Patent Application
20120302603
Alzheimer's disease (AD) is characterized by a progressive loss of cognitive function and constitutes the most common and fatal neurodegenerative disorder. See L. Minati, T. Edginton, M. G. Bruzzone, G. Giaccone, Am. J. Alzheimers Dis. 2009, 24:95-121; J. M. Schott, J. Kennedy, N. C. Fox, Curr. Opin. Neurol. 2006, 19:552-558. Genetic and clinical evidence supports the hypothesis that accumulation of amyloid deposits in the brain plays an important role in the pathology of the disease. This event is associated with perturbations of biological functions in the surrounding tissue leading to neuronal cell death, thus contributing to the disease process. The deposits are comprised primarily of amyloid (Aβ) peptides, a 39-43 amino acid sequence that self aggregates into a fibrillar β-pleated sheet motif. While the exact three-dimensional structure of the aggregated Aβ peptides is not known, a model structure that sustains the property of aggregation has been proposed. See C. A. Mathis, Y. Wang, W. E. Klunk, Curr. Pharm. Design 2004, 10:1469-1492; Y. S. Kim, J. H. Lee, J. Ryu, D. J. Kim, Curr. Pharm. Design 2009, 15:637-658. This creates opportunities for in vivo imaging of amyloid deposits that not only can help evaluate the time course and evolution of the disease but can also allow the timely monitoring of therapeutic treatments. See B. Matthews, E. R. Siemers, P. D. Mozley, Am. J. Geriat. Psychiat. 2003, 11:146-159; L. Nichols, V. W. Pike, L. S. Cai, R. B. Innis, Biol. Psychiat. 2006, 59:940-947; T. E. Golde, B. J. Bacskai, Nat. Biotechnol. 2005, 23:552-554.
Historically, Congo Red (CR) and Thioflavin T (ThT) with structures known in the art and provided below have provided the starting point for the visualization of amyloid plaques and are still commonly employed in post mortem histological analyses. See C. C. Kitts, D. A. V. Bout, J. Phys. Chem. B 2009, 113:12090-12095; M. L. Schmidt, T. Schuck, S. Sheridan, M. P. Kung, H. Kung, Z. P. Zhuang, C. Bergeron, J. S. Lamarche, D. Skovronsky, B. I. Giasson, V. M. Y. Lee, J. Q. Trojanowski, Am. J. Pathol. 2001, 159:937-943. However, due to their charge these compounds are thought to be unsuitable for in vivo applications. See C. A. Mathis, B. J. Bacskai, S. T. Kajdasz, M. E. McLellan, M. P. Frosch, B. T. Hyman, D. P. Holt, Y. M. Wang, G. F. Huang, M. L. Debnath, W. E. Klunk, Bioorg. Med. Chem. Lett. 2002, 12:295-298. To address this issue, several laboratories developed compounds with non charged, lipophilic (logP=0.1-3.5) and low molecular weight chemical structures (M.W. less than 650) that facilitate crossing of the blood brain barrier. See E. K. Ryu, X. Y. Chen, Front. Biosci. 2008, 13:777-789. Further functionalization of these compounds with radio-nuclides led to a new generation of in vivo diagnostic reagents with structure below that target plaques and related structures for imaging with positron emission tomography (PET) and single-photon emission computed tomography (SPECT), as known in the art. See K. A. Stephenson, R. Chandra, Z. P. Zhuang, C. Hou, S. Oya, M. P. Kung, H. F. Kung, Bioconjugate Chem. 2007, 18:238-246; A. Nordberg, Curr. Opin. Neurol. 2007, 20:398-402. M. Garcia-Alloza, B. J. Bacskai, Neuromol. Med. 2004, 6:65-78. Despite these advances, there is a pressing need for the design and development of new amyloid-targeting molecules with improved physical, chemical and biological characteristics. See B. J. Bacskai, W. E. Klunk, C. A. Mathis, B. T. Hyman,J. Cerebr. Blood F. Met. 2002, 22:1035-1041; D. J. Burn, J. T. O'Brien, Movement Disord. 2003, 18:S88-S95. At present, identification of new amyloid sensing molecules is based mainly on modification of existing dyes and/or screening of libraries of dyes. See E. E. Nesterov, J. Skoch, B. T. Hyman, W. E. Klunk, B. J. Bacskai, T. M. Swager, Angew. Chem. Int. Edit. 2005, 44:5452-5456; Z. P. Zhuang, M. P. Kung, H. F. Kung, J. Med. Chem. 2006, 49:2841-2844; Q. A. Li, J. S. Lee, C. Ha, C. B. Park, G. Yang, W. B. Gan, Y. T. Chang, Angew. Chem. Int. Edit. 2004, 43:6331-6335; H. F. Kung, C. W. Lee, Z. P. Zhuang, M. P. Kung, C. Hou, K. Plossl, J. Am. Chem. Soc. 2001, 123:12740-12741.
Compounds and methods for preventing or alleviating the symptoms of amyloid-associated disease, for example but not limited to, neuronal diseases and conditions associated with amyloid fibril or plaque formation, have been provided in U.S. application Ser. No. 11/487,224, filed Jul. 14, 2006, claiming priority to U.S. Prov. Appl. No. 60/699,728, filed Jul. 15, 2005, and U.S., Prov. Appl. No. 60/750,422, filed on Dec. 13, 2005, and published as U.S. Published Appl. No. 2007/0066665, published Mar. 22, 2007, all of which are incorporated herein by reference in their entireties and for all purposes. Compounds and methods for the diagnosis and treatment of amyloid associated diseases have been provided in International Appl. No. PCT/US2008/065410, filed May 30, 2008, claiming priority to U.S. Prov. Appl. No. 60/940,968, filed May 30, 2007, and published as PCT Publication No. WO 2008/15103, on Dec. 11, 2008, all of which are incorporated herein by reference in their entireties and for all purposes.
Each patent, published patent application, and reference cited herein is hereby incorporated herein in its entirety and for all purposes.
In a first aspect, there is provided a compound having the structure of Formula (I),
The term “EDG” refers to an electron donor group, as known in the art. The term “πCE” is a pi-conjugation element capable of being in conjugation with the electron donor group. The term “WSG” refers to a water soluble group.
In another aspect, there is provided a pharmaceutical composition. The pharmaceutical composition includes a compound described herein and a pharmaceutically acceptable excipient.
In another aspect, there is provided a method of detecting an Aβ peptide. The method includes contacting a compound as described herein with an Aβ peptide, and detecting a complex formed between the compound with the Aβ peptide.
In another aspect, there is provided a method of treating a disease or disorder characterized by an accumulation of amyloid deposits in the brain. The method includes administering to a subject in need of treatment an effective amount of a compound or pharmaceutical composition as described herein.
The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., —CH2O— is equivalent to —OCH2—.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e. unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).
The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkyl, as exemplified, but not limited, by —CH2CH2CH2CH2—, and further includes those groups described below as “heteroalkylene.” Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2—S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, O—CH3, —O—CH2—CH3, and —CN. Up to two heteroatoms may be consecutive, such as, for example, —CH,—NH—OCH3. Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O )2R′— represents both —C(O)2R′— and —R′C(O)2—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR, and/or —SO2R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —NR′R″ or the like, it will be understood that the terms heteroalkyl and —NR′R″ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —NR′R″ or the like.
The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” is meant to include, but not be limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
The term “acyl” means —C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together (i.e. a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e. multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent means a divalent radical derived from an aryl and heteroaryl, respectively.
For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).
The term “oxo” as used herein means oxygen that is double bonded to a carbon atom.
The term “alkylsulfonyl” as used herein means a moiety having the formula —S(O2)—R′, where R′ is an alkyl group as defined above. R′ may have a specified number of carbons (e.g. “C1-C4 alkylsulfonyl”).
Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and “heteroaryl”) are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR'R″R″′, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″′, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —CN and —NO2 in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R″, R″′ and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″ and R″″ groups when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).
Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: halogen, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R″′, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″′, —NR″C(O)2R′, —NR—C(NR′R″R″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —CN and —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″, R″′ and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″ and R″″ groups when more than one of these groups is present.
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)—(CRR′)q—U—, wherein T and U are independently —NR—, —O—, —CRR′—or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′— or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CRR′)s—X′—(C″R″′)d-, where s and d are independently integers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituents R, R′, R″ and R′″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
As used herein, the term “heteroatom” or “ring heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
A “substituent group,” as used herein, means a group selected from the following moieties:
(A) —OH, —NH2, —SH, —CN, —CF3, —NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and
(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from:
A “size-limited substituent” or “ size-limited substituent group,” as used herein means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
A “lower substituent” or “ lower substituent group,” as used herein means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
Thus, the compounds of the present invention may exist as salts, such as 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 (e.g., (+)-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.
The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
In addition to salt forms, the present invention provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those which are known in the art to be too unstable to synthesize and/or isolate.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
Where a substituent of a compound provided herein is “R-substituted” (e.g. R1-substituted), it is meant that the substituent is substituted with one or more of the named R groups (e.g. R1) as appropriate. In some embodiments, the substituent is substituted with only one of the named R groups.
The terms “treating” or “treatment” refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. For example, the certain methods presented herein successfully treat cancer by decreasing the incidence of cancer, in inhibiting its growth and or causing remission of cancer.
An “effective amount” is an amount of a compound described herein sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, or to inhibit effects of an amyloid relative to the absence of the compound. Where recited in reference to a disease treatment, an “effective amount” may also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) a disease, or reducing the likelihood of the onset (or reoccurrence) of a disease or its symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an osteoclast or leukocyte relative to the absence of the antagonist.
In one aspect, there is provided a compound having the structure of Formula (I),
The term “EDG” refers to an electron donor group, as known in the art. The term “πCE” is a pi-conjugation element capable of being in conjugation with the electron donor group. The term “WSG” refers to a water soluble group.
Examination of the chemical structures of Cmpds 1-5 (supra) reveals that the majority of these compounds contain an electron donor unit in conjugation with an electron acceptor (D-π-A motif). This motif is a typical feature in molecular rotors, a family of fluorescent probes known to form twisted intramolecular charge transfer (TICT) complexes in the excited state producing a fluorescence quantum yield that is dependent on the surrounding environment. See Z. R. Grabowski, K. Rotkiewicz, W. Rettig, Chem. Rev. 2003, 103:3899-4031; M. A. Haidekker, E. A. Theodorakis, Org. Biomol. Chem. 2007, 5:1669-1678. Following photoexcitation, this motif has the unique ability to relax either via fluorescence emission or via an internal non-radiative molecular rotation. This internal rotation occurs around the σ-bonds that connect the electronically rich π-system with the donor and acceptor groups and can modified by altering the chemical structure and microenvironment of the probe. See R. O. Loutfy, Pure & Applied Chemistry 1986, 58:1239-1248. Hindrance of the internal molecular rotation of the probe, by increasing the surrounding media rigidity or by reducing the available free volume needed for relaxation, leads to a decrease in the non-radiative decay rate and consequently an increase of fluorescence emission. In contrast, in environments of low viscosity or of high free volume the relaxation proceeds mainly via non-radiative pathways. Due to these properties, molecular rotors have been used to study polarity, free volume and viscosity changes in solvents and organized assemblies, such as liposomes, cells and polymers. See M. L. Viriot, M. C. Carre, C. Geoffroy-Chapotot, A. Brembilla, S. Muller, J. F. Stoltz, Clinical Hemorheology and Microcirculation 1998, 19:151-160; M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis, Bioorganic Chemistry 2005, 33:415-425; M. E. Nipper, S. Majd, M. Mayer, J. C. M. Lee, E. A. Theodorakis, M. A. Haidekker, Biochimica Et Biophysica Acta-Biomembranes 2008, 1778:1148; M. A. Haidekker, T. T. Ling, M. Anglo, H. Y. Stevens, J. A. Frangos, E. A. Theodorakis, Chemistry & Biology 2001, 8:123-131; M. A. Haidekker, A. G. Tsai, T. Brady, H. Y. Stevens, J. A. Frangos, E. Theodorakis, M. Intaglietta, American Journal of Physiology-Heart and Circulatory Physiology 2002, 282:H1609-H1614; M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis, Bioorganic Chemistry 2005, 33:415-425; C. Frochot, C. Muller, A. Brembilla, M. C. Cane, P. Lochon, M. L. Viriot, International Journal of Polymer Analysis and Characterization 2000, 6:109-122; C. Damas, M. Adibnejad, A. Benjelloun, A. Brembilla, M. C. Cane, M. L. Viriot, P. Lochon, Colloid and Polymer Science 1997, 275:364-371.
Intrigued by the above observations, we asked whether we could design amyloid-binding agents based on the molecular rotor motif. We envisioned that π-conjugation of, for example but not limited to, a dialkyl amino group, as the electron donor (D), with a 2-cyano acrylate unit, as the electron acceptor (A), would produce Aβ-binding molecules with inherent fluorescence properties. See S. J. Lord, N. R. Conley, H. L. D. Lee, R. Samuel, N. Liu, R. J. Twieg, W. E. Moerner, Journal of the American Chemical Society 2008, 130:9204-9205; S. J. Lord, N. R. Conley, H. L. D. Lee, S. Y. Nishimura, A. K. Pomerantz, K. A. Willets, Z. K. Lu, H. Wang, N. Liu, R. Samuel, R. Weber, A. Semyonov, M. He, R. J. Twieg, W. E. Moerner, Chemphyschem 2009, 10:55-65. Interestingly, the fluorescence properties of such motif could be fine-tuned by modifying the electronic density and extent of conjugation between the donor and acceptor units. The solubility of these amyloid-binding agents in aqueous systems can be achieved by the introduction of water solubilizing groups (WSG), such as esters of triethylene glycol monomethyl ether (TEGME) or of glycerol. The design concept is shown in Formula (I).
Important to the synthesis of compounds described herein was a Knoevenagel condensation of 1 equivalent of the appropriate aldehyde, e.g. 6, with 1.1 equivalents of the appropriate malonic acid derivative, e.g. 7. See Scheme 1. This reaction was catalyzed by piperidine (10%) and was completed within 21 hours in refluxing THF. See X. H. Chen, Z. J. Zhao, Y. Liu, P. Lu, Y. G. Wang, Chemistry Letters 2008, 37:570-571; M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis, Journal of the American Chemical Society 2006, 128:398-399. After a standard chromatographic purification on silica gel, the desired product 8 was isolated in excellent yields (Table 1). Reagents and conditions for Scheme 1: (a) 1.0 equiv 6, 1.1 equiv 7, 0.1 equiv piperidine, THF, 50° C., 21 h.
Naphthalene-based Cmpd 11 was synthesized by treatment of commercially available methoxy naphthaldehyde 9 with 8 equivalents of lithiated piperidine and Knoevenagel condensation of the resulting aldehyde 10 with cyano ester 7 (Scheme 2, 29% combined yield). See H. M. Guo, F. Tanaka, J. Org. Chem. 2009, 74:2417-2424. Scheme 2 reagents and conditions: (a) 8.0 equiv piperidine in benzene/HMPA: 1/1, 0° C., 8.0 equiv nBuLi, 0° C., 15 min, then 1.0 equiv 9, 25 ° C., 12 h, 35%; (b) 1.0 equiv 10, 1.1 equiv 7, 0.1 equiv piperidine, THF, 50° C., 21 h, 82%.
Cmpd 14 was prepared by condensation of aldehyde 6a with α-cyano ester 12, followed by an acid-catalyzed deprotection of the acetonide unit (Scheme 3, 68% combined yield). See M. A. Haidekker, T. P. Brady, S. H. Chalian, W. Akers, D. Lichlyter, E. A. Theodorakis, Bioorg. Chem. 2004, 32:274-289. Scheme 3 reagents and conditions: (a) 1.0 equiv 6a, 1.1 equiv 12, 0.1 equiv piperidine, THF, 50° C., 21 h, 91%; (b) 1.5 mmol 13, 0.10 g DOWEX-H+,1:1 THF/MeOH, 25° C., 20 h, 75%.
Stilbene-based Cmpd 19 was synthesized in four steps that included: (a) conversion of benzyl bromide 15 to phosphonate 16; (b) Horner-Emmons olefination of 16 with aldehyde 6a to form 17; (c) lithiation of bromide 17 and formylation to produce aldehyde 18; and (d) Knoevenagel condensation of the resulting aldehyde 18 with cyano ester 7 (Scheme 4, 42% combined yield). See H. Meier, E. Karpuk, H. C. Hoist, Eur. i J. Org. Chem. 2006, 2609-2617; L. Viau, O. Maury, H. Le Bozec, Tetrahedron Lett. 2004, 45:125-128. Scheme 4 reagents and conditions: (a) 1.0 equiv 15,15 equiv triethyl phosphite, 90° C., 19 h, 98%; (b) 1.0 equiv 16, 1.0 equiv NaOMe, 1.0 equiv 6a, excess DMF, 25° C., 24 h, 74%; (c) 1.0 equiv 17, 1.0 equiv n-BuLi, 1.33 equiv DMF, THF, κ78° C., 60%; (d) 1.0 equiv 18, 1.1 equiv 7, 0.1 equiv piperidine, THF, 50° C., 21 h, 97%.
In some embodiments, the EDG (Formula I) is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, —OR1, —NHC(O)R2, or —NR3R4. In some embodiments, R1, R3 and R4 are independently H, or substituted or unsubstituted alkyl; and R2 is substituted or unsubstituted alkyl.
In some embodiments, the pi-conjugation element (Formula I) is substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, the pi-conjugation element is unsubstituted aryl. In some embodiments, the pi-conjugation element is substituted aryl.
In some embodiments, the water soluble group (Formula I) includes ethylene glycol. In some embodiments, the water soluble group includes polymeric ethylene glycol.
In some embodiments, a compound is provided which further includes a detectable label. In some embodiments, the detectable label is a radiolabel. In some embodiments, the detectable label is a fluorescent label.
In some embodiments, each substituted group described herein in a compound of Formula I is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene described herein in the compounds of Formula I is substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. Alternatively, at least one or all of these groups are substituted with at least one lower substituent group.
In other embodiments of the compounds of Formula I, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene substituted or unsubstituted C4-C8 cycloalkylene, and each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 4 to 8 membered heterocycloalkylene.
Alternatively, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene substituted or unsubstituted C5-C6 cycloalkylene, and each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 5 to 7 membered heterocycloalkylene.
In some embodiments, a compound of Formula I is a compound within the scope of Formula I set forth in Table 2 below:
8a
8c
Inspired by the structures of the currently used amyloid-binding agents, we have evaluated the possibility to design new Aβ binding fluorescent compounds useful as probes based on the molecular rotor motif. We found that the molecular rotors bind to the aggregated AP peptide with low micromolar affinity. We hypothesize that this binding is a result of hydrophobic interactions between the rotor and the amyloid peptide. This binding reduces the free volume around the rotor resulting in an increased fluorescence emission. See R. O. Loutfy, B. A. Arnold, J. Phys. Chem. 1982, 86:4205-4211; A. K. Doolittle, J. Appl. Phys. 1952, 23:236-239. A similar effect has been reported for the binding of molecular rotors to actin, albumin and other proteins. See T. Lio, S. Takahashi, S. Sawada, J. Biochem. 1993, 113:196-199; T. Iwaki, C. Torigoe, M. Noji, M. Nakanishi, Biochem. 1993, 32:7589-7592. We have demonstrated that these molecules can be readily synthesized and have no significant cytotoxicity. In addition, we have shown that both the physical properties and fluorescence profile of these fluorescent compounds can be fine-tuned by modifying their chemical structure. Notably, changes of the substitution at the electron donor group can affect the intensity of fluorescence emission, while changes at the π-system can affect the emission wavelength. These effects can be implemented for the construction of multicolored dyes and can lead to potential applications for in vitro and in vivo imaging. See C. J. Sigurdson, K. Peter, R. Nilsson, S. Hornemann, G. Manco, M. Polymenidou, P. Schwarz, M. Leclerc, P. Hammarstrom, K. Wuthrich, A. Aguzzi, Nat. Methods 2007, 4,:023-1030. Interestingly, a recent report describes the identification of CRANAD-2, a small molecule containing two electron-donating groups connected simultaneously via π-conjugation to a single difluoroboronate acceptor. See C. Ran, X. Xu, S. B. Raymond, B. J. Ferrara, K. Neal, B. J. Bacskai, Z. Medarova, A. Moore, J. Am. Chem. Soc. 2009, 131:15257-15261. This compound has a high affinity for Aβ aggregates (Kd=38.0 nM) and suitable near-infrared fluorescence properties for in vivo imaging, further validating our proposed concept of exploring the molecular rotor motif for the development of new amyloid-imaging compounds. These findings demonstrate that the D-π-A motif of molecular rotors (Formula I) is a useful scaffold and represents an important first step for the rational design of new diagnostic tools for Alzheimer's disease and related amyloid-based neurodegenerative disorders.
In another aspect, the present invention provides pharmaceutical compositions (i.e., formulations) comprising a compound described herein in combination with a pharmaceutically acceptable excipient (e.g., carrier). The pharmaceutical compositions include optical isomers, diastereomers, or pharmaceutically acceptable salts of the inhibitors disclosed herein. For example, in some embodiments, the pharmaceutical compositions include a compound of the present invention and citrate as a pharmaceutically acceptable salt. The compound included in the pharmaceutical composition may be covalently attached to a carrier moiety, as described above. Alternatively, the compound included in the pharmaceutical composition is not covalently linked to a carrier moiety.
A “pharmaceutically acceptable carrier,” as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent. 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 that do not deleteriously react with the compounds of the invention.
The compounds of the invention can be administered alone or can be coadministered to the subject. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). The preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
The compounds can be prepared and administered in a wide variety of oral, parenteral, and topical dosage forms. Thus, the compounds of the present invention can be administered by injection (e.g. intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally). Also, the compounds described herein can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. It is also envisioned that multiple routes of administration (e.g., intramuscular, oral, transdermal) can be used to administer the compounds of the invention. Accordingly, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and one or more compounds of the invention.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substance that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from 5% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
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. Ampoules 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 include those 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.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
The quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.
Some compounds may have limited solubility in water and therefore may require a surfactant or other appropriate co-solvent in the composition. Such co-solvents include: Polysorbate 20, 60, and 80; Pluronic F-68, F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Such co-solvents are typically employed at a level between about 0.01% and about 2% by weight.
Viscosity greater than that of simple aqueous solutions may be desirable to decrease variability in dispensing the formulations, to decrease physical separation of components of a suspension or emulsion of formulation, and/or otherwise to improve the formulation. Such viscosity building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose, chondroitin sulfate and salts thereof, hyaluronic acid and salts thereof, and combinations of the foregoing. Such agents are typically employed at a level between about 0.01% and about 2% by weight.
The compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides, and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.
Pharmaceutical compositions provided by the present invention include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. For example, when administered in methods to treat cancer, such compositions will contain an amount of active ingredient effective to achieve the desired result (e.g. decreasing the number of cancer cells in a subject).
The dosage and frequency (single or multiple doses) of compound administered can vary depending upon a variety of factors, including 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; presence of other diseases or other health-related problems; kind of concurrent treatment; and complications from any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of the invention.
For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays, as known in the art.
Therapeutically effective amounts for use in humans may be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. In one embodiment of the invention, the dosage range is 0.001% to 10% w/v. In another embodiment, the dosage range is 0.1% to 5% w/v.
Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.
The ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl et al., In T
In one aspect, there is provided a method of detecting an Aβ peptide. The method includes contacting a compound as described herein with an Aβ peptide, and detecting a complex formed between the compound with the Aβ peptide, as described herein and known in the art. The method of detection can employ spectroscopic (i.e., UV-visible, fluorescence, and the like), radiographic, and other detection methods known in the art.
In a further aspect, there is provided a method of treating a disease or disorder characterized by an accumulation of amyloid deposits in the brain. The method includes administering to a subject in need of treatment a compound as described herein. In some embodiments, the disease is Alzheimer's disease. The term “subject” as used herein refers to a mammal to which a pharmaceutical composition or formulation is administered. Exemplary subjects include humans, as well as veterinary and laboratory animals such as horses, pigs, cattle, dogs, cats, rabbits, rats, mice, and aquatic mammals.
General notes: All the reagents were obtained (Aldrich, Acros) at highest commercial quality and used without further purification except where noted. Air- and moisture-sensitive liquids and solutions were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation below 45° C. at approximately 20 mmHg. All non-aqueous reactions were carried out under anhydrous conditions. Yields refer to chromatographically and spectroscopically (1H NMR, 13C NMR) homogeneous materials, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates (60E-254) and visualized under UV light and/or developed by dipping in solutions of 10% ethanolic phosphomolybdic acid (PMA) or p-anisaldehyde and applying heat. E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash chromatography. Preparative thin-layer chromatography separations were carried out on 0.25 or 0.50 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Varian Mercury 300 or 400 MHz instruments and calibrated using the residual undeuterated solvent as an internal reference. The following abbreviations were used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, b=broad. High resolution mass spectra (HRMS) were recorded on a VG 7070 HS mass spectrometer under electron spray ionization (ESI) or electron impact (EI) conditions. Fluorescence spectroscopy data were recorded on a MD-5020 Photon Technology International Spectrophotometer at 25° C.
General Procedure for the Preparation of Fluorescence Compounds. To a round bottom flask containing a solution of aldehyde (5.0 mmol) and 2-(2-(2-mcthoxycthoxy) ethoxy)ethyl 2-cyanoacetate (5.5 mmol) in 20 ml of THF was added 0.50 mmol of piperidine and the mixture was heated at 50° C. The reaction was monitored by TLC and was completed within 21 hours. The crude mixture was concentrated under reduced pressure and the product was purified via flash chromatography (10-30% ethyl acetate in hexane).
Example 1. Compound Synthesis and Analysis. Results of analysis of compounds described herein are provided in Examples 1a-1m following.
Example 1a. (E)-2-(2-(2-methoxyethoxy)ethoxy)ethyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate (8a). 98% ; yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.93 (d, 2H, J=9.0 Hz), 6.69 (d, 2H, J=9.1 Hz), 4.41 (m, 2H), 3.81- 3.79 (m, 2H), 3.73-3.65 (m, 6H), 3.56 -3.54 (m, 2H), 3.37 (s, 3H), 3.10 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 164.2, 154.7, 153.6, 134.1, 119.3, 117.4, 111.4, 93.6, 71.9, 70.8, 70.6, 70.5, 68.9, 65.0, 59.0, 40.0; HRMS Calc for C19H26N2O5 (M)+ 362.1836 found 362.1841.
Example 1b. (E)-2-(2-(2-methoxyethoxy)ethoxy)ethyl2-cyano-3-(4-(dimethylamino)-2-methoxyphenyl)acrylate (8b). 98% yield ; yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.64 (s,1H), 8.39 (d, 1H, J=9.2 Hz), 6.63 (dd, 1H, J=2.3 Hz, J=9.2 Hz), 6.01 (s, 1H), 4.40 (m, 2H), 3.87 (s, 3H), 3.81-3.78 (m, 2H), 3.73- 3.65 (m, 6H), 3.56- 3.53 (m, 2H), 3.36 (s, 3H), 3.10 (s, 6H); 13C NMR (400 MHz, CDCl3) δ 165.0, 162.2, 155.9, 148.5, 131.3, 118.4, 109.7, 105.4, 93.0, 92.0, 72.2, 71.1, 70.9, 70.8, 69.2, 65.1, 59.3, 55.6, 40.4; HRMS Calc for C20H28N2O6(M+Na)+ 415.1840 found 415.1836.
Example 1c. (Z)-2-(2-(2-methoxyethoxy)ethoxy)ethyl 2-cyano-3-(4-(diethylamino)phenyl)acrylate (8c). 90% yield; orange liquid; 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.92 (d, 2H, J=9.1 Hz), 6.67 (d, 2H, J=9.2 Hz), 4.42 (m, 2H), 3.82-3.79 (m, 2H), 3.73-3.72 (m, 2H), 3.69-3.65 (m, 4H), 3.57-3.54 (m, 2H), 3.45 (q, 4H, J=7.1 Hz), 3.37 (s, 3H), 1.23 (t, 6H, J=7.1 Hz); 13CNMR (100 MHz, CDCl3) δ 164.7, 154.8, 151.9, 134.8, 119.0, 117.8, 111.4, 93.0, 72.2, 71.1, 70.9, 70.8, 69.2, 65.2, 59.3, 45.0, 12.8; HRMS Calc for C21H30N2O5(M+Na)+ 413.2047 found 413.2053.
Example 1d. (Z)-2-(2-(2-methoxyethoxy)ethoxy)ethyl-2-cyano-3-(4-(dibutylamino)phenyl)acrylate (8d). 78% yield; yellow liquid; 1HNMR (400 MHz, CDCl3) 8.00 (s, 1H), 7.87 (d, 2H, J=9.0 Hz), 6.60 (d, 2H, J=9.2 Hz), 4.38 (m, 2H), 3.78-3.76 (m, 2H), 3.71-3.69 (m, 2H), 3.66-3.62 (m, 4H), 3.53-3.51 (m, 2H), 3.34-3.30 (m, 7H), 1.57 (m, 4H), 1.34 (m, 4H), 0.94 (t, 6H, J=7.3 Hz); 13CNMR (100 MHz, CDCl3) δ 164.7, 154.7, 152.2, 134.6, 118.9, 117.9, 111.5, 92.8, 72.1, 71.0, 70.8, 69.1, 65.2, 59.2, 51.1, 29.5, 20.4, 14.1; HRMS Calc for C25H38N2O5(M+Na)+ 469.2673 found 469.2677.
Example 1e. 6-(piperidin-1-yl)-2-naphthaldehyde (10). To a 50 ml round bottom flask containing benzene (3 mL), HMPA (3 mL) and piperidine (1.65 ml, 16.7 mmol) n-BuLi (1.6 M in hexane, 10.4 mL, 16.7 mmol) was added via syringe, at 0° C. After stirring for 15 min, the reaction mixture was treated with a solution of 6-methoxy-2-naphthaldehyde (390 mg, 2.09 mmol) in benzene: HMPA 1:1 (2 ml). The reaction mixture was warmed to room temperature, left stirring for 12 hours and then it was poured into cold 5% aqueous NaCl (30 ml). The mixture was extracted with diethyl ether (3×20 mL), dried over MgSO4 and concentrated. The product was purified via flash chromatography (20% EtOAc in hexanes) to give compound 9. 9: 35% yield, yellow solid; 1H NMR (300 MHz, CDCl3) δ 10.02 (s, 1H), 8.14 (s, 1H), 7.88-7.73 (m, 2H), 7.67 (d, 1H, J=8.6 Hz), 7.32 (dd, 1H, J=2.5 Hz, J=9.1 Hz), 7.08 (d, 1H, J=2.4 Hz), 3.42-3.32 (m, 4H), 1.85-1.57 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 192.2, 152.2, 138.8, 134.7, 131.6, 130.7, 127.5, 126.5, 123.6, 119.7, 109.0, 49.8, 25.8, 24.6; HRMS calc for C16H17NO (M+H)+ 240.1383 found 240.1387.
Example 1f. (E)-2-(2-(2-methoxyethoxy)ethoxy)ethyl-2-cyano-3-(6-(piperidin-1-yl) naphthalen-2-yl)acrylate (11). 82% yield; red liquid; 1H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 8.22 (d, 1H, J=1.2 Hz), 8.10 (dd, 1H, J=1.8 Hz, J=8.8 Hz), 7.76 (d, 1H, J=9.2 Hz), 7.65 (d, 1H, J=8.8 Hz), 7.29 (dd, 1H, J=2.4 Hz, J=9.2 Hz), 7.05 (d, 1H, J=2.2 Hz), 4.47 (m, 2H), 3.85-3.82 (m, 2H), 3.74- 3.66 (m, 6H), 3.57- 3.54 (m, 2H), 3.42-3.38 (m, 4H), 3.37 (s, 3H), 1.74-1.67 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 163.4, 155.5, 151.9, 137.8, 134.7, 130.6, 127.3, 126.4, 126.0, 125.7, 119.3, 116.4, 108.4, 98.7, 71.9, 70.8, 70.6, 70.5, 68.8, 65.4, 59.0, 49.4, 25.5, 24.3; HRMS Calc for C26H32N2O5(M+H)+ 453.2384 found 453.2390.
Example 1g. (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyanoacetate (12). To a solution of 2-cyanoacetic acid (1.02 g, 12 mmol), the acetal (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1.32 g, 10 mmol) in 5 ml of DCM and DMAP (61 mg, 0.50 mmol) was added dropwise at 0° C. Finally, EDC 1.86 g (12 mmol) was added and the reaction mixture was stirred at 0° C. for 6 hours. The reaction was diluted with 15 mL of DCM and the formed DCU was filtered off. The filtrate was dried over anhydrous MgSO4 and the solvents were removed under reduced pressure. The residue was purified by flash chromatography (Hex: EtOAc; 10:1) to give compound 12. 12: 71% yield; colorless liquid; 1H NMR (400 MHz, CDCl3) δ 4.34-4.32 (m, 1H), 4.28-4.17 (m, 2H), 4.07 (dd, 1H, J=6.5 Hz, J=8.5 Hz), 3.75 (dd, 1H, J=5.8 Hz, J=8.5 Hz), 3.51 (s, 2H), 1.41 (s, 3H), 1.34 (s, 3H); HRMS Calc for C9H13NO4(M+H)+ 200.0923 found 200.0931.
Example 1h. (E)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl2-cyano-3-(4-(dimethylamino)phenyl)acrylate (13). To a round bottom flask containing a solution of aldehyde 6a (0.75 g, 5.0 mmol) and compound 12 (1.2 g, 5.5 mmol) in 20 ml of THF was added 0.50 mmol of piperidine and the mixture was heated at 50° C. The crude mixture was concentrated under reduced pressure and the product was purified via flash chromatography (10-30% ethyl acetate in hexane) to give compound 13. 13: 91% yield; yellow solid; 1HNMR (400 MHz, CDCl3) δ 8.08 (s, 1 H), 7.94 (d, 2H, J=9.0 Hz), 6.69 (d, 2H, J=9.2 Hz), 4.42- 4.29 (m, 3H), 4.13 (dd, 1H, J=6.2 Hz, J=8.6 Hz), 3.89 (dd, 1H, J=5.9 Hz, J=8.5 Hz), 3.11 (s, 6H), 1.46 (s, 3H), 1.38 (s, 3H); 13CNMR (400 MHz, CDCl3) δ 164.3, 155.3, 153.9, 134.5, 119.5, 117.5, 111.7, 110.1, 93.3, 73.7, 66.7, 65.6, 40.3, 26.9, 25.7; HRMS Calc for C18H22N2O4(M+H)+ 331.1658 found 331.1691.
Example 1i. (E)-2,3-dihydroxypropyl 2-cyano-3-(4-(dimethylamino)phenyl) acrylate (14). Compound 13 (0.5 g, 1.5 mmol) was dissolved in a mixture of THF/ MeOH (1:1) and DOWEX-H+ resin (0.10 g) was added and the heterogeneous mixture was stirred for 20 hours. The DOWEX-H+ resin was removed by filtration and triethylamine (50 mg, 0.5 mmol) was added and the solvent was removed under reduced pressure. The residue was purified by flash chromatography (100% ether) to give compound 14. 14: 75% yield; bright yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.94 (d, 2H, J=9.1 Hz), 6.69 (d, 2H, J=9.2 Hz), 4.42-4.32 (m, 2H), 4.05 (m, 1H), 3.80-3.70 (m, 2H), 3.12 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 199.0, 164.8, 155.5, 154.0, 134.6, 119.4, 117.9, 111.8, 111.7, 92.8, 70.3, 70.2, 66.9, 66.8, 63.6, 63.5, 40.3, 40.2; HRMS Calc for C15H18N2O4 (M+H)+ 291.1345 Found 291.1361.
Example 1j. Diethyl 4-bromobenzylphosphonate (16). 1-bromo-4-(bromomethyl) benzene (5.0 g, 20 mmol) and triethyl phosphite (51 mL, 300 mmol) were mixed in a round bottom flask and refluxed at 90° C. for 19 hours. Excess triethyl phosphite was removed under reduced pressure and the product purified by flash chromatography (1:1 Hexane/ EtOAc) to give compound 16. 16: 98% yield ; colorless liquid; 1H NMR (400 MHz, CDCl3) δ 7.30 (d, 2H, J=7.5 Hz), 7.05 (d, 2H, J=7.6 Hz), 3.99-3.88 (m, 4H), 2.99 (s, 1H), 2.94 (s, 1H), 1.12 (t, 6H, J=6.9 Hz); 13C NMR (100 MHz, CDCl3) δ 131.7, 131.6, 131.5, 121.0, 62.3, 34.0, 32.0, 16.5; HRMS Calc for C11H16BrO3P (M+H)+307.0097 found 307.0093.
Example 1k. (E)-4-(4-bromostyryl)-N,N-dimethylaniline (17). DMF (anhydrous) (10.5 mL) was added to sodium methoxide (176 mg, 3.26 mmol) and the color was changed to pink To the above solution diethyl 4-bromobenzylphosphonate (1.0 g, 3.26 mmol) in DMF (6.5 ml) was added dropwise over 2 minutes, followed by 4 (dimethylamino)benzaldehyde (486 mg, 3.26 mmol). The reaction mixture was stirred at room temperature for 24 hours. Deionized water (17 mL) was added. The product was filtered out through vacuum filtration and recrystallized with DCM/ hexane to give compound 17. 17: 74%. Yield; tan solid; 1H NMR (400 MHz, CDCl3) δ 7.47-7.32 (m, 6H), 7.04 (d, 1H, J=12.5 Hz), 6.83 (d, 1H, J=16.3 Hz), 6.71 (d, 2H, J=8.9 Hz), 2.99 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 150.5, 137.4, 136.1, 132.1, 131.8, 129.7, 128.3, 128.2, 127.9, 127.7, 125.5, 123,2, 120.3, 112.6, 40.7; HRMS Calc for C16H16BrN 302.0541 found 302.0539.
Example 1L. 4-(4-(dimethylamino)styryl)benzaldehyde (18). To a round bottom flask compound 17 (300 mg, 1 mmol) was transferred followed by THF (5 mL). The heterogeneous solution was cooled at −78 ° C. and n-BuLi (1.6M in hexane, 1 mmol) was added dropwise over 5 min, followed by DMF (1.5 mL). The reaction mixture was stirred at −78° C. for 3 hours then it was quenched by water (1 mL) and the mixture was extracted with ether (2×25 mL). The combined organic extracts were washed with brine, dried over MgSO4 and concentrated under reduced pressure to give compound 18. 18: 60% yield; yellow powder; 1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 7.83 (d, 2H, J=8.2 Hz), 7.60 (d, 2H, J=8.2 Hz), 7.44 (d, 2H, J=8.8 Hz), 7.22 (d, 1H, J=16.2 Hz), 6.94 (d, 1H, J=16.2 Hz), 6.72 (d, 2H, J=8.8 Hz), 3.01 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 191.8, 150.8, 144.7, 134.7, 134.6, 132.7, 130.4, 128.4, 126.4, 124.9, 122.8, 112.4, 40.5; HRMS calc for C17H17NO 252.1384 found 252.1383.
Example 1m. (E)-2-(2-(2-methoxyethoxy)ethoxy)ethyl-cyano-3-(4-(4(dimethylamino)styryl)phenyl)acrylate (19). 97% yield; red solid; 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.98 (d, 2H, J=8.4 Hz), 7.57 (d, 2H, J=8.4 Hz), 7.45 (d, 2 H, J=8.7 Hz), 7.20 (d, 1H, J=16.2 Hz), 6.92 (d, 1H, J=16.2 Hz), 6.72 (d, 2H, J=8.7 Hz), 4.47 (m, 2H), 3.84-3.82 (m, 2H), 3.74-3.72 (m, 2H), 3.70-3.66 (m, 4H), 3.57-3.55 (m, 2H), 3.37 (s, 3H), 3.02 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 154.9, 133.0, 132.2, 128.6, 128.5, 126.7, 122.7, 112.4, 72.2, 71.1, 70.8, 69.0, 65.8, 59.3, 40.6, 40.5, 29.9, 28.2; HRMS calc for C27H32N2O5(M+Na)+ 487.2203 found 487.2201.
Example 2. Detection Studies. An initial study to determine whether a compound can associate with aggregated Aβ is to compare its fluorescence spectra before and after mixing with the Aβ aggregates. See E. E. Nesterov, J. Skoch, B. T. Hyman, W. E. Klunk, B. J. Bacskai, T. M. Swager, Angew. Chem. Int. Edit. 2005, 44:5452-5456; Z. P. Zhuang, M. P. Kung, H. F. Kung, J. Med. Chem. 2006, 49:2841-2844; Q. A. Li, J. S. Lee, C. Ha, C. B. Park, G. Yang, W. B. Gan, Y. T. Chang, Angew. Chem. Int. Edit. 2004, 43:6331-6335; H. F. Kung, C. W. Lee, Z. P. Zhuang, M. P. Kung, C. Hou, K. Plossl, J. Am. Chem. Soc. 2001, 123:12740-12741. Typically, a fluorescent amyloid-binding agent displays a significant fluorescence intensity increase after binding to Aβ aggregates as compared to its native fluorescence in solution. See H. LeVine III, Protein Sci. 1993, 2:404-410. Along these lines we measured the fluorescent properties of each compound at 4 μM concentration before and after mixing with preaggregated Aβ(1-42) peptides (5 μM, aggregated in PBS buffer for 3 days at 25° C.).
In all cases, a 1.3-9.4 fold fluorescence intensity increase was observed in the presence of aggregated Aβ, indicating that these compounds bind to the peptide (Table 2). In most cases a modest blue-shift (6-20 nm) was observed upon binding. Only in the case of the naphthalene-based Cmpd 11 was a significant red shift of 76 nm observed upon binding to preaggregated Aβ (
Cmpds 8a and 8b exhibited similar fluorescence characteristics suggesting that addition of a methoxy group on the phenyl group does not alter the binding properties of the compound as a probe. On the other hand, it is worth noting that increasing the size of the alkyl groups of the nitrogen leads to a significant increase in the fluorescence intensity after binding (Table 2, 8a, 8c, 8d). This is likely a result of the decreased rotational freedom of the molecules upon binding to the aggregated forms of Aβ peptide. See W. Schuddeboom, S. A. Jonker, J. M. Warman, U. Leinhos, W. Kuehnle, K. A. Zachariasse, J. Phys. Chem. 1992, 96 :10809-10819; Y. V. Il'chev, W. Kuehnle, K. A. Zachariasse, J. Phys. Chem. 1998, 102 :5670-5680. Interestingly, no increase of fluorescence intensity was observed upon mixing of these compounds with monomeric Aβ peptide. This supports the notion that these compounds bind selectively to aggregated forms of Aβ. The fluorescence profile of 8d (excitation and emission) is shown in
Similarly, the fluorescence excitation spectra of Cmpds 8a, 8b, 8c, 14 and 19 are depicted in
Example 3. Binding Studies. We also measured the apparent binding constants (Kd) of the compounds (in concentrations of 10, 5, 2.5 and 1.25 μM) to 5.0 μM pre-aggregated Aβ(1-42) peptide. The Kd can be measured from the double reciprocal of the fluorescent maximum and the concentration of the compound. See H. LeVine III, Protein Sci. 1993, 2:404-410. All Kd values were measured between 1.4 and 5.3 μM (Table 2). It is remarkable that, despite the structural differences, these compounds display similar Kd values suggesting that they bind in a similar fashion to aggregated Aβ. Moreover, these values are similar to the reported Kd values for ThT (2 μM).[22, ] See LeVine, Id.; Lockhart, L. Ye, D. B. Judd, A. T. Merritt, P. N. Lowe, J. L. Morgenstern, G. Z. Hong, A. D. Gee, J. Brown, J. Biol. Chem. 2005, 280:7677-7684; M. Biancalana, K. Makabe, A. Koide, S. Koide, J. Mol. Biol. 2008, 383:205-213; M. Biancalana, K. Makabe, A. Koide, S. Koide, J. Mol. Biol. 2009, 385:1052-1063. The double reciprocal plot of fluorescence intensity versus concentration of Cmpds 8d and 11 are shown in
The association of the synthesized compounds with aggregated Aβ peptides was tested using a semi-quantitative ELISA based assay developed by Yang and co-workers. See P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006, 16:1076-1079; P. Inbar, C. Q. Li, S. A. Takayama, M. R. Bautista, J. Yang, ChemBioChem 2006, 7:1563-1566; P. Inbar, M. R. Bautista, S. A. Takayama, J. Yang, Anal. Chem. 2008, 80:3502-3506. The assay is based on screening for molecules that inhibit the interaction of the aggregated Aβ peptide with a monoclonal anti-Aβ IgG raised against residues 1-17 of Aβ. Table 2 provides the concentrations of the compounds corresponding to 50% inhibition (IC50) of the IgG-Aβ interactions as well as the maximal percentage of the IgG's inhibited from binding to the fibrils. All compounds exhibited IC50 values at μM levels, the lowest value being measured for 8b (IC50=1.17 μM). The maximum inhibition, a measure of the extent of surface coating of the aggregated peptide by the compounds, was determined to be between 40-98% (Table 2). See P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006, 16:1076-1079; P. Inbar, C. Q. Li, S. A. Takayama, M. R. Bautista, J. Yang, ChemBioChem 2006, 7:1563-1566; P. Inbar, M. R. Bautista, S. A. Takayama, J. Yang, Anal. Chem. 2008, 80:3502-3506. Comparison of these data indicates that the surface coating increases by decreasing the size of the compound or the extent of the π system. Specifically, while the maximum inhibition is between 81-98% for the phenyl compounds, it decreases to 58% for the longer naphthalene compound 11 and to 40% for the more conjugated stilbene 19. Representative graphs for Cmpds 8d and 11 are shown in
The logP values for all the compounds were calculated to be between 1.07 and 4.62 (Table 2) indicating that most of these compounds meet the solubility criteria and should be able to cross the blood brain barrier. See P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006, 16:1076-1079; P. Inbar, C. Q. Li, S. A. Takayama, M. R. Bautista, J. Yang, ChemBioChem 2006, 7:1563-1566; P. Inbar, M. R. Bautista, S. A. Takayama, J. Yang, Anal. Chem. 2008, 80:3502-3506; C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Deliver. Rev. 1997, 23:3-25. LogP values were calculated using the Molinspiration Chem—informatics software.
Example 4. Fluorescence Studies with Aggregated Aβ: Aggregated Aβ peptide was prepared by dissolving Aβ(1-42) in PBS pH 7.4 to a final concentration of 100 μM. This solution was magnetically stirred at 1200 rpm for 3 days at room temperature. The 100 μM Aβ(1-42) stock solution in PBS was aliquoted and frozen at −80° C. for up to 4 weeks without noticeable change in its property. 150 μL of pre-aggregated Aβ(1-42) was added to 2.85 mL of compound to attain a final concentration of 5 μM Aβ(1-42) and 4 μM of compound. The solution was transferred to 3 mL cuvette and the fluorescence measured at 25° C. As shown in
Example 5. Determination of Binding Constant: Pre-aggregated Aβ(1-42) (5 μM final concentration) was mixed with various concentrations of compounds described herein (10, 5, 2.5, 1.25 μM) in PBS buffer (pH 7.4) and their fluorescence was measured. The negative inverse of the x-intercept of the linear regression, that was drawn between the double reciprocal of the fluorescence intensity maximum and concentration of the compound, represents the compound binding constant (Kd) to Aβ(1-42).
Example 6. Determination of Kd from Fluorescence Method. In order to quantify the dissociation constants (Kd's) for the binding of fluorescent compounds with aggregated (β-amyloid peptides, we used the method described by LeVine (see H. LeVine III, Protein Sci. 1993, 2, 404-410). This method is similar to the method described by Benesi-Hildebran (see C. Yang, L. Liu, T. W. Mu, Q. X. Guo, Anal. Sci. 2000, 16, 537-539). Here, the fluorescence of the compound was measured with and without the addition of the aggregated peptides in solution. The relative fluorescence enhancement of the compound upon binding to aggregated (β-amyloid peptides was determined by taking the difference between F (fluorescence after the addition of aggregated peptides) and FO (fluorescence before the addition of aggregated peptides).
In order to estimate the binding constant (Kd) for the compound-Aβ complexes from the fluorescence studies, we made the following assumptions:
1. All compounds are completely in solution and free of any significant competing binding process such as self-aggregation.
2. The concentration of unbound compounds can be approximated as close to the total concentration of the compounds.
3. The binding sites in the aggregated Aβ peptides are not completely occupied at the concentration of Aβ-binding compounds used for the fluorescence studies (i.e., the experiments are carried out under non-saturated binding conditions).
According to the Beer- Lambert law (see J. W. Robinson, “Atomic spectroscopy”, 1996), one can obtain two expressions that relate the concentration of bound compound ([HG]), free compound ([G]), and free amyloid peptides ([H]) with either 1) the measured fluorescence of the compound in solution before the addition of the aggregated peptides (FO), or 2) the measured fluorescence of the compound in the presence of the amyloid peptides (F):
FO=εGl[GO] (1)
F=ε
HG
l[HG]+ε
H
l[H]+ε
G
l[G] (2)
where [GO]=total concentration of compound
[HG]=compound- Aβ complex concentration
[HO]=total concentration of aggregated peptide
[II]=unbound aggregated peptide concentration.
εG=absorption coefficient of G
εHG=absorption coefficient of HG
εH=absorption coefficient of H
l=path length
Substituting into equation 1, and making the approximation that εHGl[HG]+εGl[G]>>εHl[H], one can arrive at a simplified expression for the relative fluorescence of bound compound (ΔF):
ΔF=F−FQ=εHGl[HG]+εGl[G]−εGl[G]−εGl[HG] (3)
or ΔF=Δεl[HG] (4)
where Δε=εHG−εG.
In order to obtain a relationship between the change in measured fluorescence of the compound (ΔF) with the binding constant of the compound to aggregated β-amyloid peptides (Kd's), we used the standard equation for a binding isotherm to obtain a relationship between [HG] and Kd:
Combining equation 4 and 5, we obtained a relationship between ΔF and Kd:
In order to estimate the Kd of the compound bound to aggregated Aβ peptides from the measured change in fluorescence, we take the reciprocal of the equation 6 to give:
Equation 7 suggests that a double reciprocal plot of ΔF and [G] should yield a straight line with x-intercept equal to −1/Kd.
Example 7. ELISA Assay: Aggregated Aβ peptides were generated from synthetic Aβ(1-42) peptides by dissolving 30 μg of peptide in 90 μL of nanopure water (pH 5-6) and incubating at 37° C. for ≧72 h without agitation. Each well of a 96-well plate (well volume 0.4 mL; clear, flat bottom polypropylene) was coated for 3 h at 25° C. with 50 μL of 1.3 μM solution of Aβ peptides in phosphate-buffered saline (PBS, 10 mM NaH2PO4/Na2HPO4, 138 mM NaCl, 2.7 mM KCl, pH 7.4). After removal of the excess sample, 50 μL solutions of compounds in
PBS buffer (various concentrations were obtained by diluting a stock solution with PBS buffer) were incubated in the wells for 12 h. Compounds that did not dissolve in PBS buffer were dissolved in DMSO and diluted in PBS buffer to give a final solution of 5% DMSO in PBS buffer. The excess solutions were then removed and all wells were blocked for 30 min by adding 300 μL of a 1% (w/v) solution of bovine serum albumin in PBS buffer (BSA/PBS). On occasion, an additional blocking step was performed prior to incubation with solutions of small molecules. The blocking solution was discarded and the wells were washed once with 300 μL of PBS buffer. Wells were incubated for 1 h with 50 μL of a 1.1 nM solution (in 1% BSA/PBS, dilution 1:6000) of anti-Aβ IgG (clone 6E10, monoclonal, mouse), followed by removal of the solution. The wells were washed twice with 300 μL of PBS buffer and incubated for 60 min with 50 μL of the secondary IgG (anti- mouse IgG H+L, polyclonal, rabbit) conjugated with alkaline phosphatase (6.8 nM in 1% BSA/PBS, dilution 1:1000). The solution was discarded, and the wells were washed twice with 300 μL PBS buffer. Bound secondary IgGs were detected by the addition of 50 μL of a p-nitrophenyl phosphate solution (2.7 mM, in 100 mM diethanol amine/0.5 mM magnesium chloride, pH 9.8). Absorbance intensities were determined at 405 nm using a UV-vis spectroscopic plate reader (Sprectramax 190, Molecular Devices, Sunnyvale, Calif.). Each run was performed five times and averaged. Error bars represent standard deviations. Graphs were plotted and fitted with the sigmoid curve fitting.
Example 8. Fluorescence Studies with Monomeric Aβ. Aβ (Biopeptide, Inc.) was initially solubilized in hexafluoroisopropanol at 1 mM concentration, vortexed, sonicated and vortexed. The vial was covered in foil and was incubated for 21 hours at 25° C. on a shaker, with 3 times of vortexing throughout the incubation period. The solution was sonicated and vortexed again then diluted with cold nanopure water (2:1 H2O:HFIP), fractionated in desired amounts into small glass vials, and immediately frozen in a CO2/acetone bath. Each fraction was covered with parafilm that was punctured to allow solvent vapors to escape. The fractions were lyophilized for 2 days to obtain monomeric Aβ (91% monomer by 12% Tris-bis PAGE gel analysis). 1.8 μL (8.42 μM) of this monomeric Aβ(1-42) was added to 3 μL of 4 μM concentration of small molecules that was prepared by dissolving in PBS buffer pH 7.4 to attain a final concentration of 5 μM of Aβ(1-42) and 4 μM of the compound. The solution was transferred to 3 mL cuvettes and the fluorescence was measured at 25° C.
Example 9. Evaluation of Rigid Rotors for Cytotoxic Activity Against SHSY-5Y human neuroblastoma cells (MTT assay): SHSY-5Y human neuroblastoma cells, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation kit, Eagle's Minimum Essential Medium (EMEM), Ham's F12 nutrient mixture, and Fetal Bovine Serum (FBS) were all purchased from ATCC (Manassas, Va.). Briefly, SH-SY5Y cells (in 1:1 EMEM:Ham's F-12 with 10% FBS) were seeded on 96-well plates at a density of 5×104 cells/well. Plates were incubated overnight (in a humidified atmosphere of 95% air, 5% CO2, at 37° C.) to promote attachment of cells to the wells. Cells were then treated with various concentrations of compound 8a, 8b, 8c, 8d, 11, or 14 and incubated for 24 hours (humidified atmosphere of 95% air, 5% CO2, at 37° C.). MTT reagent (20 μL) was added to the medium and incubated for additional 4 hours. After incubation, 100 μL of detergent reagent was added and the plates were covered with aluminum foil and left at room temperature overnight. The amount of solubilized MTT formazan was measured by spectrophotometric absorbance at 570 nm (Spectramax 190, Molecular Devices, Sunnyvale, Calif.). MTT assay was not performed on compound 19 due to its poor solubility in aqueous media. All data are presented as the mean±S.D, N=3 for each concentration. The Student's t-test was employed for all analyses. A p-value of <0.05 was considered statistically significant compared to control cells. As shown in
Example 10. NMR Spectra. NMR spectra of compounds described herein are provided in Appendix 1.
The subject matter of this application was made with Government support under Grant No. 1E21RR025358 awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention(s) provided herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/59952 | 12/10/2010 | WO | 00 | 8/9/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61285470 | Dec 2009 | US |
