2SO4, and purified by column chromatography (10% EtOAc in hexanes) to yield 27 (89%).
After the standard EDCI/HOBt couplings (Section IV) to produce MMI-101, the benzyl protecting group was removed by hydrogenation following the standard hydrogenation procedure described previously (Section II, Step F).
A. Isosteres having an Inverted Hydroxyl Group (MMI-003, MMI-113, MMI-133)
The methyl/hydroxy diastereomer of the Leucine-Alanine isostere (MMI-133) was synthesized using the minor product from the ethylpropiolate addition step (Section I, Step C) following the regular sequence for the Leucine-Alanine isostere.
MMI-133 was produced from the above diastereomer by the following sequence to invert the methyl chiral center:
To a solution of iPr2NEt (0.07 mL, 0 5 mmol) in THF (2 mL) at 0° C. was added BuLi (0.32 mL, 1.6 M in hexanes, 0.51 mmol) and stirred for 30 minutes. The above solution was cooled to −78° C. and 28 (28.3 mg, 0.1 mmol) in THF (1 mL) followed by HMPA (0.1 mL, 0.55 mmol) were added. After stirring for 1 hour at −78° C. and 1.5 hours at −42° C., the reaction was cooled to −78° C. and dimethylmalonate (0.11 mL, 1.0 mmol) was added. The reaction was allowed to warm to room temperature, diluted with EtOAc, washed with saturated aqueous NH4Cl, brine, dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (15% EtOAc in hexanes) to yield 29 (86%). 29 was then carried through the normal procedures for the production of the isostere of MMI-133 (see Section I).
B. Hydroxyethylamine Isostere Used to Prepare MMI-061, MMI-062, MMI-092, MMI-106:
To a solution of known epoxide 30 (Tetrahedron Lett., 1995, 36, 2753-2756) (229 mg, 1.0 mmol) in MeOH (5 mL) was added methylamine (2.5 mL, 2.0 M solution in MeOH, 5.0 mmol) and the resulting mixture was stirred for 4-5 hours at room temperature. The solvent was removed under reduced pressure and the residue was purified by column chromatography (50% EtOAc in hexanes) to afford 31 (90% yield).
C. Isostere Wherein P1′ and R4 Form a Pyrrolidin-2-One Ring (MMI-181, MMI-185, MMI-187, MMI-191, MMI-192):
To a solution of 4 (2.41 g, 8.89 mmol) in THF (50 mL) was added lithium hexamethyldisilazane (1.0 Min THF, 19.56 mL, 19.56 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, allyl iodide (0.89 mL, 9.78 mmol) was added dropwise at −78° C. and the resulting mixture was stirred at −78° C. for 15 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (15% EtOAc in hexanes) to give 7 (1.94 g, 70%).
To a solution of 7 (1.5 g, 4.82 mmol) in THF (30 mL) was added lithium hexamethyldisilazane (1.0 M in THF, 10.60 mL, 10.60 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, methyl iodide (0.39 mL, 6.26 mmol) was added dropwise at −78° C. and the resulting mixture was warmed to 0° C. for 1 hour. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (12% EtOAc in hexanes) to give 32 (0.684g, 44%).
Ozone was bubbled through a solution of 32 (250 mg, 0.768 mmol) in CH2Cl2/MeOH (1:1, 20 mL) at −78° C. until the blue color persisted. The solution was then flushed with N2 for 10 minutes. Me2S was added (0.31 mL, 4.23 mmol) slowly and the reaction mixture was allowed to warm to room temperature. After being stirred for 12 hours, the solvent was removed under reduced pressure and the resulting residue was purified by column chromatography (30% EtOAc in hexanes) to give 33 (155 mg, 62%).
Incorporation of compound 34 into the isostere used to prepare MMI-191 is described as an example:
To a solution of the trifluoroacetic acid (TFA) salt of leucine N-butyl amide (0.092 mmol) and sodium acetate (10 mg, 0.73 mmol) was added compound 34 (20 mg, 0.061 mmol) at room temperature. The mixture was stirred at room temperature for 15 minutes followed by addition of NaBH3CN (5.4 mg, 0.086 mmol). The resulting mixture was stirred at room temperature for 24 h, poured into H2O, and extracted with EtOAc. The organic layer was washed with brine and dried with MgSO4. Concentration under reduced pressure afforded a residue which was chromatographed (40% EtOAc in hexanes) to give compound 13 (30 mg, 99%).
D. Phenylalanine-Methionine Isostere Used to Prepare MMI-201:
To a solution of 35 (635 mg, 2.08 mmol) in THF (15 mL) was added lithium hexamethyldisilazane (1.0 M in THF, 4.57 mL, 4.57 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 30 minutes. After this period, allyl iodide (0.21 mL, 2.29 mmol) was added dropwise at −78° C. and the resulting mixture was stirred at −78° C. for 15 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (15% EtOAc in hexanes) to give 36 (413 mg, 58%).
Ozone was bubbled through a solution of 36 (400 mg, 1.158 mmol) in CH2Cl2/MeOH (1:1, 30 mL) at −78° C. until the blue color persisted. The solution was then flushed with N2 for 10 minutes. Triphenylphosphine (334 mg, 1.274 mmol) was added slowly and the reaction mixture was allowed to warm to room temperature. After being stirred for 15 minutes, the solvent was removed under reduced pressure and the resulting residue was purified by column chromatography (40% EtOAc in hexanes) to give 37 (336 mg, 84%).
To a solution of aldehyde 37 (300 mg, 0.86 mmol) in MeOH (10 mL) was added NaBH4 (49 mg, 1.3 mmol) at −78° C. The reaction was allowed to warm to 0° C. and was stirred at that temperature for 20 minutes. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Concentration under reduced pressure afforded a residue that was chromatographed (60% EtOAc in hexanes) to yield the corresponding alcohol (200 mg, 66%).
To a solution of the above alcohol (170 mg, 0.49 mmol) in CH2Cl2 (5 mL) was added imidazole (83 mg, 1.22 mmol), Ph3P (319 mg, 1.22 mmol) and iodine (247 mg, 0.97 mmol) at 0° C. The reaction was stirred for 15 minutes at 0° C., poured into saturated aqueous Na2SO4, and extracted with CH2Cl2. The organic layer was washed with brine, dried over MgSO4, concentrated under reduced pressure, and chromatographed (30% EtOAc in hexanes) to give 38 (124 mg, 55%).
To a solution of iodide 38 (124 mg, 0.27 mmol) in DMF (5 mL) was added sodium thiomethoxide (23 mg, 0.32 mmol) at 0° C. The reaction was stirred for 10 minutes, poured into saturated aqueous NH4Cl, and extracted with diethylether. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Concentration under reduced pressure gave a residue which was chromatographed (30% EtOAc in hexanes) to give 39 (66 mg, 64%). The lactone was then hydrolyzed with LiOH and the resulting free alcohol protected as in the synthesis of the Leucine-Alanine isostere.
E. Isostere Having Dimethyl Groups at the P1′ Position (MMI-218):
To a solution of 4 (625 mg, 2.19 mmol) in THF (20 mL) was added lithium hexamethyldisilazane (1.0 M in THF, 4.8 mL, 4.8 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 1 hour. After this period, methyl iodide (0.15 mL, 2.41 mmol) was added dropwise at −78° C. and the resulting mixture was warmed to −45° C. for 1 hour. After this period, to the reaction mixture was added lithium hexamethyldisilazane (1.0 M in THF, 4.8 mL, 4.8 mmol) dropwise at −78° C. The resulting mixture was stirred at −78° C. for 1 hour. After this period, methyl iodide (0.15 mL, 2.41 mmol) was added dropwise at −78° C. and the resulting mixture was warmed to −45° C. for 1 hour. The reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (15% EtOAc in hexanes) to give 40 (416 mg, 63%).
To a solution of 40 (416 mg, 1.389 mmol) in CH2Cl2 (8 mL) was added trifluoroacetic acid (2 mL) at 0° C. and the resulting mixture was stirred at 0° C. for 3.5 hours. After this time, the reaction was concentrated under reduced pressure to obtain the crude amine. To this crude amine in CH2Cl2 (15 mL) was added iPr2NEt (0.8 mL, 4.58 mmol) and benzylchloroformate (0.22 mL, 1.53 mmol) at −78° C. The reaction was stirred for 1 hour at −78° C., poured into saturated aqueous NH4Cl, and extracted with CH2Cl2. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (20% EtOAc in hexanes) to give 41 (408 mg, 88%).
To a solution of 41 (408 mg, 1.22 mmol) in THF (15 mL) was added 1N aqueous LiOH solution (9.8 mL, 9.8 mmol) at room temperature. The resulting mixture was stirred at room temperature for 15 hours. After this period, the reaction was concentrated under reduced pressure and the remaining aqueous residue was cooled to 0° C. and acidified with 25% aqueous citric acid to pH 4. The resulting acidic solution was extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (70% EtOAc in hexanes) to give 42 (110 mg, 37%).
42 was coupled with Valine-N-nbutyl amide under standard EDCl/HOBt coupling conditions (Section IV) to afford 43.
To a solution of 43 (81 mg, 0.20 mmol) in THF (4 mL) was added Et3N (0.032 mL, 0.224 mmol), Boc2O (53 mg, 0.245 mmol), and dimethylaminopyridine (5 mg, 0.041 mmol) at 0° C. After being stirred at room temperature for 3 hours, the reaction mixture was poured into saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvents under reduced pressure gave a residue which was purified by column chromatography (5% MeOH in CHCl3) to give the corresponding Boc-protected oxazolidinone (99 mg, 98%).
To the above Boc-protected oxazolidinone (74 mg, 0.149 mmol) in MeOH (4 mL) was added Cs2CO3 (97 mg, 0.297 mmol) at room temperature. After stirring at room temperature for 20 hours, the reaction mixture was neutralized with 1 N aqueous HCl and extracted with EtOAc. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (2% MeOH in CHCl3) to give the corresponding amino alcohol (38 mg, 54%).
To the above amino alcohol (38 mg, 0.081 mmol) in CH2Cl2 (2 mL) were added t-butyldimethylsilyl trifluoromethanesulfonate (0.022 mL, 0.097 mmol) and iPr2NEt (0.034 mL, 0.193 mmol) at −78° C. After being stirred at −78° C. for 15 minutes, the reaction mixture was poured into saturated aqueous NH4Cl and extracted with CH2Cl2. The organic layer was washed with brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (1.5% MeOH in CHCl3) to give 44 (42 mg, 89%).
B. Isosteres Having P1 Amino Acid Side Chains Other Than Leucine Side Chain
Inhibitors with a different amino acid-based side-chain in P1 were produced by substitution the appropriate Boc-protected amino acids for N-(t-butyloxycarbonyl)-L-Leucine (Boc-Phe: MMI-040, MMI-048, MMI-201; Boc-Ser: MMI-155) in Section I, Step A.
C. Isostere Having Non-Natural P1 Amino Acid Side Chains (MMI-178, MMI-179, MMI-170, MMI-172)
To a solution of NaH (4.8 g, 0.12 mol) in THF (150 mL) was added triethylphosphonoacetate (23.8 mL, 0.12 mol) dropwise at 0° C. for 10 minutes. To the stirred mixture was added cyclobutanone (7.5 mL, 0.10 mol) (for q=2, cyclopentanone was added instead of cyclobutanone). After 1 hour at room temperature, the reaction mixture was poured into saturated aqueous NH4Cl and was extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (5% EtOAc in hexanes) to give 46 (13.66 g, 96%).
Compound 46 was hydrogenated at 40 psi with Pd/C in ethanol (EtOH) to afford compound 47 in 84% yield.
Compound 47 was reduced to an aldehyde with diisobutylaluminum hydride (DIBAL-H) at −78° C. and the aldehyde was reacted with vinylmagnesium bromide at −20° C. to yield compound 48 (39% for two steps).
To a solution of compound 48 (2.158 g, 17.1 mmol) and 1,5-hexadiene (1.52 mL, 12.83 mmol) in CH2Cl2 was added SOBr2 (2.0 mL, 25.65 mmol) at 0° C. After the mixture was stirred at 0° C. for 45 min, the reaction was quenched by the addition of H2O and stirred at 0° C. for 15 minutes. The mixture was extracted with CH2Cl2. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (hexanes) to give compound 49 (2.85 g, 88%).
To a solution of compound 49 (2.4 g, 12.69 mol) in acetone (40 mL) was added NaI (2.47 g, 16.50 mmol). After 1 hour at room temperature, the reaction was quenched by the addition of H2O. The mixture was concentrated under reduced pressure and the remaining aqueous residue was extracted with EtOAc. The organic layer was washed with saturated aqueous Na2S2O3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (hexanes) to give compound 50 (2.43 g, 81%).
Compound 30 was prepared from compound 50 in 71% yield following Evan's protocol (J. Med. Chem. 33:2335-2342 (1990)).
To a solution of compound 52 (2.1 g, 6.15 mol) in ethylene glycol dimethyl ether (DME)/H2O (1:1, 40 mL) was added N-bromosuccinamide (NBS) (1.2 g, 6.77 mmol) at 0° C. After stirring for 45 minutes at 0° C., the reaction was quenched by the addition of H2O and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3, brine and dried over MgSO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (10% EtOAc in hexanes) to give compound 52 (727 mg, 45%).
The reaction of compound 52 with NaN3 in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) at room temperature for 3 days yielded compound 32 (65%). Completion of the isostere synthesis was accomplished by hydrolysis of the lactone with LiOH, TBS protection of the resulting alcohol (see Part I, step F), and hydrogenation of the azide following the standard hydrogenation procedure described previously (Section II, Step F).
H. Isosteres in MMI-162, MMI-163, MMI-168, MMI-169 are Described in the Following Scheme:
The synthesis of MMI-162 and MMI-163 used one isomer of compound 58, and the synthesis of MMI-168 and MMI-169 used the other isomer of compound 58.
Amide bonds in inhibitors of the invention were generally created through 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt)-mediated coupling of the appropriate carboxylic acid and amine. An example is given below for the coupling of isostere 6 and amine-containing compound 62.
Boc-protected amine compound 62 (71 mg, 0.10 mmol) was dissolved in CH2Cl2 (3 mL) and TFA (0.75 mL) was added at room temperature. The reaction mixture was stirred for 30 minutes followed by concentrating under reduced pressure to provide the free amine (61 mg, quantitative). Leucine-alanine isostere 6 (42 mg, 0.1 mmol) was dissolved in dichloromethane (DCM) (2 mL). To this solution, HOBt (20 mg, 0.15 mmol) and EDCI (29 mg, 0.15 mmol) were added successively at room temperature and stirred for 5 minutes. To this solution was added dropwise a solution of the above free amine (41 mg, 0 2 mmol) and diisopropylethylamine (0.2 mL) and the resulting mixture was stirred overnight. The mixture was poured into H2O and extracted with EtOAc, dried over Na2SO4 and concentrated under reduced pressure. Flash column chromatography (20% EtOAc in hexanes) yielded compound 63 (57 mg, 95%). 1H-NMR (500 MHZ, CDCl3) δ 0.09 (s, 3H), 0.10 (s, 3H), 0.91 (s, 9H), 0.92-0.98 (m, 12H), 1.10 (d, 3H, J=6.7 Hz), 1.25 (m 1H), 1.44 (m, 1H), 1.46 (s, 9H), 1.63 (m, 1H), 1.74 (br s, 1H), 1.80 (m, 1H), 2.18 (m, 1H), 2.56 (m, 1H), 3.62-3.78 (m, 2H), 4.13 (m, 1H), 4.48-4.56 (m, 3H), 6.35 (br d, 1H, J=8.5 Hz), 6.41 (br s, 1H), 7.26-7.40 (m, 5H).
A. General Synthetic Methods
Inhibitor MMI-138 (also referred to herein as MMI-138, OM-138, GT-138) was synthesized employing a N-(3,5-dimethylpyrazole-1-methoxy carbonyl)-L-methionine and Boc-Leu-Ψ-Ala-Val-NHCH2Ph according to a procedure described by Ghosh, et al. 2001 (Ghosh, A. K., et al., J. Med. Chem. 44:2865-2868 (2001), the teachings of which are incorporated herein by reference in their entirety. N-(3,5-dimethylpyrazole-1-methoxy carbonyl)-L-methionine was prepared by alkoxycarbonylation of methionine methyl ester with commercially available 3,5-dimethylpyrazole-1-methanol (Aldrich Chemical) followed by saponification with aqueous lithium hydroxide (36% overall) as described by Ghosh, et al. 1992 (Ghosh, A. K., et al., Tetrahedron Letter 22:781-84 (1992), the teachings of which are incorporated herein by reference in their entirety). Removal of the Boc (t-butoxycarbonyl) group of compound 43 shown below (Ghosh, A. K., et al., J. Med. Chem. 44:2865-2868 (2001), the teachings of which are incorporated herein by reference in their entirety) by treatment with trifluoroacetic acid in dichloromethane gave the corresponding amine which was reacted with N-(3,5-dimethylpyrazole-1-methoxy carbonyl)-L-methionine in the presence of N-ethyl-N′-(dimethylaminopropyl)-carbodiimide hydrochloride, diisopropylethylamine and 1-hydroxybenzotriazole hydrate in dichloromethane to compound MMI-138 in 50% yield.
Other compounds of the invention in which R1 or R18 is a heteroazaaralkoxy group were prepared using the above-described method in which the various heteroazaaralkyl-alcohols in Table 4 were used instead of 3,5-dimethyl-pyrazole-1-methanol.
In a typical procedure as outlined in Scheme XVII, L-methionine methyl ester hydrochloride in methylene chloride was added, in the presence of a tertiary base, to a solution of triphosgene in methylene chloride (molar ratio 1:0.37) over a period of 30 minutes using a syringe pump to form an isocyanate intermediate. The alcohol component was then added to the above solution and stirred for 12 hours to provide the
urethane-methyl ester which was hydrolyzed with LiOH in 10% aqueous THF to give the corresponding acid.
(a) L-Methionine methylester hydrochloride, Et3N, CH2Cl2, 23° C., 30 minutes;
(b) 3,5-Dimethylpyrazol-1-yl)-methanol, CH2Cl2, 23° C., 12 hours;
(c) LiOH, 10% aqueous THF, 23° C. 3 hours.
Other heteroaralkyl-alcohols that may be employed in the synthesizing shown in Scheme XVII are listed in Table 5.
Table 6 lists memapsin inhibitors of the invention that were prepared that have a heteroazaaralkoxy R1 group. A representative example of the general synthesis of various inhibitors listed in Table 6 is outlined in Scheme XVIII.
Thus, valine derivative 67 (Scheme XVIII) was reacted with the known dipeptide isostere 6 (see Part I, step F) in the presence of N-ethyl-N′-(dimethylaminopropyl) carbodiimide hydrochloride, diisopropylethylamine, and 1-hydroxybenzotriazole hydrate in a mixture of DMF and CH2Cl2 to generate amide derivative. Compound 68 was initially exposed to trifluoroacetic acid (TFA) in CH2Cl2 to remove the Boc and silyl groups. Coupling of the resulting aminol with the compound 66 generated inhibitor MMI-138. All the other inhibitors containing different R1, P2′, and R3 groups were prepared following analogous procedures using the corresponding substituted heteroazaaralkoxy urethanes and valine (or leucine) derivatives.
The inhibitor MMI-139 was synthesized by the oxidation of MMI-138 with OXONE® in a mixture (1:1) of methanol and water at 23° C. for 12 hours as depicted on Scheme XIX.
A. Representative Synthesis of MMI-138 and MMI-139
To a stirred solution of triphosgene (132 mg, 0.44 mmol) in methylene chloride (2 mL) at 23° C., a solution of L-methionine methyl ester hydrochloride 50 (242 mg, 1.21 mmol) and triethylamine (0.42 mL, 3.03 mmol) in methylene chloride (4 mL) was added slowly over a period of 30 minutes using a syringe pump. After further 5 minutes of stirring, a solution of (3,5-dimethyl-pyrazol-1-yl)-methanol 49 (152 mg, 121 mmol) in methylene chloride was added in one portion. The reaction mixture was stirred for 12 hours, diluted with ethyl acetate, washed with water, brine dried over NaSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (50% EtOAC /Hexane) to give 143 mg (36%) of the compound 65. 1H-NMR (300 MHZ, CDCl3): δ 1.94-2.20 (2H, m), 2.0 (3H, s), 2.18 (3H, s), 2.26 (3H, s), 2.41 (2H, m), 3.74 (3H, s), 4.48 (11 μm), 5.50 (1H, br s), 5.82 (1H, s), 5.90 (2H, s).
In general, carbamates linkages of inhibitors of the invention were synthesized by the above method of coupling a compound having an alcohol group with a compound having an amine group using triphosgene. Urea linkages in inhibitors of the invention were formed by an analogous method in which triphosgene is used to couple two compound that have amine groups using the procedure described above.
To a stirred solution of above ester 65 (140 mg, 0.43 mmol) in a mixture of 10% aqueous THF (3 mL) was added LiOH (27 mg, 0.65 mmol). The mixture was stirred for 3 hours. After this period, solvents were removed and the residue was acidified with aqueous 1N HCl to pH-4. The white solid was extracted twice with ethyl acetate and the combined extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide compound 66 (134 mg, quantitative) which was carried on to the next step without further purification. 1H-NMR (300 MHZ, CDCl3): δ 1.94-2.20 (2H, m), 2.0 (3H, s), 2.18 (3H, s), 2.26 (3H, s), 2.48 (21 μm), 3.74 (3H, s), 4.40 (1H, m), 5.50 (1H, br s), 5.82 (1H, s), 5.90 (2H, s).
To a stirred solution of N-Boc-Valine (500 mg , 2.3 mmol) and benzylamine (0.50 mL, 4.60 mmol) in a mixture of CH2Cl2 (20 mL) and DMF (2 mL), HOBt (373 mg, 2.8 mmol), EDC (529 mg, 2.8 mmol) and diisopropylethylamine (2.4 mL, 13.8 mmol) were added successively at 0° C. After the addition, the reaction mixture was allowed to warm to 23° C. and it was stirred overnight. The mixture was poured into aqueous NaHCO3 solution and the mixture was extracted with 30% EtOAc/hexane. The organic layer was washed with brine and dried over Na2SO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by flash column chromatography (30% EtOAc/hexane) to give 442 mg (63%) of coupled product. The resulting amine was dissolved in CH2Cl2 (20 mL), and TFA (4 mL) was added at 23° C. The reaction mixture was stirred for 30 minutes and then it was concentrated under reduced pressure to provide the compound 67 (297 mg, quantitative). 1H-NMR (500 MHZ, CDCl3): δ 0.87 (31-1, d, J=6.9 Hz), 1.02 (3H, d, J=6.9 Hz), 2.00 (2H, br s), 2.37 (1H, m), 3.36 (1H, br s), 4.43-4.52 (2H, m), 7.27-7.37 (5H, m), 7.70 (1H, brs).
Dipeptide isostere 6 (42 mg, 0.1 mmol) and compound 67 (41 mg, 0.2 mmol) were dissolved in DMF (2 mL) To this solution, HOBt (20 mg, 0.15 mmol), EDC (29 mg, 0.15 mmol) and diisopropylethylamine (0.2 mL) were added successively at 0° C. After the addition, the reaction mixture was allowed to warm to 23° C. and it was stirred overnight. The mixture was poured into aqueous NaHCO3 and it was extracted with 30% EtOAc/hexane. The organic layer was washed with brine and dried over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (20% EtOAc/hexane) to give 55 mg (95%) of compound 68. 1H-NMR (500 MHZ, CDCl3) δ 0.09 (3H, s), 0.10 (3H, s), 0.91 (9H, s), 0.92-0.98 (12H, m), 1.10 (3H, d, J=6.7 Hz), 1.25 (1H, m), 1.44 (1H, m), 1.46 (9H, s), 1.63 (1H, m), 1.74 (1H, br s), 1.80 (1H, m), 2.18 (1H, m), 2.56 (1H, m), 3.62-3.78 (2H, m), 4.13 (1H, m), 4.48-4.56 (3H, m), 6.35 (1H, br d, J=8.5 Hz), 6.41 (1H, br s), 7.26-7.40 (5H, m).
To a solution of 68 (37 mg, 0.06 mmol) in CH2Cl2 (1 mL) was added TFA (0.4 mL) at 23° C. The resulting mixture was stirred at 23° C. for 1 hour, the concentrated under reduced pressure and the residue was dissolved in DMF (2 mL). To this solution, compound 66 (18 mg, 0.06 mmol), HOBt (8 mg, 0.06 mmol), EDC (11 mg, 0.06 mmol) and diisopropylethylamine (0.2 mL) were added successively at 0° C. After the addition, the reaction mixture was allowed to warm to 23° C. and it was stirred overnight. The mixture was poured into aqueous NaHCO3 and it was extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (2% MeOH/CHCl3) to provide the inhibitor MMI-138 (16 mg; 40%). 1H-NMR (300 MHZ, CD3OD): δ 0.80-0.97 (12H, m), 1.10 (3H, d, J=6.7 Hz), 1.20-2.38 (8H, m), 2.0 (3H, s), 2.18 (3H, s), 2.24 (3H, s), 2.41 (3H, t, J=6.4 Hz), 2.60 (1H, m), 3.41 (1H, m), 3.80 (1H, m), 4.15 (1H, m), 4.20-4.32 (3H, m), 5.80 (3H, s) 7.17-7.30 (5H, m).
To a solution of MMI-138 (10 mg, 0.015 mmol) in MeOH—H2O (1:1) (2 mL), were added NaHCO3 (11.6 mg, 0.12 mmol) and potassium peroxymonosulfate (OXONE®) (27 mg, 0.05 mmol) and stirred for 12 hours. The reaction was then diluted with ethyl acetate, washed with water and dried over anhydrous Na2SO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (4% MeOH/CHCl3) to provide the inhibitor MMI-139 (6.8 mg, 65%). 1H-NMR (300 MHZ, CD3OD): δ 0.72-0.92 (12H, m), 1.20 (3H, d, J=6.0 Hz), 1.15-2.06 (6H, m), 2.16 (3H, s), 2.24 (3H, s), 2.58 (3H, s), 2.82 (3H, s), 3.30 (2H, m), 3.60 (1H, m), 3.78 (1H, m), 4.0 (2H, m), 4.22 (1H, m), 4.34-4.38 (3H, m), 5.80 (3H, s), 7.18-7.36 (5H, m).
C. Other Inhibitors of the Invention
The following memapsin inhibitors of the invention were prepared via a method analogous to the method of preparing MMI-138 and MMI-139. The various R1 groups of the inhibitors listed below were obtained by substituting the appropriate heteroazaaryalkyl-alcohol listed in Table 1 for 3,5-dimethylpyrazol-1-yl)-methanol in the method of preparing the urethane portion of the molecule (see Scheme 10). A leucine side chain was obtained at the P2′ position in the inhibitors listed below by substituted N-Boc-leucine for N-Boc-valine in the method described in Section V-B(iii). Other natural and non-natural Boc-protected amino acids may be substituted for N-Boc-valine in the method described in Section V-B(iii) to obtain other P2′ groups in the inhibitors of the invention. Inhibitors having 2-methylprop-1-yl or 1-methyleth-1-yl R3 groups were obtained by substituting 2-methylpropyl amine or 1-methylethyl amine for benzylamine in the synthesis described Section V-B(iii). Other compounds containing amine groups may also be substituted for benzyl amine in the synthesis described in Section V-B(iii). For example, aliphatic amines, aryl amines, aralkyl amines, heterocycle amines, heterocycloalkyl amines, heteroaryl amines, heteroaralkyl amines, peptide or a carrier molecule containing amine groups may be used instead of benzylamine in the synthesis describe in Section V-B(iii). In addition, heterocycles or heteroaryl compounds that have secondary amines may be used instead of benzylamine in Section V-B(iii).
1H-NMR (300 MHZ, CD3OD): δ 0.80-0.90 (18H, m), 1.20 (3H, d, J=6.6 Hz), 1.18-2.04 (8H, m), 2.0 (3H, s), 2.17 (3H, s), 2.24 (3H, s), 2.42 (3H, t, J=6.2 Hz), 2.50 (1H, m), 2.80-3.30 (m, 2H), 3.41 (1H, m), 3.78 (1H, m), 3.90 (1H, d, J=6.8 Hz), 4.18 (1H, t, J=6.3 Hz), 5.80 (3H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.80-0.97 (12H, m), 1.40 (9H, m,), 1.18-2.20 (8H, m), 2.0 (3H, s), 2.18 (3H, s), 2.26 (3H, s), 2.50 (3H, m), 3.42 (1H, m), 3.80 (1H, m), 3.90 (2H, m), 4.20 (1H, m), 5.80 (3H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.80-0.96 (18H, m), 1.20 (3H, d, J=6.7 Hz), 1.06-2.20 (8H, m), 2.0 (3H, s), 2.17 (3H, s), 2.23 (3H, s), 2.38-2.60 (3H, m), 3.0 (2H, m), 3.42 (1H, m), 3.78 (1H, m), 4.2 (3H, m), 4.38 (1H, s), 5.80 (3H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.80-1.0 (19H, m), 1.10 (3H, d, J=6.2 Hz), 1.20-2.26 (8H, m), 2.0 (3H, s), 2.18 (3H, s), 2.3 (3H, s), 2.5 (2H, m), 2.6 (3H, m), 3.40 (1H, m), 4.10 (1H, m), 4.20 (1H, m), 4.44 (1H, s), 5.84 (3H, s).
1H-NMR (500 MHZ, CD3OD): δ 0.78-0.85 (18H, m), 1.10 (3H, d, J=6.2 Hz), 1.20-2.0 (9H, m), 1.93 (3H, s), 2.11 (3H, s), 2.15 (3H, s), 2.42 (2H, t, J=5.1 Hz), 2.55 m), 2.80 (1H, m), 3.10 (1H, m), 3.40 (1H, m), 3.80 (2H, m) 3.90 (1H, m), 4.10 (2H, m), 4.2 (2H, m), 5.7 (1H, s).
1H-NMR (500 MHZ, CD3OD): δ 0.75-0.81 (18H, m), 1.0 (3H, d, J=6.8 Hz), 1.1 (9H, s), 1.2 (9H, s), 1.10-2.0 (9H, m), 1.90 (3H, s), 2.35 (2H, t, J=5.3 Hz), 2.60 (1H, m), 2.80 (1H, m), 2.90 (1H, m), 3.30 (1H, m), 3.60 (1H, m) 3.90 (1H, m), 4.10 (1H, m), 4.20 (2H, m), 5.70 (1H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.82-1.15 (18H, m), 1.19 (3H, d, J=6.2 Hz), 1.21 (6H, s), 1.23 (6H, s), 1.22-2.60 (8H, m), 2.30 (3H, s), 2.54 (2H, t, J=5.0 Hz), 2.60 (1H, m), 2.82-3.18 (3H, m), 3.60 (1H, m), 3.82 (1H, m) 4.12 (1H, m), 4.2 (2H, m), 4.4 (2H, m), 5.82 (1H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.77-0.85 (18H, m), 1.10 (3H, d, J=6.0 Hz), 1.16-2.0 (9H, m), 1.98 (3H, s), 2.42 (2H, t, J=5.6 Hz), 2.50 (1H, m), 2.84 (1H, m), 3.00 (1H, m), 3.40 (1H, m), 3.72 (1H, m), 3.78 (3H, s), 3.94 (1H, m) 4.18 (1H, m), 5.0 (2H, s), 6.20 (1H, s), 7.36 (1H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.77-0.85 (18H, m), 1.20 (3H, d, J=6.4 Hz), 1.18-2.05 (9H, m), 2.03 (3H, s), 2.16 (3H, s), 2.4-2.6 (3H, m), 2.84-2.98 (2H, m), 3.44 (1H, m), 3.57 (3H, s), 3.80 (1H, s), 3.98 (1H, s), 4.20 (1H, s), 5.0 (2H, s), 7.31 (1H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.78-0.92 (18H, m), 1.05 (3H, d, J=5.8 Hz), 1.20-2.05 (9H, m), 2.03 (3H, s), 2.13 (3H, s), 2.4-2.6 (3H, m), 2.84-3.40 (2H, m), 3.44 (1H, m), 3.47 (3H, s), 3.78 (1H, m), 3.98 (1H, m), 4.20 (1H, m), 5.05 (2H, s), 6.65 (1H, s).
1H-NMR (300 MHZ, CD3OD): δ 0.78-0.92 (18H, m), 1.05 (3H, d, J=6.6 Hz), 1.18-2.02 (9H, m), 2.02 (3H, s), 2.15 (3H, s), 2.46 (2H, t, J=5.8 Hz), 2.56 (1H, m), 2.84-2.96 (1H, m), 3.00 (1H, m), 3.44 (1H, m), 3.72 (3H, s), 3.78 (1H, m), 3.98 (1H, s) 4.22 (1H, m), 4.97 (2H, s), 5.99 (1H, s).
Synthesis of compounds used in the preparation of inhibitors of the invention that are not commercially available are described below.
A. Synthesis of Starting Material for Inhibitors Having Heteroazaaralkyl R1 Groups
General procedure (J. Gen. Chem. (UUSR) 33:511 (1963)): A mixture of 1,3-dimethylpyrazole (395 mg, 4.11 mmol) and 2-methyl acrylic acid methyl ester (1.0 mL) were heated in a sealed tube at 200° C. for 4 hours. The reaction was cooled to room temperature, the solvent was removed under reduced pressure and the residue was chromatographed (35% EtOAc in hexanes) to afford 51 (470 mg, 58%) which was used to prepare inhibitors MMI-195, MMI-196, MMI-214, and MMI-226. Pyrazoles 72 and 73 were synthesized using analogous procedures. Compound 72 was used to prepare inhibitors MMI-194 and MMI-213, and compound 73 was used to prepare inhibitors MMI-204, MMI-225, MMI-228 and MMI-229. Hydrolysis of the methyl esters was accomplished by stirring the ester in a room temperature saturated solution of LiOH in 10% aqueous THF, for 3-48 hours.
B. Synthesis of Starting Material for Inhibitors Having Heteroazaaralkoxy R1
A solution of 2-hydroxyethylhydrazine (1.02 mmol) in absolute ethanol (1 mL) was added dropwise to a solution of the corresponding diketone (1.0 mmol) at 0° C. The mixture was warmed to room temperature and stirred for 1 hour. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 and washed with water. The organic layer was dried with Na2SO4, concentrated, and purified by flash chromatography (60% EtOAc in hexanes) to yield the product.
To a solution of compound 77 (184 mg, 1 mmol) in acetone/H2O (3:1, 20 mL) was added N-methyl morpholine N-oxide (292 mg, 2.5 mmol) followed by OsO4 (0.38 mL, 2 wt % in t-BuOH, 0.03 mmol) and stirred overnight. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 and washed with water. The organic layer was dried with Na2SO4, concentrated under reduced pressure and chromatographed (4% MeOH in CHCl3) to yield compound 78 (110 mg, 51%).
Methylmagnesium bromide (5.4 mL, 1.4 M in THF, 7.6 mmol) was added dropwise to a solution of (3,5-dimethylpyrazole-1-yl)-acetic acid ethyl ester (J. Med. Chem., p. 1659 (1983)) (compound 79, 554 mg, 3.04 mmol) in THF at 0° C. After 30 minutes the reaction was quenched with saturated aqueous NH4Cl and extracted with EtOAc. The organic layer was dried with Na2SO4, concentrated, and purified by column chromatography (40% EtOAc in hexanes) to yield 276 mg (65%) of compound 80.
C. Synthesis of Additional Starting Materials for Inhibitors Having Heteroazaaralkoxy R1 Groups
The following heteroazaaralkyl-alcohol starting materials were synthesized via the method described in the cited reference.
D. Synthesis of Boc-Protected Non-Natural Amino Acid Having a Tetrahydrofuranylmethyl Side Chain Used to Form P1 Substituent of Inhibitors MMI-013, MMI-014, MMI-019, MMI-020, MMI-034, MMI-035, MMI-205 and MMI-215:
To a solution of compound 81 (J. Med. Chem., p. 495-505 (1997)) (1.17 g, 4.8 mmol) in diethylether (20 mL) at −78° C. was added dropwise allylmagnesium bromide (7.5 mL, 1.0 M in diethylether, 7.5 mmol). After stirring for 30 min, the reaction was quenched with saturated aqueous NH4Cl at −78° C. The mixture was warmed to room temperature and the layers were separated. The organic layer was dried with Na2SO4 and concentrated under reduced pressure. The diastereomers were separated by flash column chromatography (25% EtOAc in hexanes) to yield 500 mg (37%) of the faster isomer and 630 mg (46%) of the slower isomer. The remainder of the synthesis was carried out on each of the isomers separately to prepare non-natural amino acid used to form inhibitors MMI-205 and MMI-215.
Non-natural amino acids used to prepare inhibitors MMI-013, MMI-014, MMI-019, MMI-020, MMI-034, MMI-035 were synthesized by the same protocol using the appropriate aldehyde with one less methylene (Bioorg. Med. Chem. Lett. 8:179-182 (1998)).
9-borabicyclo[3.3.1]nonane (9-BBN) (3.86 mL, 0.5 M in THF, 1.93 mmol) was added to a solution of the product from Step 1 (500 mg, 1.75 mmol) in TIM (5 mL) and stirred for 12 h, after which time the reaction mixture was cooled to −20° C. and MeOH (0.13 mL), 3 N NaOH (0.87 mL), and 30% H2O2 (0.87 mL) were added sequentially. The reaction mixture was warmed to 60° C. and stirred for 1 hour. The resulting clear solution was poured into brine (25 mL), extracted with diethylether, dried with Na2SO4, concentrated, and purified by flash column chromatography (70% EtOAc in hexanes) to yield 280 mg (53%) of the product.
To a solution of the product from step 2 (112 mg, 0.34 mmol) in CH2Cl2 (3 mL) was added triethylamine (0.1 mL, 0.74 mmol), p-toluene sulfonyl chloride (78 mg, 0.41 mmol), dimethylaminopyridine (9 mg, 0.07 mmol) sequentially and the reaction was stirred at room temperature for 12 hours, after which it was diluted with CH2Cl2 and washed with saturated aqueous NH4Cl, dried with Na2SO4, concentrated under reduced pressure and purified by column chromatography (20% EtOAc in hexanes) to yield 83 mg (86% of the corresponding tetrahydrofuran.
To a stirred solution of the tetrahydrofuran prepared in step 3 in MeOH (3 mL) was added p-toluene sulfonic acid hydrate (13 mg, 0.07 mmol) and stirred at room temperature for 1 hour. The reaction was then quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was dried with Na2SO4, concentrated and chromatographed (50% in EtOAc in hexanes) to yield compound 82 (55 mg, 65%).
The compound 82 was oxidized to the corresponding carboxylic acid using H5IO6/CrO3 in wet CH3CN via the following procedure (Tetrahedron Lett., p. 5323 (1998)): A stock solution of H5IO6/CrO3 was prepared by dissolving H5IO6 (11.4 g, 50 mmol) and CrO3 (23 mg, 1.2 mol %) in wet CH3CN (0.75 v % water) to a volume of 114 mL (complete dissolution typically required 1-2 h). The H5IO6/CrO3 solution (0.7 mL) was then added to a solution of compound 82 (30 mg, 0.12 mmol) in wet CH3CN (1 mL) over a period of 30 minutes 0° C. The reaction was quenched by adding aqueous Na2HPO4. The mixture was extracted with diethylether and the organic layer was washed with brine, aqueous Na2HPO4, brine, dried with Na2SO4, and concentrated under reduced pressure. The crude yield of 83 was 22 mg (69%).
E. Synthesis of Cbz-Protected Non-Natural Amino Acid Having a Methoxymethoxyethyl Side Chain Used to Form P2 Substituent of Inhibitor MMI-190:
To a solution of Cbz-protected homoserine (J. Org., Chem., 5442 (1997)) (60, 140 mg, 0.52 mmol) in CH2Cl2 (3 mL) at 0° C. were added diisopropylethylamine (DIPEA) (0.28 mL, 1.6 mmol) and chloromethylmethylether (MOMCI) (0.05 mL, 0.62 mmol). After stirring for 3 h, the reaction was quenched with saturated aqueous NH4Cl and extracted with diethylether. The organic layer was dried with Na2SO4, concentrated under reduced pressure and chromatographed (30% EtOAc in hexanes) to yield 116 mg (71%) of compound 85. Removal of the Cbz protecting group by hydrogenation provided the free amine for coupling.
F. Synthesis of Boc-Protected Non-Natural Amino Acid Having a Methoxyethyl Side Chain Used to Form P2 Substituent of Inhibitors MMI-079, MMI-185, MMI-228:
To a solution of Boc-protected homoserine (86, 400 mg, 1.83 mmol) in DMF (8 mL) at 0° C. were added NaH (60%, 155 mg, 4.02 mmol) followed by MeI (0.45 mL, 7.3 mmol). The reaction was stirred for 12 hours at room temperature. The DMF was removed under reduced pressure. The residue was dissolved in EtOAc and washed with saturated aqueous NH4Cl, dried with Na2SO4, concentrated under reduced pressure and chromatographed (20% EtOAc in hexanes) and yielded 323 mg (72%) of compound 87. Compound 87 was hydrolyzed with LiOH (as described above) to quantitatively yield the free acid.
G. Synthesis of Starting Materials for Macrocyclic Inhibitors MMI-149, MMI-150, MMI-152, MMI-153, MMI-174, and MMI-175 and Macrocyclic Inhibitor Precursors MMI-148, MMI-151, and MMI-173
EDCI/HOBt coupling of Boc-Asp methyl ester with allyl amine (see Section IV) was followed by TFA removal of the Boc protecting group and coupling with various Val derivatives 89 (these carbamates were produced by triphosgene coupling of Val methyl ester with various alcohols—allyl alcohol, 4-butenol, and 5-pentenol) (see Section V-B(i). The compounds represented by structure 90 were incorporated into inhibitors MMI-148, MMI-151, and MMI-173 by hydrolysis followed by coupling of the free acid. The macrocycles were formed using ring-closing olefin metathesis to form the macrocyclic group in inhibitors MMI-149, MMI-150, MMI-152, MMI-153, MMI-174, and MMI-175. A representative procedure for the formation of the macrocyclic group follows:
To a 0.002 M solution of the diene (90) in CH2Cl2 was added Grubbs's catalyst (20 mol %). The flask was flushed with Argon and stirred at room temperature for 12 hours. The solvent was removed under reduced pressure and the residue was chromatographed (2% MeOH in CHCl3) to yield approximately 75% of the desired macrocycle. This metathesis step was followed by LiOH hydrolysis and yielded the free acid for further coupling. This produced ligands for MMI-149, MMI-152, and MMI-174.
To prepare inhibitors MMI-150, MMI-153, and MMI-175 inhibitors MMI-149, MMI-152, and MMI-174, respectively, were hydrogenated following the standard hydrogenation procedure described previously (Section II, Step F).
H. Synthesis of 2-methyl-1-(tetrahydrofuran-2-yl)-propylamine and 2-methyl-1-(tetrahydro-pyran-2-yl)-propylamine Used to Form R3 Substituent of Inhibitors MMI-154 and its Pyran Derivative:
To a solution of compound 92 (Angew. Chem., Int. Ed. Engl. 11:1141 (1988)) (530 mg, 1.64 mmol) in THF at 0° C. were added NaH (60%, 130 mg, 3.28 mmol) and allyl iodide (0.23 mL, 2.46 mmol) and stirred for 12 hours at room temperature. The reaction was quenched with saturated aqueous NH4Cl, extracted with diethylether, dried with Na2SO4, concentrated under reduced pressure, and chromatographed (2% EtOAc in hexanes) to yield 530 mg (90%) of allyl ether 93.
To a solution of compound 93 (200 mg, 0.55 mmol) in 100 mL of CH2Cl2 was added Grubbs's catalyst (20 mg, 5 mol %) and the mixture was refluxed under argon for 2 hours. The solvent was removed under reduced pressure and the residue was chromatographed (3% EtOAc in hexanes) to provide 171 mg (93%) of dihydropyran 94.
A mixture of compound 94 (135 mg, 0.4 mmol) and Pd(OH)2/C (20%, 20 mg) in MeOH was stirred under an H2 atmosphere for 5 hours. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to yield compound 95 quantitatively.
I. Synthesis of 3-(1-amino-2-methyl-propyl)-5-benzyl-cyclohexanone (100) and 1-(3-benzyl-cyclohexyl)-2-methyl-propylamine (101) used to form the R3 Substituent of Inhibitors MMI-140, MMI-141, MMI-146, and MMI-147:
To a solution of 96 (930 mg, 2.8 mmol) in CH2Cl2 (10 mL) at 0° C. were added Et3N (1.2 mL, 8.64 mmol) and acryloyl chloride (0.3 mL, 3.74 mmol). The reaction was stirred at room temperature for 1 hour and quenched with saturated aqueous NH4Cl. The aqueous layer was extracted with diethylether and the combined organic layers were dried with Na2SO4, concentrated under reduced pressure and chromatographed (4% EtOAc in hexanes) to yield lactone 97 (700 mg, 66%).
Ring-closing olefin metathesis following the same procedure as previously described in Section VI, Part G was performed and yielded compound 98 in 89% yield.
To a solution of compound 98 (75 mg, 0.2 mmol) in diethylether was added CuCN (2 mg, 10 mol %). The mixture was cooled to −78° C. and PhCH2MgCl (0.24 mL, 1.0 M in diethylether, 0.24 mmol) was added dropwise. The reaction was allowed to warm to room temperature over a period of 1 hour and quenched with saturated aqueous NH4Cl, extracted with diethylether. The organic layer was dried with Na2SO4, concentrated under reduced pressure, and chromatographed (25% EtOAc in hexanes) to yield compound 98 (47 mg, 50%).
Hydrogenation of compound 99 to remove the benzyl protecting groups as previously described (Section II, Step F) led to 3-(1-amino-2-methyl-propyl)-5-benzyl-cyclohexanone (100) which was used to prepare inhibitors MMI-140 and MMI-141.
DIBAL-H (1.28 mmol, 1.0 M in hexanes, 1.28 mmol) was added to a solution of compound 99 (225 mg, 0.57 mmol) in toluene (3 ml.) at −78° C. and stirred for 30 minutes. The reaction was quenched with aqueous Na-K-tartrate, warmed to room temperature, and extracted with diethylether. The organic layer was dried with Na2SO4 and concentrated under reduced pressure to yield the crude lactol.
The crude lactol was dissolved in CH2Cl2 (5 mL), cooled to 0° C., and Et3SiH (0.12 mL, 0.75 mmol) and BF3.OEt2 (0.07 mL, 0.55 mmol) were added successively. After 30 minutes, the reaction was quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was dried with Na2SO4, concentrated under reduced pressure, and chromatographed to afford the corresponding tetrahydropyran (175 mg, 80%) which was hydrogenated to remove the benzyl protecting groups as previously described to afford compound 101. Compound 101 was used to prepare inhibitors MMI-146 and MMI-147.
J. Synthesis of 4-amino-6-methyl-1-phenyl-heptan-3-ol used to form the P2′-P3′ Substituents of Inhibitor MMI-091:
To a mixture of know oxazolidinone 81 (J. Org. Chem. 63:6146-6152 (1998)) (80 mg, 0.33 mmol) and 10% Pd/C (15 mg) in MeOH (4 mL) was stirred under an H2 atmosphere for 1 hour. The catalyst was filtered off and the filtrate was concentrated under reduced pressure and chromatographed (40% EtOAc in hexanes) to yield 48 mg (61%) of the saturated product.
To a solution of the product of step 1 (48 mg, 0.19 mmol) in EtOH/H2O (1:1, 4 mL) was added KOH (45 mg, 0.78 mmol) and stirred for 12 hours. The reaction was then acidified to pH 3 with 1 M HCl, extracted with CHCl3, dried with Na2SO4, and concentrated under reduced pressure to yield 35 mg (83%) of 82.
K. Preparation of Sulfone Ligand of MMI-003, MMI-007, MMI-009, MMI-016, MMI-018, MMI-024, MMI-026, MMI-035, MMI-043, MMI-045, MMI-047, MMI-052, MMI-054, MMI-056, MMI-058, MMI-060, MMI-067, MMI-069, MMI-071, MMI-073, MMI-082, MMI-088, MMI-090, MMI-096, MMI-098, MMI-100, MMI-105, MMI-122, MMI-123, MMI-126, MMI-128, MMI-129, MMI-135, MMI-136, MMI-137, MMI-139:
Inhibitor MMI-139: To a solution of MMI-138 (10 mg, 0.015 mmol) in MeOH—H2O (1:1) (2 mL), were added NaHCO3 (11.6 mg, 0.12 mmol) and Oxone® (potassium peroxymonosulfate) (27 mg, 0.05 mmol) and stirred for 12 hours. The reaction was diluted with ethyl acetate, washed with water and dried with Na2SO4. Evaporation of the solvent under reduced pressure gave a residue which was purified by column chromatography (4% MeOH in CHCl3) to provide the inhibitor MMI-139 (6.8 mg, 65%). 1H-NMR (300 MHZ, CD3OD): δ 0.72-0.92 (12H, m), 1.20 (3H, d, J=6.0 Hz), 1.15-2.06 (6H, m), 2.16 (3H, s), 2.24 (3H, s), 2.58 (3H, s), 2.82 (3H, s), 3.30 (2H, m), 3.60 (1H, m), 3.78 (1H, m), 4.0 (2H, m), 4.22 (1H, m), 4.34-4.38 (3H, m), 5.80 (3H, s), 7.18-7.36 (5H, m).
L.
M. Literature References for Other Starting Materials:
The following starting materials were prepared as described in the cited references. The teachings of all of the references cited below are incorporated herein by reference.
All other fragments needed for the synthesis of inhibitors of the invention are commercially available and were coupled using the appropriate procedures described above.
An aliquot of the inhibitor of known concentration in DMSO was diluted into 1.8 ml 0.1 M NaOAc, pH 4.0. DMSO and added to a final concentration of 10% (v/v), and memapsin 2 (final concentration of 80 nM), followed by a 20 minute equilibration at 37° C. Compounds were evaluated for the ability to inhibit memapsin 1 and memapsin 2 at concentrations between about 10 nM and about 10 μm of inhibitor. Proteolytic activity in presence of inhibitor was measured by addition of 20 μl of 300 μM substrate FS-2 dissolved in DMSO and increase in fluorescence intensity measured as previously described (Ermolieff, J., et al., Biochemistry 39:12450-12456 (2000), the teachings of which are incorporated herein by reference in their entirety).
The Kiapp (apparent Ki) values of inhibitors against memapsins 1 and 2 were determined employing previously described procedures (Ermolieff, J., et al., Biochemistry 39:12450-12456 (2000), the teachings of which are incorporated herein by reference in their entirety). The relationship of Ki (independent of substrate concentration) to Kiapp is a function of substrate concentration in the assay and the Kin for cleavage of the substrate by either memapsin 1 or memapsin 2 by the relationship: Kiapp=Ki (1+[S]/Km).
Memapsin 1 is a protease that is closely homologous to memapsin 2 (also referred to herein as BACE, ASP2, β-secretase). Memapsin 2 catalyzes cleavage of β-amyloid precursor protein (APP) to produce β-amyloid (Aβ) peptide (also referred to herein as β-amyloid protein or β-amyloid peptide). Accumulation of Aβ peptide is associated with Alzheimer's disease. Memapsin 1 hydrolyzes the β-secretase site of APP, but is not significantly present in the brain. Further, there is no direct evidence the memapsin 1 activity is linked to Alzheimer's disease. The residue specificity of eight memapsin 1 subsite is: in positions P4, P3, P2, P1, P11, P2′, P3′ and P4 of the substrate, the most preferred residues are Glu, Leu, Asn, Phe, Met, Ile, Phe and Trp; while the second preferred residues are Gln, Ile, Asp, Leu, Leu, Val, Trp and Phe. Other less preferred residues can also be accommodated in these positions of the substrates. Some of the memapsin 1 residue preferences are similar to those of human memapsin 2, as described above. One embodiment of Applicants' invention is an N-terminal blocking group at P3 of the inhibitor to attain the selectivity of the inhibitor for memapsin 2 activity over memapsin 1 activity. For example, compound MMI-138 with a dimethylpyrazole group at P3 resulted in an inhibitor with a Ki value about 60 times lower for memapsin 2 relative to memapsin 1 (see Table 1).
Determination of Side Chain Preference in Memapsin 1 Subsites
The relative hydrolytic preference of memapsin 1 at all eight positions of the peptide substrate is depicted in
Farzan, et al. (Proc. Natl. Acad. Set., USA 97:9712-9717 (2000), the teachings of which are incorporated herein by reference in their entirety) reported that memapsin 1 hydrolyzes APP preferentially at two sites in the sequence, between phe-phe and phe-ala in the sequence KLVFFAED (SEQ ID NO: 42). Based on specificity data described herein, either cleavage site has the most favored residue Phe at P1 and medium or high ranking residues at P2, Pi', P2′ and P3′. P2, P4 and one of the P4 residues are clearly unfavorable (
The screening of memapsin 1 binding to a combinatorial inhibitor library produced about 30 darkly stained beads. The sequences of fourteen of the darkest ones produced consensus residues in three of the four randomized positions on the substrate: P3, Leu>Ile; P2, Asp>Asn/Glu; P2′ Val (Table 5). Side chain P3′ did not produce clear consensus. Leu and Trp and Glu, which appeared more than once, are also preferred in substrate hydrolysis (
Comparison on Subsite Preferences of Memapsin 1 and Memapsin 2
As discussed above, the overall substrate specificity of memapsin 1 subsites is similar to that for memapsin 2. As shown in Table 4, the top side chain preferences are either identical (for P4) or differ only in the order of preference (for P1, P2, P3 and P2′).
The two memapsins differ in residue preferences at the least specific P3′ and P4 positions. The close similarity in consensus inhibitor residues at positions, P3, P2 and P2′ are also seen from the inhibitor library (Table 7). In contrast to the preference of Glu and Gln in memapsin 2 sub-site S3′, memapsin 1 failed to show a preference in this sub-site. The P3′ side chain may interact poorly with memapsin 1 S3′ site. Poor binding of both P3′ and P4′ has been observed for the binding of inhibitor OM99-2 to memapsin 2.
Implications on the Design of Selectivity for Memapsin 2 Inhibitors
β-secretase, also referred to herein as memapsin 2 or Asp 2, has been implicated in Alzheimer's disease since it cleaves the β-secretase site of β-amyloid precursor protein (APP) to generate β-amyloid (Aβ) protein which is localized in the brain. Memapsin 1 is a weak β-secretase enzyme compared to memapsin 2 and is not localized in the brain. Differences in the tissue distribution and β-secretase activity of memapsin 1 and memapsin 2 indicates they have different physiological functions.
The capping group (also referred herein as “blocking group”) at position P3 in memapsin 2 inhibitors of the invention was evaluated to create selectivity of memapsin 2 inhibition. Small, yet potent, memapsin 2 inhibitors can be achieved by the elimination of the P4 and the substitution of P3 residue with a capping group as described above. New inhibitor MMI-138 (also referred to herein as “GT-1138”), which differs from inhibitor compound MMI-017 by a dimethylpyrazole group instead of a Boc at the N-terminus, produced a Ki for memapsin 2 about 60 fold lower than the Ki for memapsin 1 (Table 9, a blank space in the Table indicating that the value was not determined). Other inhibitors containing P3 pyrazole capping group exhibited similar selectivity toward memapsin 2 (Table 9, % Inh (Ml/M2)), whereas compounds with standard amino acid side chains in the P3 position did not (Table 9).
The data depicted in
For effective penetration of the blood-brain barrier, memapsin 2 inhibitor drugs should be small in size. In view of the close similarity in inhibitor specificity of memapsin 1 and memapsin 2, a P3 blocking group and other blocking groups to enhance binding and selectivity of memapsin 2 inhibitors were employed to design selective memapsin 2 inhibitors with desirable characteristics (e.g., appropriate size to penetrate the blood brain barrier, minimal peptide bonds, maximal hydrophobicity). The synthesis of inhibitors is described above and then Ki, Kiapp and relative selective inhibition are listed in Tables 1 and 9.
aAmino acid residues are shown in one-letter code
bMemapsin 2 (amino acid residues 43-456 of SEQ ID NO: 8 (FIG. 11) and amino acid residues 45-456 of SEQ ID NO: 8 (FIG. 11)).
aLibrary template: Gly-P3-P2-Leu*Ala-P2-P3′-Phe-Arg-Met-Gly-Gly-resin (SEQ ID NO: 21). The asterisk “*” denotes a hydroxyethylene
bNot determinable
cNegative controls are randomly selected beads with no memapsin 1 binding capacity
aHeteroaralkyl or heteraaralkoxy derivative attached at P3 carbonyl, or standard P3 amino acid side chain
bValues of 1 or less indicate inhibition which is greater towards memapsin 2 than memapsin 1
cLarger values indicate inhibitors with greater affinity to memapsin 2 than to memapsin 1
dPercentage of inhibition of proteolytic activity measured under conditions where the enzyme concentration equals the compound concentration
e“Mep 2” and “M2” refer to memapsin 2
f“Mep 1” and “M1” refer to memapsin 1
The hallmark of the Alzheimer's disease (AD) is a progressive degeneration of the brain caused by the accumulation of amyloid beta peptide, as referred to herein as β-amyloid protein (Selkoe, D. J., Physiol Rev 81:741-66 (2001)). The first step in the production of β-amyloid protein is the cleavage of a membrane protein called amyloid precursor protein (APP) by a protease known as the β-secretase, which has been identified as a membrane anchored aspartic protease termed memapsin 2 (or BACE or ASP-2). A first-generation inhibitor OM99-2 (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000)) was designed based on substrate information (Lin, X., et al., Proc Natl Acad Sci USA 97:1456-60 (2000), the teachings of which are incorporated herein by reference in their entirety) which is an eight-residue transition-state analogue, EVNL*AAEF (SEQ ID NO: 20) with Ki near 1 nM (Ermolieff, J., et al., Biochemistry 39:12450-6 (2000)). A 1.9-Å crystal structure of the catalytic unit of memapsin 2 bound to OM99-2 (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety) revealed that the conformation of the protease and the main features of its active site are those of the aspartic proteases of the pepsin family. All eight residues of OM99-2 were accommodated within the substrate-binding cleft of memapsin 2. The locations and structures of six memapsin 2 subsites for the binding of residues P4 to P2′ of OM99-2 were clearly defined in the structure (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety). This part of the inhibitor assumed an essentially extended conformation with the active-site aspartyls positioned near the transition-state isostere between P1 and P1′. Unexpectedly, the backbone of the inhibitor turned at P2′ Ala, departing from the extended conformation, to produce a kink. With less defined electron density, the side chains of P3′ Glu and P4′ Phe appeared to be located on the molecular surface and to have little interaction with the protease. These observations led to the idea that the S3′ and S4′ subsites in memapsin 2 were not well formed and perhaps contributed little to the interaction with substrates and inhibitors (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety).
The detailed subsite preferences of memapsin 2 was determined as described above and by using preferred binding residues selected from a combinatorial inhibitor library, a second-generation inhibitor OM00-3, Glu-Leu-Asp-Leu*Ala-Val-Glu-Phe SEQ ID NO: 23 was designed and found to have a Ki of 0.3 nM as described below. The structure of the catalytic unit of memapsin 2 in complex with OM00-3 is described herein. The new structure defines the locations and structures of sub-sites S3′ and S4′, redefines subsite S4 and provides new insight into their functions. Novel inhibitor/enzyme interactions were also observed in other sub-sites.
Promemapsin 2-T1 (amino acid residues 1-456 of SEQ ID NO: 8 (
For data collection at 100° K., a crystal was first cryoprotected by transferring to well solution containing 20% (v/v) glycerol and then quickly frozen with liquid nitrogen. Diffraction data were collected on a Mar 345 image plate mounted on a Msc-Rigaku RU-300 X-ray generator with Osmic focusing mirrors. The data were processed using the HKL program package (Otwinowski, Z., et al., W. Methods in Enzymol. 276:307-326 (1997), the teachings of which are incorporated herein by reference in their entirety). Statistics are shown in Table 10.
Molecular replacement solutions were obtained for both crystals with the program AmoRe (Navaza, J., Acta Crystallogr D Biol Crystallogr 57:1367-72 (2001), the teachings of which are incorporated herein by reference in their entirety) using the previously determined memapsin 2 structure (Identifier Code: PDB ID 1FKN) as the search model. Translation search confirmed the two crystal forms are isomorphous in space group P212121 (Table 7) with two memapsin 2/inhibitor complexes per crystallographic asymmetric unit. The refinement was completed with iterative cycles of manual model fitting using graphics program O (Jones, T. A., et al., Acta Crystallogr A 47:110-9 (1991), the teachings of which are incorporated herein by reference in their entirety) and model refinement using CNS (Brunger, A. T., et al., Acta Crystallogr D Biol Crystallogr 54:905-21 (1998), the teachings of which are incorporated herein by reference in their entirety). Water molecules were added at the later stages of refinement as identified in |Fo|-|Fc| maps contoured at 3 σ level. Ten percent of the diffraction data were excluded from the refinement at the beginning of the process to monitor the Rfree values. The two memapsin 2/inhibitor complexes in the crystallographic asymmetric unit were found to be essentially identical. The coordinates for the structure reported here have been deposited in the Protein Data Bank (Accession Code 1M4H).
The measurement of relative kcat/Km values for the determination of residue preference at P3′ and P4′ were carried out as described above. Two template substrate sequences, WHDREVNLAAEF (SEQ ID NO: 28) and WHDREVNLAVEF (SEQ ID NO: 44) were used. The former had a P3′ Ala and the latter a P3′ Val. Four N-terminal residues, WHDR (SEQ ID NO: 29), were added to the substrate to facilitate the analysis using mass spectrometry. For each template, two each of peptide mixtures containing a total of 11 representative residues (in single letter code: A, D, E, F, L, M, R, T, V, W and Y) each at either P3′ or P4 were designed and synthesized. The initial velocities for memapsin 2 hydrolysis of each peptides in the mixtures were determined in MALDI-TOF mass spectrometer as described above. The internal standards and the calculation of relative kcat/Km values were also as described above.
The crystal structure of OM99-2 bound to memapsin 2 is previously described in monoclinic space group P21 (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety). In this study, the structures of OM99-2 and OM00-3/memapsin 2 complexes were solved and compared in the same space group —P212121 (Table 10).
OM00-3 was designed based the crystal structure data of OM99-2 bound to memapsin 2 and the binding of memapsin 2 to a combinatorial inhibitor library as described above. Three amino acid residues are different in OM00-3 relative to OM99-2: P3 Val to Leu, P2 Asn to Asp, and P2′ Ala to Val. These modifications improve the Ki by 5.2 fold as shown above. The crystal structure of the OM00-3/memapsin 2 complex shows conformational changes for both the inhibitor and the enzyme. The most significant changes on the inhibitor can be observed at P4 Glu.
In the OM99-2 structure, the P4 Glu side chain carboxylate forms a strong hydrogen bond with the P2 Asn side chain amide nitrogen (bond distance 2.9 A). This conformation stabilizes the inhibitor N-terminus, but the P4 side chain makes little contacts with the enzyme. The P2 change from Asn to Asp in OM00-3 introduces electrostatic repulsion between the P2 and P4 side chains and eliminates the hydrogen bond between them. For the same reason, there is a rotation of the P4 Glu main chain torsion of about 152 degrees, which places the P4 side chain in a new binding pocket. At this position, the carboxylate oxygen atoms of P4 Glu form several ionic bonds with the guanidinium nitrogen atoms of the Arg30 (SEQ ID NO: 9 (
The memapsin 2 residues contacting the P3 Leu, P1 Leu, P2′ Val, and P4′ Phe (distance less than 4 Å) are shown in bold cased letters. The salt linkages (ion pairs) are likely to significantly increase the binding energy contributions of P4 Glu to memapsin 2; yet, P4 has increased mobility compared to that of the OM99-2 as indicated by their crystallographic B factors, whereas the average B factor differences between the two inhibitors from P3 to P2′ are insignificant (
OM99-2 was designed based on the Swedish Mutation of APP (SEVNLDAEFR; SEQ ID NO: 11) (Ghosh, A. K., et al., J Med Chem 44:2865-8 (2001), the teachings of which are incorporated herein by reference in their entirety). In its complex with memapsin 2, the side chains of P2 Asn and Arg235 (SEQ ID NO: 9 (Figure' 12)) form hydrogen bonds, which may contribute to enhanced proteolysis and subsequently elevated Aβ production, leading to the early onset of Alzheimer's Disease (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety). In the OM00-3 structure, the P2 side chain is changed to Asp, and the Arg235 side chain adopts a new conformation, forming two salt linkages to the P2 Asp side. These new ionic bonds make additional contributions to the inhibitor binding.
The effect of Val to Leu change at P3 is subtle and involves adding and rearranging of hydrophobic interactions. The longer side chain of Leu at P3 allows it to make van der Waals contacts with that of the P1 Leu. The interactions between P1 and P3 side chains make them fit better into the corresponding hydrophobic binding pockets of the enzyme. Conformational changes are observed on the enzyme at Leu+.
In the OM99-2 structure, the Lee (SEQ ID NO: 9 (
Structural flexibilities of the substrate binding sites of memapsin 2, such as the variations of side chain positions of Arg235 (SEQ ID NO: 9 (
The third residue change of OM00-3 from OM99-2 is at the P2′ from Ala to Val. While the P2′ is Ala in the pathogenic substrate APP, Val is a considerably better choice. The crystal structure indicates that the energetic benefit comes from the added van der Waals interactions in this hydrophobic pocket. The larger Val side chain has 5 more van der Waals contacts with the enzyme than the smaller Ala side chain (Table 11). There are 15 more van der Waals enzyme/inhibitor contacts in OM00-3 than that of OM99-2 because of the structure changes at P3, P1 and P2′ (inter-atomic distances<4 Å).
aResidue describes the amino acid residue and number of memapsin 2 (according to SEQ ID 9 (FIG. 12)) and whether side chain (sc) or backbone (bb) atoms of memapsin 2 are described in the interaction. Residues are grouped by subsite. Amino acid of compound OM00-3 appears in bold next to the subsite with which it is interacting.
bResidue describes the amino acid residue and number of memapsin 2 (according to SEQ ID 8 (FIG. 11)).
cType of interaction: Phobic, hydrophobic or van der Waal contact; Hbond, hydrogen bond; ionic, ion pair between ionizable functional groups of opposite charges.
dInteracting group of the compound OM00-3 is either side chain (sc) or backbone atoms (bb).
Glu and Phe comprise P3′ and P4′ for both of the inhibitors. Unlike the results obtained in space group P21 (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety), the positions of P3′ and P4′ are better defined by electron density in space group P212121. However,
The crystal structure of the memapsin 2/OM99-2 indicates of an S5 substrate binding site on the enzyme. The N-terminal nitrogen of OM99-2 points to a hydrophilic opening on memapsin 2, which comprises Lys9, Ser10, Gly11, Gln12, Pro160, and Pro308 (SEQ ID NO: 9 (FIG. 12)), and can potentially be used as a substrate or inhibitor binding pocket beyond S4. The N-terminal nitrogen of OM00-3 points to the inside of the enzyme and does not likely mimic the extending N-terminal position of a protein substrate. On the contrary, the orientations of C-terminal carboxyl groups of both inhibitors indicate that the next residue would be pointing away from the enzyme surface and no additional binding sites can be found beyond S4′.
The crystal structure of memapsin 2 and the compound, OM00-3, was compared with a crystal structure of memapsin 2 and the inhibitor compound OM99-2 in the same space group. New enzyme/inhibitor interactions have been identified in several binding pockets. These include both electrostatic and van der Waals contacts. A possible substrate binding site beyond S4 was also identified.
The structure of the catalytic domain of human memapsin 2 bound to an inhibitor OM00-3 (ELDL*AVEF; SEQ ID NO: 23, Ki=0.3 nM, * denotes hydroxyethylene transition-state isostere) has been determined at 2.1 Å resolution. Uniquely defined in the structure are the locations of S3′ and S4′ sub-sites, which were not identified in the previous structure of memapsin 2 in complex with inhibitor OM99-2 (EVNL*AAEF; SEQ ID NO: 20 ICH nM). Different binding modes for P2 and P4 side chains are also identified. The structural and kinetic data demonstrate that the replacement of the P2′ alanine in OM99-2 with a valine in OM00-3 stabilizes the binding of P3′ and P4′.
The structure of the OM00-3/memapsin 2 complex in space group P212121 was determined at 2.1 Å using the molecular replacement method. The structure of the enzyme, the interactions of the P1/P1′ (Leu*Ala) region of OM00-3 with the substrate binding cleft of memapsin 2 and the backbone conformation of the inhibitor from P3 to P2′ are essentially the same as in the structure of the OM99-2/memapsin 2 complex. However, the current structure shows different side-chain configurations within the S4, S3 and S2 sub-sites when compared to those of the OM99-2 structure (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety). In addition, the locations and nature of S3′ and S4 binding pockets are defined.
The new S4 pocket in the current structure involves memapsin 2 residues Gly11, Gln73, Thr232, Arg307 and Lys321. The Arg307 and Lys321 (SEQ ID NO: 9 (
In the OM00-3 structure, Leu30 (SEQ ID NO: 9 (
In the S2 pocket, the P2 Asp of OM00-3 forms two ionic bridges to the Arg235 (SEQ ID NO: 9 (
In contrast to the OM99-2/memapsin 2 structure, the conformation of the P3′ and P4′ side chains is well defined by electron density in the OM00-3/memapsin 2 structure. The backbone at P3′ and P4′ of OM00-3 assumes an extended conformation which is stabilized by a hydrogen bond from P3′ backbone carbonyl to Arg128 (SEQ ID NO: 9 (
Inhibitors OM99-2 and OM00-3 have identical P3′ and P4 residues. It was therefore unexpected that the P3′ and P4 are better defined for the latter structure. Kinetics studies have shown that, compared to the other subsites, subsites that bind P3′ and P4′ have a considerably broader range of amino acid preference (
Ten representative residues were chosen for each of the P3′ and P4′ positions in addition to the native residue. The relative kcat/Km values of these eleven substrates in a single mixture were determined by their relative initial hydrolytic rate using a mass spectrometric method as described above. The results show that the differences in residue preferences at subsites that bind P3′ (
The template sequence EVNLAAEF (SEQ ID NO: 15) employed to discern the amino acid residue preference (
A number of interactions are noted between the inhibitor compounds of the invention and memapsin 2. As shown in Table 11, there are hydrophobic contacts between the side chains of P3, P1 and P2′. In addition, salt bridges and hydrogen bonds from the P4 and P2 side chains and the P3′ and P4′ backbone of the inhibitor are also observed.
There is no shift of preference at P3′ and P4′ side chains toward Glu and Phe, respectively, when P2′ is Val; yet, peptide substrates with Val at P2′ have on average about 30% higher kcat/Km values than their counterparts with Ala at P2′. To determine which kinetic parameter contributes to this difference, the individual kcat and Km values for two substrates differing at only P2′ by Val or Ala was measured. Substrate EVNLAVEFWHDR (SEQ ID NO: 30) produced a Km of 83±8.9 mM and a kcat of 1,007±106 s-1 (n=3) while substrate EVNLAAEFWHDR (SEQ ID NO: 31) had a Km of 125±11 mM and a kcat of 274±23 s-1 (n=2). The differences in kinetic parameters between P2′ Val and P2′ Ala substrates are much greater in kcat (˜4 fold) than in Km (˜1.5 fold). Thus, compared with P2′ Ala, P2′ Val primarily improves the transition-state binding of P3′ and P4′ residues, but does not alter their specificity.
The first structure of memapsin 2 catalytic domain complexed to inhibitor OM99-3 (Hong, L., et al., Science 290:150-3 (2000), the teachings of which are incorporated herein by reference in their entirety) has been shown to be useful in the structural based design of smaller and potent memapsin 2 inhibitors (Table 1). The new structure described here provides improved versatility for inhibitor design. Memapsin 2 inhibitors with clinical potentials should be potent, selective and small enough to penetrate the blood-brain barrier. It is known that HIV protease inhibitor drug indinavir, 614 Da, can cross the blood-brain barrier (Martin, et al., Aids 13:1227-32 (1999), the teachings of which are incorporated herein by reference in their entirety). A memapsin 2 inhibitor of similar size would bind to about five sub-sites consecutively. Inhibitors with Ki at low nM range can be designed without evoking binding at the P3′ and P4′ subsites (Table 1). The new binding modes at P4 and P2 can be utilized for the design of inhibitors of this type. The new sub-site structures of S3′ and S4′ described above can be incorporated in the design of inhibitors with P3′ and P4′ but without P4 and P3 residues. Such a design is predicted to have a strong binding side chain, such as Val, at P2′.
Compound MMI-138 selectively inhibits memapsin 2 over memapsin 1, evident as the Ki value for the former are 60-fold lower than that of the latter. Moreover, other compounds that have a functional group containing pyrazole as the R1 group of formula II likewise demonstrate selectivity based upon their relative Vi/Vo measurements (Table 9). To determine the structural features of MMI-138 that contribute to the selectivity of the inhibitor, a crystal structure of memapsin 2 in complex with MMI-138 was determined. The structure reveals the pyrazole group was bound to the enzyme in the S3 subsite, forming hydrogen bonds. A peptide bond in memapsin 2 was flipped relative to its orientation in the crystal structures of complexes between memapsin 2 and either OM99-2 (Hong, L., Turner, R. T., 3rd, Koelsch, G., Shin, D., Ghosh, A. K., Tang, J., “Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor ° M00-3,” Biochemistry 41:10963-10967 (2002); and Hong, L., Koelsch, G., Lin, X., Wu, S., Terzyan, S., Ghosh, A. K., Zhang, X. C., Tang, J., “Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor,” Science 290:150-153 (2000)) or OM00-3 (Hong, L., Turner, R. T., 3rd, Koelsch, G., Shin, D., Ghosh, A. K., Tang, J., “Crystal structure of memapsin 2 (beta-secretase) in complex with an inhibitor OM00-3,” Biochemistry 41:10963-10967 (2002)). Modeling of the memapsin 1 structure in the vicinity of the pyrazole binding region suggests that such an orientation is unfavorable for memapsin 1. The possibility of other energetic or structural features that impart selectivity are not excluded by the model.
Promemapsin 2-T1 was expressed as outlined in Example 1 and purified. The memapsin 2 used in the crystallization procedure was obtained by activation of promemapsin 2-T1 (SEQ ID NO 8 as shown in
The memapsin 2/MMI-138 crystals were obtained by a replacement or “soaking” procedure (Munshi, S., Chen, Z., Li, Y., Olsen, D. B., Fraley, M. E., Hungate, R. W. and Kuo, L. C., “Rapid X-ray diffraction analysis of HIV-1 protease-inhibitor complexes: inhibitor exchange in single crystals of the bound enzyme,” Acta Cryst.D54:1053-1060 (1998); see procedure below). In this procedure, a complex is obtained between the protein and a compound of affinity less than the compound of interest (in this case MMI-138). This crystal is then placed in a solution of the compound of interest (i.e., “soaked”) to allow the compound of interest to diffuse and exchange with the compound of weaker affinity present in the proteins of the crystal. Therefore, for crystals of memapsin 2 in complex with MMI-138, crystals first had to be obtained with a complex of memapsin 2 and a compound of weaker affinity. The compound of weaker affinity used in the procedure was designated OM01-1 (Ki=126 nM):
OM01-1 was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 25 mg/ml. Memapsin 2 (amino acids 60-456 of SEQ ID NO: 8 (
To obtain crystals of a complex between memapsin 2 and compound MMI-138, a replacement or “soaking” procedure was followed (Munshi, et al. 1998). OM01-1 synthesized using standard solid-phase peptide synthesis using an FMOC-protected hydroxyethylene isostere established by our lab (Ghosh, et al. 2000). Crystals of memapsin 2 in the presence of OM01-1 were obtained by the above mentioned crystallization procedure and were transferred to a 10 μl volume of a solution of the crystallization buffer containing 10% DMSO and 2 mg/ml of compound MMI-138, as well as memapsin 2 protein, present at a concentration of no more than one-half the molar concentration of MMI-138, but preferably one-fifth the molar amount of MMI-138, for the purpose of stabilizing the crystal during the soaking procedure. The solution was incubated at 20° C. for 48 hours to allow the compound OM01-1 present in the crystal to equilibrate with the compound MMI-138, resulting in an exchange between OM01-1 in complex with memapsin 2 in the crystals for compound MMI-138.
Crystals of memapsin 2 in complex with MMI-138, obtained by the above procedure were incubated in cryo-protectant buffer (crystallization buffer containing 20% glycerol) for 1-2 minutes, followed by flash-freezing in a liquid nitrogen stream. Diffraction data was collected on a Rigaku RU-300 X-ray generator with a M345 image plate at 100° K. Data was indexed and reduced with the HKL program package (Otwinowski, Z., and Minor, W., Methods in Enzymol. 276:307-326 (1997)). Molecular replacement method was used to solve the structure with the memapsin 2/0M99-2 crystal structure as the initial model. Molecular replacement solutions were obtained with the program AmoRe (Navaza, J., Acta Crystallogr D Biol Crystallogr 57:1367-72 (2001)). The refinement was completed with iterative cycles of manual model fitting using graphics program 0 (Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, Acta Crystallogr A 47:110-9 (1991)) and model refinement using CNS (Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L., Acta Crystallogr D Biol Crystallogr 54:905-21 (1998)). The data obtained is shown in Table 12.
aRmerge
bRwork
bRfree
aRmerge = ΣhklΣi | Ihkl, i − <Ihkl> Σ/Σhkl <Ihkl>, where Ihkl, i is the intensity of the ith measurement and <Ihkl> is the weighted mean of all measurements of Ihkl.
bRwork (free) = Σ || Fo| − |Fc||/Σ |Fo|, where Fo and Fc are the observed and calculated structure factors. Numbers in parentheses are the corresponding numbers for the highest resolution shell (2.18-2.1 Å). Reflections with Fo/σ(Fo) >= 0.0 are included in the refinement and R factor calculation.
The dimethylpyrazole group at the N-terminus (e.g., the R1 group of Formula II) of the compounds of the invention provides inhibition selectivity for memapsin 2 over memapsin 1 (see
However, the crystal structure likewise indicates the flip of the carbonyl oxygen of Ser10 is unfeasible for memapsin 1. The Lys9 in memapsin 2 is an Asp in memapsin 1. According to our modeled memapsin 1 structure, the Asp side chain would form a hydrogen bond with the backbone nitrogen of Are (2.9 Angstroms). This hydrogen bond and position of Asp9 side chain should stabilize the hairpin loop from Ser9 to Arg12 and prevent the peptide flip as required for the pyrazole group binding. The flip would position the Seri° carbonyl oxygen in close proximity (˜2.3 Å) to the negatively chargedAsp9 side chain and/or distort the hydrogen bond, which is not energetically favorable. It is also possible that in memapsin 1, the main chain conformation is different from that of the memapsin 2 around See), and the peptide flip would cause the main chain torsion angles to have disfavored IV and (I) combinations.
The carrier molecule peptide employed in these experiments was a peptide derived from a segment of the HIV tat protein (amino acid residues 47-57) (Schwarze, S. R., et al., Science 285:1569-1572 (1999), the teachings of which are incorporated herein in their entirety) or has an amino acid sequence Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO: 13) and an oligo-D-arginine residue (R-R-R-R-R-R-R-R-R (SEQ ID NO: 33)) (Wender, P. A., et al. Proc. Natl. Acad. Science USA 97:13003-13008 (2000), the teachings of which are incorporated herein in their entirety).
Carrier Peptide-Inhibitor conjugates are referred to herein by the designation “CPI” followed by a number, e.g., CPI-1, CPI-2 and CPI-3. CPI-1 is the OM99-2 inhibitor complexed to a carrier peptide. CPI-2 is the OM00-3 inhibitor complexed to a carrier peptide.
The structure of the carrier peptide inhibitor conjugates employed in the experiments was:
Where G is glycine; Y, R, K, Q, E, V, N, L, A, F and D are L-amino acids tyrosine, arginine, lysine, glutamine, glutarmic acid, valine, asparagine, leucine, alanine, phenylalanine and aspartic acid, respectively. Italic R represents D-arginine. 5-(and 6-) carboxyfluorescein (FAM), is linked to the amino group of the 6-aminohexanoic acid (Ahx) group. The carboxyl group of Ahx is linked by an amide bond to amino group of the first amino acid in the inhibitor moiety.
Ahx and glycine residues were employed as spacers in the complex. The square brackets enclose the carrier peptides, which are tat residues 47-57 in CPI-1 and nine D-arginine residues (Wender, P. A., et al., Proc. Natl. Acad. Sci. USA 97:13003-13008 (2000), the teachings of which are incorporated hereby in their entirety) in CPI-2, respectively. The asterisks in the inhibitor sequences represent the transition-state isostere, hydroxyethylene (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000), the teachings of which are incorporated hereby in their entirety).
The Carrier Peptide is referred to herein a “CP,” followed by a number. A fluorescein-labeled carrier peptide, CP-1, excluding a conjugated inhibitor moiety, was also designed for control experiments. The structure of CP-1 is as follows:
The peptide portions of CPI-1, CPI-2 and CP-1 were synthesized using solid-phase peptide synthesis and purified by reversed phase HPLC. Protected Leu*Ala diisostere derivative was used at a single step in the synthesis of CPI-1 and CPI-2 (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000) the teachings of which are incorporated hereby in their entirety). FAM attachment was facilitated by active ester chemistry according to procedures of the supplier (Molecular Probes).
Kinetic inhibition experiments (
The concentrations of the conjugates and control were normalized to peptide concentration either from amino acid analysis or by fluorescence values using a fluorescence spectrophotometer AMINCO-Bowman Series 2. An excitation wavelength of 492 nm and an emission wavelength of 516 nm were used to monitor the amount of fluorescence from the conjugated fluorescein.
Two- to four-month-old Cd72c mice were injected intraperitoneally (i.p.) with 0.3 to 10 nmoles of the conjugates (CPI-1 or CPI-2) or with control fluorescein, in 200 IA of PBS. Whole blood cells (with EDTA as anti-coagulant in the syringe or in the capillary tube) were isolated from anesthetized animals from the orbital artery or by heart puncture and diluted 1:10 in PBS. Prior to the harvest of other tissue samples, animals were anesthetized and perfused with 150 ml of neutral-buffered 10% formalin. Spleens were harvested intact. Brains were harvested and cerebral hemispheres separated, one for sectioning by cryostat, the other for single cell isolation for flow cytometry.
Sections of the brain hemispheres were obtained by soaking in OCT/PBS at 4° C. for overnight, recovered and frozen in Histo Prep Media. Sections (10 μm) were cut on a cryostat, fixed in 0.25% of formalin for 15 min, and histologically stained with three antibodies: (1) Alexa Fluor 488 conjugated anti-fluorescein (Molecular Probes; (2) Polyclonal goat anti-human-pro-memapsin 2 antibody; (3) followed by Cy3 conjugated anti-goat IgG antibody (Sigma, St. Louis, Mo.); and (4) Biotin-conjugated anti-bovine α-tubulin followed by Alexa Fluor 350™ conjugated to neutravidin (Molecular Probes). After mounting with anti-fade solution with a cover slip, the sections were analyzed by fluorescence confocal microscopy.
To collect single cell suspensions, spleens and brain hemispheres were homogenized through a 30 μm screen and directly analyzed by flow cytometric analysis. An alternative means to staining brain cells was first to permeabilize them in 0.2% Tween 20 in PBS, blocked with 1% normal rabbit serum, incubated with 1:50 diluted Alexa Fluor 488™ conjugated anti-fluorescein (1 mg/ml; Molecular Probes, Eugene, Oreg.) for 30 minutes, then analyzed by flow cytometric analysis.
Fluorescein was conjugated to the amino terminus of OM00-3 by incubation with NHS-fluorescein (Pierce, Rockford, Ill.) and purified to >90% by reversed-phase HPLC and dissolved in DMSO to 50 mg/ml.
Fluorescently labelled inhibitors or fluorescein (Fs) as a control were incubated with suspended cells for time intervals ranging from 10 to 30 minutes. Cells were fixed with paraformaldehyde and permeabilized in 0.2% Tween-20 in PBS for 6 minutes and incubated with anti-fluorescein-Alexa™ 488 antibody (Molecular Probes, Eugene, Oreg.) in order to enhance detection of intracellular inhibitor present from penetration. Flow cytometry (FACSCalibur™) and confocal fluorescent microscopy (Leica TCS NT™) were performed at the Flow and Image Cytometry Lab, OUHSC.
Results
The conjugated inhibitors, CPI-1 and CPI-2, readily penetrated cultured cells within minutes, as indicated by intracellular fluorescence of FAM group (
Incubation of HEK293 cells with Fs[OM99-2]tat resulted in an increase of fluorescence relative to cells incubated with fluorescein alone, as demonstrated by flow cytometry (
Entry of the CPI-1 and CPI-2 conjugates into the mammalian brain was determined. Mice, strain Cd27c, were injected i.p. with 0.3 nmol of either CPI-1, CPI-2 or CP-1 and cells and organs monitored for fluorescence due to the FAM group in the injected compounds. Flow cytometric analysis of whole blood isolated 20 minutes after i.p. injection with CPI-1 revealed a strong fluorescence signal in approximately 100% of blood cells (
Splenocytes were analyzed for the presence of CPI-1, CPI-2 and CP-1 by performing a splenectomy 2 hours after i.p. injection of the mice and isolating the splenocytes. Flow cytometric analysis revealed translocation of conjugates into all splenocytes, including T cells, B cells, and macrophages, resulting in a fluorescence peak shift in almost 100% of cells (
The uptake of the CPI-1 into brain tissue was determined. Whole brains were dissected from perfused mice 8 hours after i.p. injection of the conjugate or fluorescein as a control. Hemispheres were separated and either frozen for cryostat sectioning or for isolation of cells by homogenization on nylon mesh. Flow cytometric analysis revealed penetration of the fluorescent conjugate into all brain cells, resulting in a fluorescence peak shift (
Fluorescence confocal microscopy analysis of 10 μm hemispheric sagittal brain sections revealed a strong signal in all areas of the brain from mice injected with CPI-2, while the signal in fluorescein control injected mice remained at background levels. Eight hours after i.p. injection, the confocal microscopy result showed that fluorescein localized primarily to the nuclei of cell bodies throughout the brain section.
Inhibition of Aβ Secretion from Cultured Cells by Conjugated Inhibitors
Observations described above established that the two conjugated inhibitors, CPI-1 and CPI-2, were able to penetrate the plasma membrane of cells in vitro or the blood brain barrier (BBB) or in vivo. Inhibition of the activity of memapsin 2 in cultured cells by a conjugated inhibitor was determined. Since the hydrolysis of APP by memapsin 2 leads to the formation of Aβ and its secretion to the culture medium, the effect of conjugated inhibitor CPI-2 on APP cleavage was determined by measuring the secreted Aβ in the culture medium.
Experimental Procedure
Cultured cells, including human embryonic kidney (HEK293) cells, HeLa, and neuroblastoma line M17, purchased from American Type Culture Collection (ATCC), were stably transfected with two nucleic acid constructs that encode human APP Swedish mutant (APPsw; SEVNLDAEFR (SEQ ID NO: 11)); and human memapsin 2 (amino acid residues 14-501 of SEQ ID NO: 6 (FIG. 9)), which included leader peptide from PSEC-tag genes. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum and 1% penicillin/streptomycin. Two antibiotics, Zeocin (1 μg/ml) and G418 (250 μg/ml) were included in the media for maintenance of the stably transfected lines.
Either the parental lines (293, HeLa, or M17) or the stably transfected lines (293-D,HeLa-D, or M17-D) were plated on 6-well plates and grown in a 37° C., 5% CO2 incubator until 90% confluent. Cells were then treated with or without 10 pmole of CPI-2 overnight then labeled by using [35S]TransLabel Protein Labeling Mix (100 mCi/ml) (ICN) in methionine- and cysteine-free DMEM for an additional 18 hours. For treatment of cells with CPI-2, 10 pmole of the inhibitor conjugate was dispensed to cells 20 minutes prior to labeling, and likewise into labeling media.
Cells were lysed in 1 ml of RIPA buffer (10 mM Tris, pH 7.6, 50 mM NaCl, 30 mM sodiumpyrophosphate, 50 mM NaF, and 1% NP-40) supplemented with 1 mM PMSF, 10 μg/ml leupeptin, 2.5 mM EDTA, 1 μM pepstatin, and 0.23 U/ml aprotonin. The total cell lysates were subject to immunoprecipitation by the addition of 1 μl of 1 mg/ml of monoclonal antibody raised specifically against human Aβ17-24 (MAB 1561, Chemicon) with 20 μl of protein G-sepharose beads. Immunoprecipitated proteins were denatured in Tricine-SDS sample buffer with 2.5% β-mercaptoethanol by boiling for 5 minutes. Immunoprecipitated proteins were analyzed by using 10-20% gradient SDS-PAGE (NOVEX) and radiolabeled proteins were visualized by autoradiography. Quantitative results were obtained using the STORM™ phosphorimaging system (Amersham).
Results
Immunoprecipitation of Aβ from HEK 293 cells transfected with sw (Swedish mutation) APP and memapsin 2 (amino acid residues 14-501 of SEQ ID NO: 6 (
The structure of the carrier peptide inhibitor conjugate CPI-3 was designed as follows:
Where G is glycine; Y, R, K, Q, E, V, N, L, A, F and D are L-amino acids tyrosine, arginine, lysine, glutamine, glutarmic acid, valine, asparagines, leucine, alainine, phenylalanine and aspartic acid, respectively. Italic R represents D-arginine. The preparation of CPI-2 is described above. CPI-3 was synthesized employing a similar procedure. CPI-3 has the same amino acid sequence as CPI-2, but lacks the fluorescent FAM tag. The amino terminus of CPI-3 is a free primary amine and is not linked either to aminohexyl or to the FAM group. The asterisks in the inhibitor sequences represent the transition-state isostere, hydroxyethylene.
The peptide portion of CPI-3 was synthesized using solid-phase peptide synthesis and purified by reversed phase HPLC. Protected Leu*Ala diisostere derivative, described previously (Ghosh, A. K., et al., J. Am. Chem. Soc. 122:3522-3523 (2000), the teachings of which are incorporated hereby in their entirety), was used at a single step in the synthesis of CPI-3 as described in Ghosh, et al. (J. Am. Chem. Soc. 122:3522-3523 (2000), the teachings of which are incorporated hereby in their entirety).
Kinetic inhibition experiments using a procedure as described in Ermolieff, et al. (Biochemistry 39:12450-12456 (2000), the teachings of which are incorporated hereby in their entirety) showed that the conjugated inhibitors .CPI-3 had similar inhibition potencies as the parent inhibitor, OM00-3, with a Ki of 35 nM.
Experimental Procedure
Six-month-old tg2576 mice (n=21) were injected intraperitoneally (i.p.) 200 μg of conjugate CPI-3 or with control DMSO, in 200 μl of PBS. Plasma were collected from anesthetized animals by orbital bleed or sephaneous vein into heparinized capillary tubes and clarified by centrifugation. Plasma Aβ 1-40 levels were determined by capture ELISA (BioSource International, Camarillo, Calif.). The peptide analogue of CPI-3, with an amide group instead of the hydroxyethylene isostere was synthesized by SynPep (Camarillo, Calif.).
Results
The conjugated inhibitor CPI-2 readily penetrated cultured cells within minutes, and penetrated into the brain and other tissue within hours, as indicated by intracellular fluorescence of FAM group, as discussed above.
Since the conjugate inhibitors can cross the blood brain barrier in vivo, enter cells both in vitro and in vivo, and inhibit Aβ production in vitro, inhibition of Aβ production in vivo was determined. Tg2576 mice, expressing the Swedish mutation of the human amyloid precursor protein (including SEQ ID NO: 11) (Hsiao, K., et al., Science 274:99-102 (1996), the teachings of which are incorporated hereby in their entirety) were injected with CPI-3, which is identical in amino acid sequence to CPI-2 and lacks the amino-terminal fluorescein (FAM) derivative. Blood was collected from tg2576 animals at time intervals following injection of 400 μg of CPI-3.
At ages up to 9 months, plasma Aβ in the tg2576 mice serves as a reliable marker for brain Aβ production as a result of memapsin 2 activity (Kawarabayashi, T., et al., J. Neurosci. 21:372-381 (2001)). Nine tg2576 mice were injected intraperitoneally with various doses of inhibitor CPI-3. Two hours following the injection of CPI-3, plasma Aβ40 showed a significant dose-dependent reduction relative to Aβ40 from control mice injected with PBS (
To study the duration of inhibition, eight tg2576 mice were injected intraperitoneally with inhibitor CPI-3. The plasma Aβ40 level dropped to about one third of the initial value at 2 hours following injection (
Since the observed duration of inhibition had been relatively short, the maximal inhibition level of this inhibitor by repeated injections was determined. Experiments with four injections at 2 hour intervals significantly reduced the Aβ level to an average low of 29% (ranging 22% to 33%) of the average initial value (
Carrier molecules had previously been shown to facilitate the transport of natural macromolecules such as protein and DNA across the cell membrane. The demonstration here that carrier molecules assist the transport of synthetic inhibitors containing non-peptidic bonds across the cell membrane and the blood brain barrier (BBB) raises the possibility that carrier molecules can be employed for the delivery of Alzheimer's disease therapeutics and others targeted to the central nervous system or other tissues or organelles. The advantage of such an approach is that the parental inhibitors need not be small enough for BBB penetration so the drug can be selected from a wider repertoire of candidate compounds based on potency, selectivity and other drug properties. Drug delivery employing carriers could be considered for those targets of the for which drugs with properties suitable for cell membrane penetration are difficult to attain.
Although many of the compounds of the invention demonstrate strong inhibition of memapsin 2 (amino acid residues 43-456 of SEQ ID NO: 8 (SEQ ID NO: 62) (
The tg2576 transgenic mouse expresses the human Swedish amyloid precursor protein (APP) under control of the prion promoter to direct expression mainly in the brain (Hsiao, K., et al., Science 274:99-102 (1996), the teachings of which are incorporated hereby in their entirety). The Aβ peptide produced in the brain can be detected in plasma of these transgenic animals from ages 3-12 months (Kawarabayashi, T., et al., J. Neurosci. 21:372-381 (2001), the teachings of which are incorporated hereby in their entirety) and results from its efflux from the brain, known to occur within minutes (Ghersi-Egea, et al., J. Neurochem. 67:880-883 (1996), the teachings of which are incorporated hereby in their entirety). Thus, monitoring the plasma Aβ provides a useful continuous measurement of effective inhibition of Aβ production in the brain.
Reduction of Aβ levels in the plasma, following administration of a memapsin 2 inhibitor, is an indication that the compound inhibited Aβ production in the brain by crossing the blood brain barrier. Fluorescently-labeled memapsin 2 inhibitor conjugated to a carrier peptide (CPI-2) was shown to cross the blood brain barrier, and inhibit Aβ production, as discussed above. Employing the same experimental protocol described above, it was demonstrated that three of the inhibitor compounds of the invention, MMI-138, MMI-165, and MMI-185 penetrated the blood brain barrier in transgenic mice (strain tg2576), resulting in reduction of Aβ production.
Compounds MMI-138, MMI-165, and MMI-185 were synthesized as described above. Compounds were dissolved in 1 ml of dimethyl sulfoxide (DMSO) to a final concentration of about 1 mg/ml for MMI-165 and MMI-185, and about 10 mg/ml for MMI-138. Inhibitor OM00-3 was synthesized as described above and dissolved in DMSO to about 10 mg/ml. Inhibitors were diluted into PBS or H2O immediately prior to injection, as described below. Inhibition constants were determined by methods described by Ermolieff, et al. (Biochem. 39:12450-12456 (2000), the teachings of which are incorporated hereby in their entirety).
The tg2576 strain of mice was obtained from Taconic (Germantown, N.Y.). The APP/F strain of mice were obtained by mating the tg2576 mice with the FVB/N strain. To determine presence of the Swedish APP gene in APP/F mice, the DNA from mice was isolated according to the Qiagen Dneasy™ Tissue Kit. PCR (Qiagen kit and protocol) was used to amplify the fragment of DNA corresponding to the human Swedish APP gene. The following primers were used:
Beta actin primers were used as a positive control. After PCR was performed, the samples were analyzed on a 1% agarose gel containing 0.5 μg/ml EtBr in a 1×TAE (Tris-Acetate-EDTA) running buffer.
At the age of three months, animals of the Alzheimer's disease mouse model APP/F were injected intraperitoneally (i.p.) with about 163 nanomoles of either compounds MMI-138 (molecular weight 674 g/mole; 110 μg per animal, n=2), MMI-165 (molecular weight 626 g/mole; 102 pg per animal, n=2), or MMI-185 (molecular weight 686; 112 μg per animal, n=2). Control animals were injected with either DMSO alone (100 μl diluted into 100 μl of PBS) or 163 nmoles of inhibitor OM00-3 (Table 3) to 10 mg/ml stock in DMSO diluted into PBS, final volume 200 μl.
Heparinized capillary tubes collected blood samples from anesthetized animals from either the retro-orbital sinus or from the saphenous vein at specified intervals following injection. The blood samples were transferred to sterile 1.5 mL microcentrifuge tubes, centrifuged at 5,100 RPM for 10 minutes to recover the plasma (supernatant), and stored at −70° C. until analysis for the Aβ40 by Enzyme Linked-Immuno-Sorbent Assay (ELISA).
A sandwich ELISA (BioSource International, Camarillo, Calif.) was used to determine the levels of Aβ40 in plasma samples. The ELISA utilizes a primary antibody specific for human Aβ for the immobilization of the amino-terminus and a detection antibody specific for the carboxy-terminal amino acids of Aβ40. A conjugated secondary antibody was used to detect the ternary complex, using a stabilized chromogen substrate, quantifiable following addition of 1 M HCl, with the optical densities measured at 450 nm. The procedures were followed according to the BioSource protocol. Optical densities were converted to pg/ml quantities of Aβ40 using a linear regression of the optical densities of standards obtained from the commercial kit, to their known concentrations.
Six APP/F animals were injected intraperitoneally with one of three different memapsin 2 inhibitors, MMI-138, MMI-165, or MMI-185. Following injection, blood samples were removed at various times by bleeding the saphenous vein, and analyzed for amount of Aβ40.
Transgenic mice were injected with a single injection of 163 nM of MMI-138, MMI-165 or MMI-185 and blood collected prior to the administration of the inhibitor compound (0 hours) and 2, 4, 6 and 8 hours following the administration of the inhibitor compound. Plasma β-amyloid protein (Aβ40) was determined. Data expresses the mean±the standard error of the mean. Two animals were used in each treatment group. As shown in
Control animals treated with DMSO or inhibitor OM00-3 revealed a decrease of only 21% and 16%, respectively, at 2 hours following injection (
The extent of inhibition observed at 30 minutes (
Memapsin 2 has been identified and experimentally supported as the β-secretase enzyme involved in the pathogenesis of Alzheimer's disease, and has further been characterized as a novel membrane bound aspartic protease. As such, memapsin 2 has many of the observed characteristics of the aspartic protease family. These characteristics include: an acidic pH optimum, the conserved D T/S G catalytic aspartic acid motif, an observed large substrate binding cleft, and an extended peptide substrate specificity. These last two characteristics of the aspartic protease family have been analyzed in a number of experimental studies and across a variety of species. The consensus of these studies is that the extended substrate binding cleft facilitates the interaction of eight amino acid residues of the substrate peptide, four on either side of the scissile bond. Here, we report the observation of a catalytic effect resulting from four distal amino acid residues of its substrate, namely in positions P5, P6, P7, and P8, which are N-terminal (upstream) to the traditional catalytic binding sequence. We have further conducted a specificity analysis of these positions to determine the optimal amino acid composition for catalysis.
The peptide sequence EVNLAAEF (SEQ ID NO: 15) (described in Example 1), successfully utilized in the memapsin 2 residue preference analysis for memapsin 2 was used as the base template peptide to analyze the extended upstream interaction. For the initial series of analyses, three peptides were created using solid phase peptide synthesis (Research Genetics, Invitrogen, Huntsville, Ala.). These peptides, EVNLAAEFWHDR (SEQ ID NO: 16) (designated WHDR (SEQ ID NO: 29)), RWHHEVNLAAEF (SEQ ID NO: 17) (designated RWHH (SEQ ID NO: 53)), and EEISEVNLAAEF (SEQ ID NO: 46) (designated EEIS (SEQ ID NO: 52) (asterisk denotes the cleavage site in each peptide) were created to examine the
downstream, upstream, and native APP sequence extensions, respectively. Additionally, four peptide mixtures were synthesized based on the extended native APP sequence (P5: RTEEIxEVNLAAEF (SEQ ID NO: 47); P6: RTEExSEVNLAAEF (SEQ ID NO: 48); P7: RTExISEVNLAAEF (SEQ ID NO: 49); P8: RTxEISEVNLAAEF (SEQ ID NO: 50); where x denotes a mixture of nine amino acid residues at that position, as shown in
Substrate mixtures were prepared following the method of Example 1 to obtain an incubation mixture with memapsin 2 (SEQ ID NO: 9 (
The crystal structure of memapsin 2 bound to inhibitor OM00-3 shows eight amino acid side chains accommodated within the substrate binding cleft of the enzyme (Lin, 2000). MALDI-TOF analysis was utilized in this initial study to determine this primary specificity. For this analysis, two template peptide sequences were designed to facilitate the examination of both the upstream and downstream interacting residues. These templates, RWHHEVNLAAEF (SEQ ID NO: 17) (designated RWHH (SEQ ID NO: 53)) and EVNLAAEFWHDR (SEQ ID NO: 16) (designated WHDR (SEQ ID NO: 29)), utilized an asymmetric design to allow the separation of the common product of catalysis from the unique catalytic products, dramatically enhancing the sensitivity of the assay system. While this design allowed an extremely sensitive analysis of the specificity for the observed binding sites, a very interesting and dramatic difference was observed in the rate of catalysis between all substrate mixtures of the P side relative to the P′ side, which might have resulted from simply extending the substrate with either RWHH (SEQ ID NO: 53) upstream of the template sequence, or WHDR (SEQ ID NO: 29) downstream. This change in the rate of catalysis due to changes in the peptide sequence outside of the traditional interacting residues is a novel observation for aspartic proteases in general (Davies, 1990). Whereas this initial observation was made with independent assays, it was sought to confirm and directly measure this effect by competitive cleavage assays of a mixture of the two peptides. These data supported the initial observation revealed a 60-fold decrease in the rate of catalysis for the upstream RWHH (SEQ ID NO: 53) sequence addition when compared to the downstream WHDR (SEQ ID NO: 29) sequence addition.
An analysis of the crystal structure of memapsin 2 (Lin, 2000) and specifically of the positioning of bound inhibitor, suggests that the downstream WHDR (SEQ ID NO: 29) sequence would be sufficiently distant from the enzyme to have no effect on catalysis. The upstream RWHH (SEQ ID NO: 53) sequence addition, however, does not extend beyond the outer peptide loop insertions near the enzyme cleft and could potentially interact with two of the sequence insertions of memapsin 2. A comparison of the crystal structures of pepsin and memapsin 2 indicates these observed structural differences identified on the upstream side of the binding cleft and could therefore be supportive of a distal upstream substrate interaction. Moreover, the presence of structural features coupled with the observation of a catalytic rate difference permits a hypothesis of a distal substrate binding cleft, previously unobserved for aspartic proteases. Presence of a binding cleft implies the possibility of substrate selectivity. Based on these observations, we examined whether the observed kinetic interaction resulted from the RWHH (SEQ ID NO: 53) N-terminal sequence addition specifically, indicating a selective extended binding cleft, or whether this interaction would result from any extended upstream sequence. To this end, a third peptide was synthesized using the same eight residue template sequence, EVNLAAEF (SEQ ID NO: 51), and extending it upstream with amino acids EEIS (SEQ ID NO: 52), the native sequence from human APP. Competitive cleavage analysis of a mixture of these three peptides resulted in statistically identical rates of catalysis for the upstream EEIS (SEQ ID NO: 52) and the downstream WHDR (SEQ ID NO: 29) sequence additions, while the RWHH (SEQ ID NO: 53) sequence addition still demonstrated a 60-fold decrease in catalytic rate. This result confirmed that the change in catalytic efficiency resulted from an interaction with the upstream residues of the peptide, with particular amino acid sequence RWHH (SEQ ID NO: 53) having a negative effect. Furthermore, that the N-terminal amino acid composition altered the rate of catalysis directly, an analysis of the possibility of a residue preference in these four distal positions became the next experimental objective.
The observation that a negative effect on the catalytic efficiency was due to the specific upstream sequence extension of RWHH (SEQ ID NO: 53) suggests that a binding interaction is occurring. To further characterize this interaction, an analysis of the amino acid specificity for this change in enzyme efficiency was performed. This analysis was conducted using the MALDI-TOF/MS quantitation method as previously discussed in Example 1, utilizing a synthesized substrate mixture library to explore the distal upstream positions P5, P6, P7, and P8. The resulting substrate side-chain preferences, reported as the preference index, for these four positions are presented in
Table 13 is a summary of the nucleic acid and amino acid sequences described herein.
This application is a continuation of U.S. application Ser. No. 10/281,092, filed Oct. 23, 2002, which is a continuation-in-part of U.S. application Ser. No. 10/032,818, filed Dec. 28, 2001, and of International Application No. PCT/US01/50826, filed Dec. 28, 2001, both of which claim the benefit of U.S. Provisional Application Nos. 60/258,705, filed Dec. 28, 2000, and 60/275,756, filed Mar. 14, 2001, and U.S. application Ser. No. 10/281,092 also claims the benefit of U.S. Provisional Application Nos. 60/335,952, filed Oct. 23, 2001; 60/333,545, filed Nov. 27, 2001; 60/348,464, filed Jan. 14, 2002; 60/348,615, filed Jan. 14, 2002; 60/390,804, filed Jun. 20, 2002; 60/397,557, filed Jul. 19, 2002; and 60/397,619, filed Jul. 19, 2002, the teachings of all of which are incorporated herein by reference in their entirety.
This invention was supported, in whole or in part, by a National Institutes of Health grants AG-18933 and AI-38189. The Government has certain rights in the invention
| Number | Date | Country | |
|---|---|---|---|
| 60275756 | Mar 2001 | US | |
| 60258705 | Dec 2000 | US | |
| 60335952 | Oct 2001 | US | |
| 60333545 | Nov 2001 | US | |
| 60348464 | Jan 2002 | US | |
| 60348615 | Jan 2002 | US | |
| 60390804 | Jun 2002 | US | |
| 60397557 | Jul 2002 | US | |
| 60397619 | Jul 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10281092 | Oct 2002 | US |
| Child | 12481339 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10032818 | Dec 2001 | US |
| Child | 10281092 | US | |
| Parent | PCT/US01/50826 | Dec 2001 | US |
| Child | 10032818 | US |
