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WO2015095337A2 - Dérivés pyrazolo [1,5-a]pyrimidine carboxamide pour le traitement de troubles cognitifs - Google Patents

Dérivés pyrazolo [1,5-a]pyrimidine carboxamide pour le traitement de troubles cognitifs Download PDF

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Publication number
WO2015095337A2
WO2015095337A2 PCT/US2014/070876 US2014070876W WO2015095337A2 WO 2015095337 A2 WO2015095337 A2 WO 2015095337A2 US 2014070876 W US2014070876 W US 2014070876W WO 2015095337 A2 WO2015095337 A2 WO 2015095337A2
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hydrocarbyl
alkylamino
substituted
alkoxy
acyl
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WO2015095337A3 (fr
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Hyung Jin Ahn
J. Fraser Glickman
Sidney Strickland
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Rockefeller University
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Rockefeller University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings

Definitions

  • the invention relates to N-substituted pyrazolo[ 1 ,5-a]pyrimidine-5-carboxamides and 7-carboxamides that inhibit the the interaction between fibrinogen and amyloid- ⁇ . These compounds are useful to treat cognitive impairment and thrombotic disorders.
  • AD Alzheimer's disease
  • cerebral amyloid angiopathy which is the deposition of the beta-amyloid ( ⁇ ) peptide within cerebral blood vessels, results in degenerative vascular changes.
  • CAA cerebral amyloid angiopathy
  • Patients with both cerebral amyloid angiopathy and neurological pathology including neurofibrillary tangles and neuritic plaques have more severe cognitive impairment than patients with only Alzheimer's disease pathology or cerebral amyloid angiopathy alone, and reduction of cerebral amyloid angiopathy levels in Alzheimer's disease transgenic mice leads to memory improvement.
  • fibrinogen a primary protein component of blood clots, serves as a molecular link between the vascular and neurological abnormalities observed in Alzheimer's disease patients. Normally, fibrin(ogen) is found in the blood and is excluded from the brain via the blood brain barrier.
  • fibrin(ogen) is often localized to cerebral amyloid angiopathy in the brain's blood vessels and brain parenchyma in Alzheimer's disease patients and in mouse models of Alzheimer's disease; 2) fibrin deposition in the vasculature increases blood brain barrier dysfunction and neurovascular damage in AD mice; 3) ⁇ binds specifically to fibrinogen; and 4) fibrin clots formed in the presence of ⁇ have an abnormal structure, making them resistant to degradation by fibrinolytic enzymes.
  • any fibrin clots formed might be more persistent and may exacerbate neurovascular damage and cognitive impairment. Therefore, one might theorize that molecules that block the interaction of fibrinogen with amyloid- ⁇ , without affecting clotting in general, could restore altered thrombosis and fibrinolysis and protect against vascular damage in Alzheimer's disease patients, and could be used as therapeutic agents. In fact, this is confirmed in the experiments described below.
  • the invention relates to a method of inhibiting the interaction between fibrinogen and amyloid- ⁇ , comprising bringing amyloid- ⁇ into contact with a compound of formula I or II:
  • R 5 is (C4-C 10 ) hydrocarbyl, aryl or heteroaryl, said hydrocarbyl, aryl or heteroaryl optionally substituted with from one to three substituents chosen independently from the group consisting of halogen, acyl, hydroxy, (Ci-Ce)alkoxy, carboxy, (Ci-Ce)alkoxycarbonyl, cyano, acetoxy, nitro, amino, (Ci-C 6 ) alkylamino, and di(Ci-C 6 )alkylamino;
  • R 1 is (C 3 -C 10 ) heterocyclyl, (C 3 -C 10 ) hydrocarbyl or (C 3 -C 10 ) hydrocarbyl substituted with from one to three substituents chosen independently from the group consisting of halogen, acyl, hydroxy, (Ci-Ce)alkoxy, carboxy, (Ci-Ce)alkoxycarbonyl, cyano, acetoxy, nitro, amino, (Ci-C 6 ) alkylamino, and di(Ci-C 6 )alkylamino;
  • R is (C1-C10) hydrocarbyl or (C1-C10) hydrocarbyl substituted with from one to three substituents chosen independently from the group consisting of halogen, acyl, hydroxy, (Ci- Ce)alkoxy, carboxy, (Ci-Ce)alkoxycarbonyl, cyano, acetoxy, nitro, amino, (Ci-C 6 ) alkylamino, and di(Ci-C 6 )alkylamino, wherein a benzene ring, if present, is not directly attached to the amide nitrogen and wherein halogen, if present, is only attached to a benzene ring;
  • R is (C 1 -C 10 ) hydrocarbyl
  • R 4 is chosen from (Ci-Cg) acyl, (Ci-Cg) hydrocarbyl, and (Ci-Cg) hydrocarbyl substituted with from one to three substituents chosen independently from the group consisting of halogen, acyl, hydroxy, (Ci-Ce)alkoxy, carboxy, (Ci-Ce)alkoxycarbonyl, cyano, acetoxy, nitro, amino, (Ci-C 6 ) alkylamino, and di(Ci-C 6 )alkylamino, or, taken together, any two of R 2 and R 3 or R 3 and R 4 , together with the nitrogens to which they are attached, may form a 4-7-membered, saturated nitrogen heterocycle;
  • R 4 when R and R form a 6-membered, saturated nitrogen heterocycle and R 4 is chosen from (Ci-C 8 ) hydrocarbyl, and substituted (Ci-C 8 ) hydrocarbyl, then R 4 must be benzyl, substituted benzyl or phenyl substituted with (Ci-Ce)alkoxy; and
  • n 2, 3 or 4.
  • the invention in another aspect, relates to a method for treating cognitive disorders comprising administering to a mammal a therapeutically effective amount of a compound of formula I or II above.
  • the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of formula I or II.
  • the invention relates to compounds of formula I or II with the provisos that:
  • R and R when R and R form a 6-membered, saturated nitrogen heterocycle, then R is (Ci-C 8 ) acyl, benzyl, substituted benzyl or phenyl substituted with (Ci-Ce)alkoxy; and R 5 is not phenyl, methoxyphenyl or fluorophenyl .
  • the invention relates to inhibiting the interaction between fibrinogen and amyloid- ⁇ by bringing amyloid- ⁇ into contact with a compound of formula I or II:
  • R 5 is (C 4 -C 10 ) hydrocarbyl, for example t-butyl.
  • R 5 is aryl , wherein aryl is optionally substituted phenyl, particularly phenyl or phenyl substituted with halogen or (Ci-Ce)alkoxy.
  • R 4 is (Ci-C 8 ) acyl, particularly (Ci-C 6 ) acyl; in other embodiments R 4 is (Ci-C 8 ) hydrocarbyl.
  • R 4 is phenyl substituted with (Ci-C 6 ) alkoxy.
  • R 1 is is (C3-C 10 ) heterocyclyl, particularly (C 3 -C 6 ) heterocyclyl; in other embodiments R 1 is (C 3 -C 10 ) hydrocarbyl, particularly (C 3 -C 10 ) alkyl and (C 3 -C 10 ) cycloalkyl; in some other embodiments, R 1 is optionally substituted phenyl. In some embodiments, R 1 is (C 3 -C 6 ) oxygen
  • R is phenyl or t-butyl. In some embodiments, R
  • R 4 is as previously defined.
  • R 4 is a phenyl substituent (i.e. an aromatic (Ci-C 8 ) hydrocarbon or substituted aromatic (Ci-C 8 ) hydrocarbon in which the benzene ring is directly attached to the nitrogen as shown by the R 4 -N bond)
  • R 4 can only be alkoxy-substituted phenyl.
  • R 4 may be (Ci-C 8 ) hydrocarbyl or 2 R 3
  • R 4 is phenyl substituted with (Ci-Ce)alkoxy.
  • R 3 and R 4 are independently chosen from (Ci-C 8 ) hydrocarbyl, for example (Ci-C 4 )alkyl.
  • R 3 and R 4 together with the nitrogen to which they are attached, form a piperidine or pyrrolidine ring, r
  • m 1 or 2.
  • R 1 is chosen from (C 3 -C 10 ) alkyl, (C 3 -C 10 ) cycloalkyl and optionally substituted phenyl; R 2 and R 3 taken together form a piperazine; and R 4 is (C 1 -C 10 ) hydrocarbyl.
  • R 1 is chosen from (C 3 -C 10 )
  • the invention also relates to a compound of formula I and II, as identified above.
  • R 5 is (C 4 -C 10 ) hydrocarbyl, for example t-butyl.
  • R 5 is aryl , wherein aryl is optionally substituted phenyl, particularly phenyl or phenyl substituted with halogen or (Ci-Ce)alkoxy.
  • R 1 is is (C 3 -C 10 ) heterocyclyl, particularly (C 3 -C 6 ) heterocyclyl; in other embodiments R 1 is (C 3 -C 10 ) hydrocarbyl, particularly (C 3 -C 10 ) alkyl and (C 3 -C 10 ) cycloalkyl; in some other embodiments, R 1 is optionally substituted phenyl.
  • R 1 is (C 3 -C6) oxygen
  • R is phenyl or t-butyl.
  • R is (C 1 -C 10 ) hydrocarbyl, for example benzyl.
  • R 4 is (Ci-C 8 ) acyl; in 4 2 3
  • R is (Ci-C 8 ) hydrocarbyl.
  • R and R taken together form a 5 or 6-membered heterocycle, for example a piperazine.
  • Such compounds may be represented by the formula IV, as identified above.
  • R 4 is as previously defined.
  • R 4 is (Ci-C 8 ) acyl, benzyl, substituted benzyl or phenyl substituted with (Ci-C 6 ) alkoxy; and R 5 is not phenyl, methoxyphenyl or fluorophenyl.
  • R 4 may be (Ci-C 8 ) hydrocarbyl or (Ci-C 6 ) acyl.
  • R 3 and R 4 are independently chosen from (Ci-C 8 ) hydrocarbyl, for example (Ci-C 4 )alkyl.
  • R 3 and R 4 together with the nitrogens to which they are attached, form a piperidine or pyrrolidine ring
  • R 1 is chosen from (C 3 -C 6 ) heterocyclyl, (C 3 -C 10 )
  • R 1 is chosen from (C 3 -C6) heterocyclyl, (C 3 -C 10 ) alkyl, (C 3 -C 10 ) cycloalkyl and optionally substituted phenyl; R 2 is benzyl; and R 2 and R 3 are independently chosen from (Ci-C 4 )alkyl.
  • hydrocarbon when used as a substituent
  • hydrocarbyl includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthylethyl.
  • Hydrocarbyl refers to any substituent or scaffold comprised of hydrogen and carbon as the only elemental constituents.
  • Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched.
  • aliphatic hydrocarbons examples include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, cyclohexyl, norbornyl, etc.
  • Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
  • alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof.
  • Alkyl refers to alkyl groups from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s- butyl, t-butyl and the like.
  • Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl and the like.
  • carbocycle is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state.
  • C 3 -C 10 carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene;
  • C 8 -C 12 carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene.
  • Carbocycle if not otherwise limited, refers to monocycles, bicycles and polycycles.
  • Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • a heterocycle may be non- aromatic (heteroaliphatic) or aromatic (heteroaryl).
  • heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like.
  • heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl,
  • thiazolidinyl isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and
  • Alkoxy or alkoxyl refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms of a straight or branched configuration attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower-alkoxy refers to groups containing one to four carbons. For the purpose of this application, alkoxy and lower alkoxy include
  • halogen means fluorine, chlorine, bromine or iodine atoms. In one embodiment, halogen may be a fluorine or chlorine atom.
  • substituted refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical.
  • Oxo is also included among the substituents referred to in "optionally substituted”; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl).
  • 1, 2, or 3 hydrogen atoms are replaced with a specified radical.
  • more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine.
  • substituents are halogen, haloalkyl, alkyl, acyl, hydroxyalkyl, hydroxy, alkoxy, haloalkoxy, aminocarbonyl oxaalkyl, carboxy, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino arylsulfonyl, arylsulfonylamino, and benzyloxy.
  • halogen (Ci-C 4 )alkyl, halo(Ci-C 4 )alkyl, (Ci-C 4 )alkoxy, halo(Ci-C 4 )alkoxy, and aminocarbonyl.
  • acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. Examples include acetyl, benzoyl, propionyl, isobutyryl and the like. Lower- acyl refers to groups containing one to four carbons.
  • the double bonded oxygen, when referred to as a substituent itself is called "oxo".
  • “compound” refers to the compound or a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salt refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • the compounds of the present invention are usually basic, and salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediammetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succin
  • structures depicted herein are also meant to include all stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans isomeric) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and cis-trans isomeric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • compound 10 was synthesized as follows:
  • reaction mixture was stirred for about 15 minutes, and then was allowed to stand in a refrigerator overnight.
  • the reaction mixture was poured into a mixture of cone. HC1 solution (300 mL) and ice (c.a. 1 kg).
  • the resulting precipitate was filtered off and washed with plenty of water, finally dried in a vacuum desiccator over P2O5/KOH. Yield: 165.5 g (3) (96%) as a light yellow .powder.
  • the reaction mixture was stirred at room temperature overnight, by which time the reaction was complete.
  • the reaction mixture was evaporated to dryness, resulting in 27 g of crude regioisomer mixture, which was separated by column chromatography on silica gel with gradient elution from hexanes to 5% THF in hexanes.
  • the first regioisomer (upper spot on TLC) was obtained as a yellow powder (3 g). It was recrystallized from ethanol-water mixture and resulted 2.4 g (9 %) of ethyl 2-(tert-butyl)-5-(4-fluorophenyl)pyrazolo[l,5- aJpyrimidine-7-carboxylate as yellow crystals.
  • the second regioisomer (lower spot on TLC) was obtained as a yellow thick oil (12 g). It was crystallized with isopropyl ether, and 9 g (35 %) of ethyl 2-(tert-butyl)-7-(4-fluorophenyl)pyrazolo[l ,5-a] pyrimidine-5- carboxylate was obtained as a yellow crystalline powder.
  • N-benzyl-2-(pyrrolidin-l-yl)ethan-l-amine used in the synthesis above was obtained by reductive amination of benzaldehyde:
  • benzaldehyde In a 500 mL round bottom flask equipped with a reflux condenser and a CaCl 2 drying tube l-(2-aminoethylpyrrolidine (5.02g, 44 mmol) and benzaldehyde ( 4.7 mL, 46 mmol, 1.05 equiv.) were dissolved in ethanol (100 mL). The reaction mixture was stirred and refluxed for two hours and then allowed to stand at RT overnight.
  • the resulting solution was diluted with methanol (50 mL), cooled down to about 10 °C and sodium borohydride (1.75 g, 46 mmol, 1.05 equiv.) was added in small portions with stirring, while the flask was kept in an ice -water cooling bath. (Intense foaming!) The reaction mixture was allowed to warm up to RT and stirred further for an hour. The mixture was evaporated in a rotary evaporator to almost dryness. The residue was diluted with water (200 mL), acidified by addition of 10% aq. HC1 solution and washed with ether (3x30 mL). The solution was neutralized by addition of 10% aq.
  • Test compound (20 ⁇ ) or vehicle (0.4% DMSO) was incubated for 10 min with purified human fibrinogen and plasminogen in the presence or absence of ⁇ 42. Fibrin clot formation and degradation were analyzed by measuring turbidity immediately after adding thrombin and tissue plasminogen activator (tPA) to the mixture. In the presence of ⁇ 42, the maximum turbidity of the fibrin clot was decreased, since ⁇ altered fibrin clot structure, and the dissolution of the fibrin clot was delayed. An example of a compound of genus I was tested. The compound of example 11 restored the ⁇ -induced decrease in turbidity during fibrin clot formation and significantly reduced the delay in fibrin degradation in the presence of ⁇ .
  • tPA tissue plasminogen activator
  • the compound of example 11 did not have any effect on fibrin clot formation and degradation in the absence of ⁇ . This result confirms that a compound of the invention effectively restores ⁇ -induced altered fibrin clot structure and delayed degradation without affecting normal clot formation and fibrinolysis.
  • SPR surface plasmon resonance
  • Tg6799 mice were treated for three months with test compound in a protocol to assess contextual fear conditioning. Treatment with example 11 had no effect on baseline freezing behavior in WT and Tg6799 mice. Looking at contextual memory of Tg6799 and WT mice 24 hours after training, it is seen that vehicle-treated Tg6799 mice show a severe memory deficit compared to vehicle-treated WT mice. Tg6799 mice treated with compound 11 exhibited significantly improved memory compared to their vehicle- treated AD counterparts, while long-term treatment with example 11 in WT mice did not affect basal freezing behavior or contextual memory.
  • Another method of cognitive assessment is the Barnes maze, a behavioral test that assesses spatial learning and memory in rodents.
  • Vehicle-treated Tg6799 mice spent a significantly longer time to find the target hole compared to vehicle-treated WT and Tg6799 mice treated with example 11.
  • vehicle- treated Tg6799 mice also had significantly longer latency to reach the closed target hole and significantly fewer visits to the target hole compared to vehicle-treated WT and Tg6799 mice treated with compound 11.
  • Tg6799 mice moved significantly less than WT mice. However, there was no significant difference in distance traveled between untreated Tg6799 mice and mice treated with the compound of Example 11. This result suggests that the better performance of Example 11 -treated Tg6799 mice compared to untreated Tg6799 mice is likely due to memory improvement and not effects on locomotion.
  • example 11 was administered to 4-month-old TgCR D8 mice for three months (analyzed at 7 months-of-age). During training, treatment with test compound did not lead to
  • Tg6799 mice (Jackson Laboratory) are double transgenic mice for APP/Presenilin 1 that co-express five early onset familial AD mutations.
  • TgCRND8 mice (provided by A. Chishti and D. Westaway, University of Toronto, Canada) have three APP mutations (K670N, M671L, and V717F) driven by the human prion protein promoter.
  • Test compound was prepared in 2.5% EtOH, 4.5% Cremophor RH40 (Sigma-Aldrich), and 14% D5W (5% dextrose in water) in saline.
  • test compound or vehicle was administered to Tg6799 mice and a 25 mg/kg dose or vehicle to TgCRND8 mice subcutaneously every other day for three months.
  • Non-transgenic (wild-type; WT) littermates were used in all experiments.
  • the assigned genotype of all the mice used in the experiments was double- checked by taking tail tissue the day of sacrifice. Only male mice were used in experiments, and all animals were maintained in The Rockefeller University's Comparative Biosciences Center and treated in accordance with I ACUC -approved protocols.
  • Compounds were commercially available and were purchased from several vendors.
  • the primary assay utilized fluorescence polarization (FP) to measure the changes in the anisotropy induced by binding of 5-carboxytetramethylrhodamine (TAMRA)-labeled ⁇ 42 (Anaspec) to fibrinogen.
  • TAMRA 5-carboxytetramethylrhodamine
  • ⁇ ⁇ - ⁇ 42 (2 nM) was mixed with 300 nM fibrinogen (Calbiochem) and 20 ⁇ compounds (dissolved in 1% DMSO (final)) in 50 mM PBS, pH 7.4, 0.001% Tween 20, and 0.001% BSA as 50 ⁇ . final volume in black 384-well plates (Greiner) at room temperature. After binding reached equilibrium, polarization
  • Compounds that showed over 75% inhibition of the ⁇ -fibrinogen interaction in the FP assay were screened by AlphaLISA as a secondary assay.
  • Compounds (12.5 ⁇ ) were plated in white 384-well plates (Greiner) and were incubated with 10 nM biotinylated ⁇ 42 (Anaspec) and 1 nM fibrinogen for 30 min at RT in final volume of 10 ⁇ ⁇ assay buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, 0.1% BSA).
  • the mixture was incubated with anti-fibrinogen antibody (Dako), 20 ⁇ g/mL streptavidin-conjugated donor, and protein A-conjugated acceptor beads (Perkin-Elmer) for 90 min at RT. Samples were read by a PerkinElmer En Vision plate reader.
  • Anti-fibrinogen antibody Dako
  • 20 ⁇ g/mL streptavidin-conjugated donor 20 ⁇ g/mL
  • protein A-conjugated acceptor beads Perkin-Elmer
  • the AlphaScreen TruHits kit (PerkinElmer) was used to detect those compounds that react with singlet oxygen and thus unspecifically quench the assay.
  • the AlphaScreen TruHits kit also allows for the identification of color quenchers, light scatterers (insoluble compounds), and biotin mimetics interfering with the AlphaLISA signal. If inhibition by quenching was more than 30%> at 10 ⁇ compound, the compound was considered inactive for the purpose of the invention.
  • Compounds were then tested in a dose-response experiment at various concentrations (0.01 - 20 ⁇ ) using FP and AlphaLISA.
  • test compound dissolved in 0.4%> DMSO (final)
  • DMSO control was incubated with fibrinogen (1.5 ⁇ ) in the presence or absence of ⁇ 42 (3 ⁇ ) for 10 min and then mixed with plasminogen (0.25 ⁇ ) in 20 mM HEPES buffer (pH 7.4) with 137 mM NaCl.
  • Fibrin clot formation and degradation was analyzed measuring turbidity right after adding thrombin (0.5 U/mL), tPA (0.15 nM), and CaCl 2 (5 mM) in a final volume of 150 ⁇ .
  • Assays were performed at RT in High Binding 96-well plates (Fisher Scientific) in triplicate and were measured at 450 nm using a Molecular Devices Spectramax Plus384 reader.
  • DMSO dilutions were used as a buffer blank, and a solvent correction assay was performed to correct the difference of DMSO response between empty reference surface and protein-immobilized surface.
  • Sulindac sulfide and sulindac were used as positive control and negative controls, respectively.
  • In vivo toxicity study Maximum tolerated dose studies were carried out to determine toxicity and to identify the optimal dose for in vivo assays.
  • Single injection toxicity was performed at Absorption Systems LP (Exton, PA), and four different doses (200, 100, 50, and 20 mg/kg mouse) of test compound, along with saline and vehicle, were injected into male and female CD-I mice intravenously. Mortality and overt clinical signs of toxicity were monitored for two days. All animals dosed with 200 mg/kg were found dead after single intravenous injection of the compound of example 11, and no clinical signs of toxicity were observed after single dose of 20, 50, or 100 mg/kg for two days after injection. Therefore, the maximum tolerated dose of example 11 after single intravenous dose in mice was established as 100 mg/kg.
  • mice and WT littermates were treated with two doses (100 mg/kg and 50 mg/kg) of example 11 every other day for 3 months, and overt clinical signs of toxicity were monitored. After three months, mice were sent to the Laboratory of Comparative Pathology at Memorial Sloan-Kettering Cancer Center for complete necropsy and hematology reports to determine the effects after long-term treatment. It was found that 100 mg/kg for long-term treatment was toxic to the AD mice, but 50 mg/kg showed no clinical signs of toxicity except local chronic
  • a 2.5-mm circular craniotomy was prepared using 5-10 circular brush strokes with a fine dental drill bit, and a 4 mm plastic ring surrounding the window was attached with dental acrylic and cyanoacrylate adhesive. Sterile saline was applied periodically to protect the brain surface and prevent drying.
  • 100 ⁇ of 5 mg/ml 2 MDa FITC-conjugated dextran (Sigma) dissolved in PBS was administered by retro-orbital injection.
  • the body temperature of mice was kept at 37.5°C using a TC-1000 Mouse complete temperature control system (CWE Inc).
  • Behavioral Analysis All behavioral experiments were performed and analyzed with a researcher blinded to genotype and treatment. Forty mg/kg of test compound or vehicle was administered to 4-month-old Tg6799 mice and WT littermates and 25 mg/kg or vehicle to 4-month-old TgCRND8 mice and WT littermates subcutaneously every other day for three months (analyzed at 7 months-of-age). Mice were handled and allowed to acclimate to the testing room for 10 min per day for at least 5 days.
  • mice were placed in the center of the maze in a black starting box for 30 s. After 30 s, the box was removed, and mice were allowed to freely explore and find the target hole within 2 min. Latency to poke the target hole was recorded. If mice did not enter into the escape chamber within 2 min, they were gently guided into the escape chamber and placed in the chamber for 30 sec.
  • a probe trial was conducted 24 h and 3 days after the last training. The target hole was closed like the other 19 holes, and the escape chamber was removed. Holes were kept in the same position as during the training. Mice were placed in the center of the maze in a black starting box for 30 s. After 30 s, the box was removed, and mice were allowed to freely explore for 90 s.
  • the present invention provides a pharmaceutical composition comprising a compound together with one or more pharmaceutical carriers and optionally one or more other therapeutic ingredients.
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions for administration to patients include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and
  • intraarticular rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
  • parenteral administration will be preferred.
  • the most suitable route may depend upon the condition and disorder of the recipient.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

La présente invention concerne des composés de formule I et II: formule (I) ou formule (II) qui inhibent l'interaction entre le fibrinogène et l'amyloïde-β et qui sont utiles pour traiter les troubles cognitifs tels que la déficience de la mémoire et la maladie d'Alzheimer.
PCT/US2014/070876 2013-12-18 2014-12-17 Dérivés pyrazolo [1,5-a]pyrimidine carboxamide pour le traitement de troubles cognitifs Ceased WO2015095337A2 (fr)

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