WO2015048306A1 - Nouveaux agents ciblant cyp51 - Google Patents

Nouveaux agents ciblant cyp51 Download PDF

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WO2015048306A1
WO2015048306A1 PCT/US2014/057483 US2014057483W WO2015048306A1 WO 2015048306 A1 WO2015048306 A1 WO 2015048306A1 US 2014057483 W US2014057483 W US 2014057483W WO 2015048306 A1 WO2015048306 A1 WO 2015048306A1
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compound
nmr
mhz
dmso
alkyl
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PCT/US2014/057483
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William R. Roush
Jun Yong Choi
Larissa PODUST
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The Scripps Research Institute
The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin

Definitions

  • Chagas disease or American trypanosomiasis is a chronic tropical infection caused by the protozoan parasite Trypanosoma cruzi. The infection can be lethal if untreated. Chagas disease is the leading cause of heart failure in Latin America. 1 Although first described a century ago,- it is still a major public health challenge in South America. Furthermore, many cases have been reported in North America, Europe, and Asia due to human population movements, migration of the triatomine insect vectors, HIV-coinfections, and contaminated blood transfusion. 15
  • nifurtimox and benznidazole are the only drugs approved for treatment of Chagas disease.- Although these drugs, which date from the late 1960s, show considerable efficacy in the acute stage of Chagas disease, their efficacy is debated in the chronic stage, which involves chronic Chagas cardiomyopathy, leading to congestive heart failure, thromboembolic phenomena, severe arrhythmias, and sudden unexpected death. - Moreover, these old drugs are associated with frequent side effects such as dermatitis, gastrointestinal, and neurologic toxicities, and even a rare case of bone marrow suppression.- ⁇ Therefore, the need exists to develop new therapeutics bearing better safety profiles and improved efficacy to treat T. cruzi infections and prevent cardiovascular Chagas disease.
  • Sterol biosynthesis is a recognized target for the development of new therapeutic agents to treat T. cruzi infections - Sterol 14-demethylase (CYP51) has been successfully targeted for combating pathogenic fungal infections with azole drugs such as fluconazole, ketoconazole, and posaconazole, among others.
  • CYP51 catalyzes the oxidative removal of the 14-methyl group of lanosterol and produces A 14 15 -unsaturated intermediates in ergosterol biosynthesis.
  • the invention provides a compound of formula (I) Het
  • Het is a 5- or 6-membered heteroaryl comprising a nitrogen atom
  • R is independently at each occurrence H or (Ci-Ce)alkyl
  • R 1 is an aryl(Co-C6)alkyl or a heteroaryl(Co-C6)alkyl wherein the aryl or the heteroaryl is substituted with 0-3 J
  • R 2 is a group of formula wherein Ar 1 and Ar 2 are independently selected aryl or heteroaryl, wherein each aryl or heteroaryl is substituted with 0-3 J
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient.
  • the excipient can comprise a cyclodextrin or solutol.
  • the invention further provides, in various embodiments, a method of inhibiting a sterol C14-demethylase comprising contacting the demethylase with an effective amount or concentration of a compound of the invention, such as a compound of formula (I), i.e., of any of the various specific structural formulas disclosed and claimed herein.
  • a compound of the invention such as a compound of formula (I), i.e., of any of the various specific structural formulas disclosed and claimed herein.
  • the sterol C14 demethylase can be CYP51.
  • the invention further provides, in various embodiments, a method of treatment of Chagas disease, comprising administering to a patient afflicted therewith an effective dose of a compound of the invention.
  • the compound can be orally administered to the patient as a cyclodextrin inclusion complex.
  • the invention further provides, in various embodiments, a method of treatment of a fungal disease, comprising administering to a patient afflicted therewith an effective dose of a compound of the invention.
  • the invention provides CYP51 inhibitors of high potency and selectivity, having demonstrated bioactivity versus Chagas disease in a mouse model.
  • Various embodiments of the present invention also provide methods of inhibition of CYP51 in vitro and in vivo.
  • various embodiments provide a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition of the invention can comprise a compound of the invention and a cyclodextrin; this composition can be highly suitable for oral administration for treatment of Chagas disease, the cyclodextrin complex of the inventive compound providing for an unexpectedly high degree of oral bioavailability.
  • Figure 1 Inhibitors of 7cCYP51.
  • A Azole type CYP51 inhibitors.
  • B Pyridinyl type CYP51 inhibitors.
  • FIG. 1 X-ray co-crystal structure of the 73 ⁇ 4CYP51-14t complex.
  • A Electron density map (blue mesh) contoured at 1.0 ⁇ delineates the positions of 14t (CYP-I-181)(yellow sticks) in the active site. In purple are amino acid residues providing hydrophobic contacts within 5 A to the indol moiety of 14t plus F105. Heme is displayed as grey sticks.
  • B View of the 14t-bound CYP51 clipped by a plane through the binding site compares the binding modes of 14t (yellow) and posaconazole (cyan).
  • the structure of 73 ⁇ 4CYP51 complexed with posaconazole (PDB code: 2X2N) is superimposed on that of with 14t.
  • the active site surface is colored by hydrophobicity from orange (lipophilic) to blue (hydrophylic).
  • C View of bound inhibitors from the entrance to the active site.
  • the enzyme is represented by a gray surface.
  • the hydrophobic units of posaconazole and 14t occupy different hydrophobic tunnels in corresponding co-crystal structures.
  • the images here and otherwise were generated using PYMOU2 0 r CHIMERA ⁇ .
  • FIG. 3 Comparison of the 14t binding mode with NEE and VNF. View of the 14t- bound 73 ⁇ 4CYP51 clipped by a plane through the binding site.
  • This extension accommodates a substituted benzyl ring in NEE (purple) (PDB ID 4H60) (B) or biaryl moiety of VNF (pink) (PDB ID 3KSW) in the corresponding 7cCYP51 co-crystal structures. (C). Both structures were superimposed on the 14t-bound 73 ⁇ 4CYP51.
  • FIG. 4 Predicted binding modes of inhibitors in 7icCYP51. Binding modes of 271 (A), 27s (B), 27k (C), and 27r (D) resulting from molecular docking using Glide XP. Inhibitors are in stick mode colored by atom type: carbon in yellow, oxygen in red, nitrogen in blue, fluorine in cyan, hydrogen on the tertiary amino group of 27s is in gray. The protein is shown as a semi-transparent gray surface; heme is displayed as orange spheres.
  • FIG. 1 Comparison of the binding modes of 1 (blue) and posaconazole (red) in the active site of CYP51. Protein is shown as solvent accessible surface with M460 side chain present (A) or omitted (B) from the coordinates.
  • FIG. 1 Comparision of the binding poses of (S)-l (blue) and (R)-2 (red). Heme is shown in semi-transparent spheres.
  • B Dose-response curves for 1 (blue) and 2 (red) show >500-fold difference in hit potency.
  • C (R)-3 (red) protruding through the hydrophobic tunnel to the solvent accessible area (PDB ID 4BMM). Protein is represented by grey semi- transparent surface.
  • FIG. 7 Rationale for (R)-2 optimization.
  • Compounds are in stick mode, protein is represented by grey opaque surface.
  • Figure 10 Binding modes of S- and R-enantiomers.
  • A. An overlay between all the available 7cCYP51 co-structures with bound S- (purple) or R- (yellow) enantiomers of the N- indolyl-oxopyridinyl-4-aminopropanyl scaffold is shown. Inhibitors are in stick
  • FIG. 11 Compound 11-259 in the active site of 7icCYP51.
  • C. Slice through the binding site shows nearly perfect fit of 11-259 (yellow spheres) and the protein surface colored by hydrophobicity, hydrophobic areas are in orange and hydrophilic areas are in blue. Heme is shown in dark red spheres.
  • FIG. 12 Compound 11-71 in the active site of 7icCYP51.
  • FIG. 13 Compounds 11-250, 11-251, 11-255, 11-257, 11-258, and 11-259 are effective versus T. cruzi in mice. Luminescence assay shows the reduction in trypanosome populations in treated mice versus controls.
  • Figure 14 A 4-day mouse model oral delivery of selected compounds of the invention in 20% solutol.
  • Compounds of the invention 11-250, 11-251, 11-258, and 11-259 in 20% solutol were administered orally to mice.
  • High light emission intensity shows high trypanosome populations in the mice.
  • Figure 15 A comparison of orally administered compound 11-250 in hydroxypropyl- ⁇ - cyclodextrin (HPpCD) versus in solutol.
  • Luminescence assay shows comparative effects of orally delivered compound of the invention 11-250.
  • High light emission intensity shows high trypanosome populations in the mice.
  • mammals include, for example, humans; non-human primates, e.g. apes and monkeys; and non-primates, e.g. dogs, cats, cattle, horses, sheep, and goats.
  • Non-mammals include, for example, fish and birds.
  • CYP51 plays a role in the biochemical mechanisms involved in the disease or malcondition or symptom(s) thereof such that a therapeutically beneficial effect can be achieved by acting on CYP51.
  • Acting on” CYP51 can include binding to CYP51 and/or inhibiting the bioactivity of CYP51 and/or allosterically regulating the bioactivity of CYP51 in vivo.
  • an effective amount when used to describe therapy to an individual suffering from a disorder, refers to the amount of a compound of the invention that is effective to inhibit or otherwise act on CYP51 in the individual's tissues wherein CYP51 involved in the disorder is active, wherein such inhibition or other action occurs to an extent sufficient to produce a beneficial therapeutic effect.
  • substantially as the term is used herein means completely or almost completely; for example, a composition that is "substantially free” of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is
  • substantially pure is there are only minimal traces of impurities present.
  • Treating” or “treatment” within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder.
  • an "effective amount” or a “therapeutically effective amount” of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects.
  • phrases such as "under conditions suitable to provide” or “under conditions sufficient to yield” or the like, in the context of methods of synthesis, as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.
  • an "analog” of a chemical structure refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure.
  • a related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative.”
  • stable compound and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.
  • substituted refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non-hydrogen atom such as, but not limited to, a halogen (i.e., F, CI, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines
  • Non-limiting examples of substituents J that can be bonded to a substituted carbon (or other) atom include F, CI, Br, I, OR', OC(0)N(R') 2 , CN, NO, N0 2 , ONO2, azido, CF3, OCF3, R', O (oxo), S (thiono), methylenedioxy, ethylenedioxy, N(R , SR, SOR, SO2R, S02N(R , SO3R, C(0)R, C(0)C(0)R, C(0)CH 2 C(0)R, C(S)R, C(0)OR, OC(0)R, C(0)N(R) 2 ,
  • J is any of halo, (Cl-C6)alkyl, (Cl-C6)alkoxy, (Cl- C6)haloalkyl, hydroxy(Cl-C6)alkyl, alkoxy(Cl-C6)alkyl, (Cl-C6)alkanoyl, (Cl-
  • C6alkanoyloxy cyano, nitro, azido, R2N, RzNQO
  • R2NC(0)0, R2NC(0)NR (Cl- C6)alkenyl, (Cl-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl, (C6- C10)aryl(Cl-C6)alkyl, (C6-C10)aryl(Cl-C6)alkoxy, (C6-C10)aryloxy(Cl-C6)alkyl, (C6- C10)aryloxy(Cl-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to 9- membered)heterocyclyl(Cl-C6)alkyl, (3- to 9-membered)heterocyclyl(Cl-C6)alkoxy, (5- to 10-membered)heteroaryl, (5- to 10-membered
  • each individual integral number representing the number of carbon atoms is intended.
  • recitation of a (Ci-C4)alkyl group indicates that the alkyl group can be any of methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, or tert-butyl. It is understood that a specification of a number of carbon atoms must be an integer.
  • the heterocyclyl ring can include any of 3, 4, 5, 6, 7, 8, or 9 atoms, which can be atoms of any element capable of forming two or more bonds, e.g., carbon, nitrogen, oxygen, sulfur, and the like.
  • the number of atoms in a ring is understood to necessarily be an integer.
  • Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl.
  • Representative substituted alkyl groups can be substituted one or more times with any of the groups listed above, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci ealkyl, Ci-4alkyl, and Ci salkyl, respectively.
  • Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2 -methyl- 1 -butyl, 3-methyl-2-butyl, 2 -methyl- 1 -pentyl, 3 -methyl- 1-pentyl, 4- methyl- 1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- 1 - butyl, 3, 3- dimethyl- 1 -butyl, 2-ethyl- l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.
  • Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members.
  • a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure.
  • a heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.
  • a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquino
  • Heteroarylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above.
  • linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like.
  • branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert- butoxy, isopentyloxy, isohexyloxy, and the like.
  • Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci ealkoxy, and C2-6alkoxy, respectively.
  • Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.
  • An alkoxy group can include one to about 12-20 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms.
  • an allyloxy group is an alkoxy group within the meaning herein.
  • a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structures are substituted therewith.
  • halo or “halogen” or “halide” by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.
  • a "haloalkyl” group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro.
  • haloalkyl examples include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.
  • Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups.
  • aryl groups contain about 6 to about 14 carbons in the ring portions of the groups.
  • Aryl groups can be unsubstituted or substituted, as defined above.
  • Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6- substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.
  • Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.
  • Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.
  • Aralkenyl group are alkenyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.
  • the compounds of the invention may contain one or more chiral centers and, therefore, exist as stereoisomers.
  • stereoisomers when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols “(+),” "(-),” "R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.
  • the present invention encompasses various stereoisomers of these compounds and mixtures thereof.
  • Individual enantiomers and diastereomers of contemplated compounds can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents.
  • Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent.
  • Stereoselective syntheses a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art.
  • Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley- VCH: Weinheim, 2009.
  • the isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers.”
  • Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light.
  • Single enantiomers are designated according to the Cahn-Ingold-Prelog system.
  • the priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated as having an (R) absolute
  • the molecule is designated as having an (S) absolute configuration.
  • the Cahn-Ingold-Prelog ranking is A > B > C > D.
  • the lowest ranking atom, D is oriented away from the viewer.
  • a carbon atom bearing the A-D atoms as shown above is known as a "chiral" carbon atom, and the position of such a carbon atom in a molecule is termed a “chiral center.”
  • Compounds of the invention may contain more than one chiral center, and the configuration at each chiral center is described in the same fashion.
  • the present invention is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof.
  • Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.
  • Isolated optical isomer or “isolated enantiomer” means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula.
  • the isolated isomer is at least about 80%, more preferably at least 90% enantiomerically pure, even more preferably at least 98% enantiomerically pure, most preferably at least about 99% enantiomerically pure, by weight.
  • enantiomeric purity is meant the percent of the predominant enantiomer in an enantiomeric mixture of optical isomers of a compound. A pure single enantiomer has an enantiomeric purity of 100%.
  • Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of
  • DAICEL ® CHIRALPAK ® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.
  • Another well-known method of obtaining separate and substantially pure optical isomers is classic resolution, whereby a chiral racemic compound containing an ionized functional group, such as a protonated amine or carboxylate group, forms diastereomeric salts with an oppositely ionized chiral nonracemic additive.
  • the resultant diastereomeric salt forms can then be separated by standard physical means, such as differential solubility, and then the chiral nonracemic additive may be either removed or exchanged with an alternate counter ion by standard chemical means, or alternatively the diastereomeric salt form may retained as a salt to be used as a therapeutic agent or as a precursor to a therapeutic agent.
  • the known compound (LP10) is a mixture of two racemic diastereomers: S- and R- isomers at the tryptophan unit and cis/trans-isomers within the methylcyclohexane ring.
  • AftCYP51 which have S-enantiomers in the bound structures. 13a
  • the S-isomer 1 had two-fold higher potency (EC50) against T. cruzi in infected cells than the R-isomer. Both isomers had similar microsome stability and both were potent inhibitors of human CYP enzymes 2C9, 2D6 and 3A4 (>90% inhibition at 10 uM). Accordingly, we first pursued S- enantiomers of LP 10 analogs in the development of non-azole CYP51 leads.
  • dValues in parentheses are % inhibition of the indicated human CYPs at 1 ⁇ .
  • analogs with secondary or tertiary amine units such as 3c, 3d, 3e, and 3f, were also found to have substantially reduced biochemical and cell-based potency, presumably because they are unable to bind in the 7cCYP51 active site which accommodates the highly lipophilic natural substrate, lanosterol.
  • the microsome stability of 3c, 3d, 3e, and 3f was significantly increased (30- 120 min half-life), and inhibition of human CYP enzymes by these compounds was greatly decreased relative to LP 10. Therefore, balancing the charge or polarity distribution of inhibitor analogs was viewed as an important factor to address in the development of more active analogs.
  • Enantiomeric pure 1 was synthesized by the sequence summarized in Scheme 1. Briefly, the S-enantiomer of N-Boc tryptophan (L-tryptophan) was coupled with 4- aminopyridine to produce 5. Treatment of 5 with 4N HCl in dioxane followed by treatment of the deprotected amine with cyclohexanecarbonyl chloride produced enantiomerically pure 1. A similar sequence, starting from R-N-Boc tryptophan, was used to synthesize 2 (not shown). Analogs 3a and 3b were synthesized by replacing 4-aminopyridine with 4-amino-2- methoxypyridine and 4-amino-3,5-dimethylisoxazole, respectively.
  • CYP inhibition for this compound was performed at 10 ⁇ Fluoro, chloro, bromo and other substituents were added to the benzamide unit in attempts to block the potential soft site(s) on the phenyl ring of 14a.
  • 14b - 14k that were synthesized in this effort, several had increased activity against T. cruzi in cell culture (14b, 14h, 14j, 14k) and while retaining microsome stability.
  • Acyl groups with larger naphthyl and biaryl units were used in attempts to further improve the microsome stability (141 - 14t). Gratifyingly, many of these inhibitors had increased microsome stability, while retaining inhibitor potency.
  • Inhibitor 14t bound in the active site of CYP51 has several interesting features.
  • the pyridine nitrogen of the 4-acylaminopyridine unit is coordinated to the heme iron (Fig 2), as expected from the series of the co-structures for the LP 10 analogs bound to M YP51--.
  • the indole ring of 14t (PDB small molecule code 181) occupies the hydrophobic area enclosed largely by the heme macrocycle and the p-electron rich residues Y103, Ml 06, Fl 10, Yl 16, F290 plus A287 (Fig. 2A); this is the same area where the 2,4-difluorophenyl unit of posaconazole binds (Fig. 2B).
  • Variable F105 is >5 A away from the indole ring and within 4 A of the carbonyl group adjacent to the biaryl moiety of 14t, suggesting additional hydrophobic contacts with the inhibitor in 73 ⁇ 4CYP51 which are missing from T. cruzi ortholog.
  • the biaryl ring of 14t projects towards the solvent exposed area, as does the tail part of the posaconazole,TM but via a different hydrophobic tunnel between the FG-loop (residues 205-210) and the hairpin of the two-stranded b-sheet at the protein C-terminus (residues 459-461) (Fig. 2C).
  • a hydrophobic cavity accommodating the indole ring of 14t extends further toward Fl 10 (Fig. 3 A) to provide space sufficient to bind a substituted benzyl ring of NEE (PDB ID 4H60) (Fig. 3B ) or a rigid biaryl moiety of VNF (PDB ID 3KSW) (Fig. 3C).
  • the cavity is enclosed by Yl 16, providing stacking interactions with the 4-chloro-3,5- dimethylbenzyl unit of NEE, as well as by the aliphatic hydrophobic residues Al 15, M123, L127, L130, A287, and hydrophilic neutral Q126.
  • the bulky substituents at the 5-position of the indole ring in analog 3k bind in this cavity.
  • biaryl carboxylic acid intermediates were prepared by palladium-mediated coupling reactions of commercially available 4-bromo-2-fluorobenzoic acid 15 with various aryl boronic acids (Scheme 3, below). This reaction was performed under microwave irradiation (100 °C for 1 h) and provided products 16 in >90% yields. Intermediate 17 was obtained by the Heck reaction of 15 and 1 -chloro-4-vinylbenzene in the presence of
  • Aryl sulfonylamide 25b and N-benzylpiperazine 26b were obtained by the reactions of amine 24 with benzenesulfonyl chloride and benzyl bromide followed by ester hydrolysis under basic conditions, respectively. All benzoic acid derivatives were coupled with indole derivative 6 to generate the inhibitors 27 (Table 4) by using the reaction conditions in Scheme 2. Table 4. Biochemical and cell-based activities, microsome stability and CYP inhibition properties of inhibitors 27. a
  • Values in parentheses are % inhibition of the indicated human CYPs at 10 ⁇ .
  • the terminal phenyl ring of 14t (CYP-I- 181) was extensively modified since it is oriented toward the solvent accessible area and opportunities existed to enhance microsome stability and minimize inhibition of human CYP enzymes through such modifications.
  • Inhibitor 14t (Small molecule code 181)
  • dValues in parentheses are for highest-resolution.
  • a series of first-generation biaryl inhibitors (e.g., 14t) were synthesized and shown to have improved microsome stability and enhanced in vitro inhibitor potency (Table 2).
  • the x-ray co-crystal structure of 73 ⁇ 4CYP51 with bound 14t (CYP-I-181) was determined and employed in structure-based design of the next round of CYP51 inhibitors.
  • microsome stability of many other inhibitors containing biaryl units was improved, as was the selectivity of 27k and 27s when tested against the battery of human CYPs.
  • 73 ⁇ 4CYP51 is similar to that of posaconazole with the exception that the biaryl unit of 14t extends towards the solvent accessible area though a different hydrophobic tunnel than used by posaconazole (Fig. 2B,C).
  • the indole ring of 14t occupies the same hydrophobic cavity as the 2,4-difluorophenyl moiety of posaconazole.
  • the cavity extends beyond these groups along the heme macrocycle and has sufficient space to accommodate an alkoxy group attached to C5 of the indole nucleus (inhibitor 3k).
  • biaryl unit of CYP-I- 181 and the tail portion of posaconazole are oriented toward a solvent-accessible area, they protrude through different hydrophobic tunnels (Fig. 5).
  • An ample void space surrounding the biaryl unit of the (S)-enantiomer CYP-I- 181 or the "tail" of posaconazole suggested the possibility of a different binding
  • CYP-II-34 was synthesized starting from D-tryptophan, and its biological properties were assessed. The potency of CYP-II-34 as an inhibitor of T. cruzi in infected cells was observed to increase remarkably relative to CYP-I-181, from EC50 of 1.3e-6 M to 2.2e-9 M. Compound CYP-II-34 also retained microsome stability and had an acceptable profile for inhibition of human CYPs (14/91/30/60% inhibition of CYP1A2/2C9/2D6/3A4 at 1 ⁇ ).
  • the x-ray co-crystal structure of the (R)-enantiomer CYP-II-34 with T. cruzi CYP51 was determined to a resolution of 3.1 A (PDB ID 4BY0), indicating that binding of the 4- acylaminopyridine and indole moieties of the R-enantiomer are same as for the (S)- enantiomer CYP-I-181, but the biaryl unit of the (R)-enantiomer is oriented toward the hydrophobic tunnel accommodating the tail of posaconazole (Fig. 6).
  • CYP-II-34 is distinguished by an L-shape in the active site of T.
  • Table 6 provides a summary of X-ray crystallographic data obtained in this crystal structure determination.
  • Inhibitor (R)-enantiomer CYP-II-34 was further optimized to increase potency against T. cruzi in infected cells.
  • a flexible piperazine ring was introduced at the position of the terminal phenyl ring of CYP-II-34 to probe binding interactions in the solvent accessible area.
  • the new inhibitors exhibit similar inhibition potency against T. cruzi in infected cells, compared to (R)-enantiomer CYP-II-34.
  • CYP-II-1 1 1, CYP-II-123, and CYP-II- 154 have substantially improved microsome stability, and are weaker inhibitors of human CYP enzymes except for CYP 2C9.
  • Table 7 Stability and Activity versus Human CYP for Selected (RVenantiomers Compound Stability Stability Stability % Inhib. % Inhib. % Inhib. % Inhib.
  • mice were infected with T. cruzi for three days, and starting on day 4 the infected mice were treated with 40 mg/kg of test compounds via intraperitoneal injection for four consecutive days b.i.d.
  • T. cruzi luminescent signal in the mice was read. It was found that the parasite load in the untreated animals significantly increased. Posaconazole used as a positive control produced >99% inhibition of parasitemia.
  • the parental hit, LP 10 showed little efficacy under these treatment conditions, while the new, rationally designed analogs 11- 123, 11-142, and 11-154 suppressed parasite load by >97% over the 4-day treatment period.
  • the compounds II-l 1 1 (84%) and 11-158 reduced the infection by 84% and 52% respectively over the 4-day treatment period..
  • Table 8 below provides a summary of the EC50 values, % inhibition in vivo, stability, and % inhibition of human CYPs at 1 ⁇ concentration for the (R)-enantiomer II- 34 and the seven related compounds 11-64, II-l 1 1, 11-123, 11- 142, 11- 154, and 11-158 discussed above, compared to LP 10.
  • Table 8 In vitro properties and in vivo efficacy of hits in the 4-day mouse model upon intraperitoneal (i.p.) or oral (p.p.) administrations
  • the plasma concentration-time curves following oral administration of a single 50 mg/kg dose orally in 20% HP CD were obtained for the five hits which suppressed parasite load in mice >98%. These compounds had notably different pharmacokinetic behavior.
  • Compound 11-259 had the longest half-life (6.9 h) followed by 11-258 (5.2 h). The highest maximum plasma concentration was achieved by compounds 11-251 and 11-258 followed by 11-259. These three compounds had the highest AUC values and the lowest clearance. The shortest half-life, lowest Cmax and highest clearance were observed for 11-257.
  • Tissue distribution was assessed for all five hits after 2 h and 8 h of exposure following oral administration.
  • Compounds 11-251 , 11-257 and 11-259 accumulated in intestines at high concentrations, particularly 11-259, whose intestine concentration remained high even after 8 h exposure (Table 10).
  • Formulation 50mg/kg in 10 mg/ml suspension in 20% HP CD Given that no non-dissolved materials were observed in the intestines, compounds likely permeated the mucosa of the gut from the apical to the basolateral side. Compounds II- 250, 11-251 , 11-258, and 11-259 penetrated lever, lung and heart tissues. Compounds 11-251 , II- 258, and 11-259 were also detected in skeletal muscles after 8 h exposure. Compound 11-257 efficiently crossed the blood-brain barrier where it was detected after 2 h exposure.
  • the x-ray structures at resolution of 2.0 A determined in this work provided atomic details of drug-target interactions for two N-indolyl-oxopyridinyl-4-aminopropanyl compounds containing piperazine ring in the structure of the longest substituent.
  • One is a low nanomolar orally bioavailable hit, compound 11-259, (R)-N-(3-(lH-indol-3-yl)- l -oxo- l - (pyridin-4-ylamino)propan-2-yl)-4-(4-(2,4-difluorophenyl)piperazin- l -yl)-2- fluorobenzamide.
  • the other is a hit belonging to a sulfonyl-containing subset of the R- stereoisomers synthesized and tested over the course of hit- to-lead optimization (Table 1 1, above).
  • the piperazine ring of the variable substituent at the chiral carbon center faces the cleft between the a- and ⁇ -domains and is the only part of the inhibitor molecule loosely surrounded by protein amino acid residues.
  • the latter allows piperazine to flex in an opposite direction in the complex of compound 11-71 to accommodate a -90° turn in the molecular skeleton enabled by the sulfonyl group pointing into the cleft between the a- and ⁇ -domains (Fig. 12).
  • the 259 substituent extends flat along the ⁇ -sheet saddle, serving as a latch fastening the cleft and holding both domains together.
  • the 4-fluoro substituent of the terminal 2,4-difluorophenyl ring is at van der Waals distances of 170 and 172, and within 5 A of V77, 179 and F55.
  • the 2-fluoro substituent is within 5 A of 145 and F48, all residues are part of the ⁇ -domain.
  • the 2-fluoro substituent of the benzamide ring in both inhibitors points toward a crevice formed by the a-domain residues Y103, 1105, Ml 06 and M480 residing at van der Waals distances of Y 103 and 1105.
  • the 2- fluoro substituent was retained earlier in the hit-to-lead optimization for the increased half- life of hits in the macrosome stability assays.
  • Binding mode of the variable longest substituent created ambiguity in binding of the invariable indole ring in 11-71 which, as demonstrated previously (11-34), contributes an order of magnitude into hit binding affinity.
  • This is in contrast to 11-259 which apparently has little space for additions to the skeleton (Fig. 12C).
  • compound 11-259 is characterized by oral bioavailability, relatively long terminal half- life, slow clearance and efficient distribution into the tissues.
  • a clear drawback of the sub-scaffold B analogs, including compound 11-259 is attenuated stability, particularly in the human microsome fraction
  • Table 13 shows structures of specific compounds prepared and tested according to the methods described herein.
  • N-phenyl rings of the compounds are depicted in orientations deduced based on the x-ray structure analysis and SAR.
  • b Each measurement performed in triplicate (see SI);
  • c Each measurement is an average of 5 mice treated 25 mg/kg (20% Kolliphor), p.o., b.i.d., for 4 days;
  • d Each measurement is an average of 5 mice treated 10 mg/kg (20% Kolliphor), p.o., b.i.d., for 4 days; Stability of compounds in human (h), rat (r) and mouse (m) liver
  • aEach measurement is an average of three mice received a singe 25 mg/kg dose of test compound at 5 mg/ml suspension in 20% Kolliphor.
  • cardiomyopathy including an overview on history, pathology, and other proposed pathogenic mechanisms.
  • Proton nuclear magnetic resonance (3 ⁇ 4 NMR) spectra and carbon ( 13 C) NMR spectra were recorded on a commercially available NMR spectrometer at 400 MHz and 100 MHz, respectively.
  • the proton signal for non-deuterated solvent ⁇ 7.26 for CHCb or ⁇ 2.50 for DMSO was used as an internal reference for 3 ⁇ 4 NMR chemical shifts.
  • Coupling constants (J) are reported in Hertz (Hz). 13 C chemical shifts are reported relative to the ⁇ 77.16 resonance of CDCb or the ⁇ 39.52 resonance of DMSO-d6.
  • Analytical thin layer chromatography was performed using glass plates precoated with a 0.25-mm thickness of silica gel. The TLC plates were visualized with UV light.
  • Column chromatography was performed using a Biotage® Isolera flash purification system using Biotage® SNAP HP-SIL cartridge (30 ⁇ silica, 10 g to 100 g size). Unless noted otherwise, all compounds isolated by chromatography were sufficiently pure by 3 ⁇ 4 NMR analysis for use in subsequent reactions.
  • Polar compounds were purified using preparative high performance liquid chromatography (HPLC) using SunFire column (30 mm x 250 mm) with a linear gradient elution at 60 mL/min.
  • DMSO-de ⁇ 171.49, 150.35, 145.44, 137.75, 136.05, 129.07, 127.16, 126.90, 123.91, 121.49, 121.35, 120.95, 1 18.46, 118.25, 1 13.92, 1 13.42, 109.31, 55.08, 27.42.
  • Microsome stability was evaluated by incubating 1 ⁇ compound with 1 mg/mL hepatic microsomes (human, rat, or mouse) in 100 mM potassium phosphate buffer, pH 7.4 at 37 °C with continuous shaking. The reaction was initiated by adding NADPH, 1 mM final concentration. The final incubation volume was 300 ⁇ ⁇ and 40 ⁇ ⁇ aliquots were removed at 0, 5, 10, 20, 40, and 60 minutes. The aliquots were added to 160 ⁇ ⁇ acetonitrile to stop the reaction and precipitate the protein. NADPH dependence of the reaction is evaluated in parallel incubations without NADPH.
  • the samples are centrifuged through a 0.45 micron filter plate (Millipore Solventer low binding hydrophilic plates, cat# MSRLN0450) and analyzed by LC-MS/MS. The data were log transformed and results are reported as half-life.
  • Cytochrome P450 inhibition was evaluated in human liver microsomes using four selective marker substrates (CYP1A2, phenaceten demethylation to acetaminophen;
  • CYP2C9 tolbutamide hydroxylation to hydroxytolbutamide
  • CYP2D6 bufuralol hydroxylation to 4'-hydroxybufuralol
  • CYP3A4 midazolam hydroxylation to - hydroxymidazolam
  • concentration of each marker substrate is approximately its Km. - Furafylline
  • the homology model of 7cCYP51 was generated based on the x-ray co-crystal structure of 73 ⁇ 4CYP51 complexed with 14t (PDB ID code: 4BJK) by using the homology model module implemented in Molecular Operating Environment (MOE).
  • the homology model was refined with Protein Preparation Wizard implemented in Maestro 9.3.
  • a receptor grid was generated from the refined structure using default values except for positional constraint at the nitrogen of 4-acylaminopyridine (radius: 0.8).
  • the structure of 14t was docked into the active site of 7cCYP51 by using Glide5.5 in extra precision (XP) mode with the predefined positional constraint (ligand feature: neutral acceptor).
  • the structures of 27k, 271, 27r, and 27s were subsequently docked to the model structure of 7cCYP51 by applying the same parameters to predict their binding poses in the 7cCYP51 active site.
  • Aobs is the absorption shift determined at any ligand concentration
  • a ma x is the maximal absorption shift obtained at saturation
  • KD is dissociation constant for the inhibitor- enzyme complex
  • S is the ligand concentration
  • Et is the total enzyme concentration.
  • Recombinant 73 ⁇ 4CYP51 mutant V34M/D249A/D250A/D251 A modified by inserting a Hiss-tag at the C-terminus and replacing the first 31 residues upstream of P32 with the fragment MAKKTSSKGKL was used to obtain co-crystal structure with 14t.
  • Concentrated purified protein stored at -80°C was diluted to 0.1 mM prior to crystallization by mixing with water supplemented with 14t to reach 1 : 1 protein:inhibitor ratio. Crystallization conditions were determined using commercial high-throughput screening kits available in deep-well format (Hampton Research), a nanoliter drop-setting Mosquito robot (TTP LabTech) operating with 96-well plates, and a hanging drop crystallization protocol.
  • Crystals were further optimized in 96-well plates for diffraction data collection and harvested directly from the 200-nL drops. Prior to data collection, crystals were cryo-protected by plunging them into a drop of reservoir solution supplemented with 20% ethylene glycol, then flash frozen in liquid nitrogen.
  • Diffraction data were collected at 100- 1 10 K at Beamline 8.3.1, Advanced Light Source, Lawrence Berkeley National Laboratory, USA. Data indexing, integration, and scaling were conducted using MOSFLM- and the programs implemented in the ELVES software suite.
  • the final model was built using COOT- and refinement was performed by using REFMAC5 software- until R and Rfiee converged to 19.4% and 27.4%, respectively. Data collection and refinement statistics are shown in Table 5, above.
  • chain A Only one of the four protein chains (chain A) constituting an asymmetric unit contained electron density corresponding to the whole molecule of 14t; 14t was assigned PDB code 181. In three other chains, only the N-indolylpyridinyl portion of 14t could be unambiguously placed. Thus, coordinates for the disordered biaryl moiety in chains B, C and D were omitted from the PDB entry.
  • Trypanosoma cruzi, Y luc strain, episomally expressing the firefly luciferase gene was developed as described elsewhere (36). Cultured trypomastigotes were obtained by weekly infection of C2C12 myoblasts, with trypomastigotes being released in the supernatant 4 to 7 days post infection, collected by centrifugation for 15 min at 3300 rpm. Without selective antibiotic pressure, the luciferase expression in the parasite is detectable for about as long as 7 passages in mammalian culture. To maintain high titer of luciferase marker in parasite population, the pressure of G418 antibiotic was applied to epimastigote form.
  • the epimastigotes were cultivated in LIT medium (Camargo et al., 1964), supplemented with 10% fetal bovine serum and 200 ⁇ g/ml of G418, at 28° C.
  • LIT medium Limargo et al., 1964
  • myoblast cultures were infected with 2-4 week old epimastigotes enriched with the metacyclic trypomastigotes.
  • the epimastigotes were removed from the medium by successive washing of cultures with phosphate buffer saline (PBS). Seven days post-infection, trypomastigote population enriched with the transgenic parasites expressing luciferase was released to the medium. 4-day dosing mouse model of T. cruzi infection.
  • mice Eight-week-old female Swiss Webster albino mice (average weight 20 g) were obtained from the Simonsen Labs. All animal procedures were approved and carried out in accordance with the guidelines established by the Institutional Animal Care and Use Committee from UCSF (Approval number AN087605-01). Mice were housed at a maximum of 5 per cage and kept in a specific -pathogen free (SPF) room at 20 to 24° C under a 12-h light/12-h dark cycle and provided with sterilized water and chow ad libitum. To infect the mice, trypomastigotes of T. cruzi Y luc strain were harvested from culture supernatant and injected intraperitonealy (i.p.), 10 5 trypomastigotes per mouse.
  • SPPF specific -pathogen free
  • mice were anesthetized by inhalation of isofluorane (controlled flow of 1.5% of isofluorane in air was administered through a nose cone via gas anesthesia system).
  • Mice were injected i.p. with 150 mg/kg D-luciferin potassium salt (Gold Biotechnology) dissolved in PBS and imaged after 5 min using IVIS Spectrum Pre-clinical In Vivo Imaging System (Perkin Elmer, Waltham, MA) and the data acquisition and analysis software Livinglmage V4.1 (Perkin Elmer, Waltham, MA). Only mice with detectable luminescence were used for treatment. The compounds potency was evaluated following oral (o.p.) administration.
  • mouse C2C12 myoblasts (ATCC #CRL- 1772) used to harbor parasites were cultivated in Dulbecco's Modified Eagle's Medium H-21 containing 4.5 g/1 glucose (DMEM H-21), supplemented with 5% fetal bovine serum (FBS), 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ streptomycin. T.
  • trypomastigotes were obtained from infected-culture supernatants after 4-7 days of infection. Cultures were maintained at 37°C with 5% CO2. Trypomastigotes and C2C 12 cells concentration was determined using a Neubauer hemocytometer. Sterile, black 384-well plates with clear-bottom wells (Greiner Bio-One) were seeded with 500 cells/well and then were infected with 2500 parasites/well in a final volume of 50 ⁇ /well. Culture plates were incubated at 37°C with 5% CO2 for 24-hours. After that, culture medium was removed and test compounds were added in fresh medium.
  • an intermediate plate (384-well plate) was prepared by serial dilution (10 mM, 2 mM, 400 ⁇ , 80 ⁇ , 16 ⁇ , 3 ⁇ , 128 ⁇ , 25.6 ⁇ , 5.1 ⁇ ) for all the compounds in 100 % DMSO. Then, 50 nl of each sample were diluted in 50 ⁇ media (DMEM H-21) and added to the experimental plate followed by incubation at 37°C with 5% CO2 for 72h. Wells containing non-infected cells were used as a positive control (100% cell survival), while T. crwzz ' -infected but untreated cells (0% cell survival) were used as a negative control. Cells were then fixed for 2 h with 4% paraformaldehyde, and rinsed with a solution of 150 mM
  • the organic layer was then dried under nitrogen gas and subsequently treated with 75 ⁇ ⁇ ⁇ , ⁇ - bis(trimethylsilyl)-2,2,2-trifluoroacetamide (BSTFA) for 2 h at 37 °C to facilitate chemical derivatization with trimethylsilyl (TMS) groups (BSTFA, Sigma-Aldrich).
  • TMS trimethylsilyl
  • BSTFA Sigma-Aldrich
  • the TMS- derivatized lipid mixture was analyzed by injecting 3 ⁇ ⁇ directly into an Agilent HP5790 gas chromatography system outfitted with a DB5-MS analytical column (30 m, 0.25 mm i.d., 0.33 ⁇ film thickness, Agilent) coupled to a mass selective detector.
  • the lipids were separated on the analytical column using a temperature profile that begins at 200 °C for 1 min, increases by 15 °C/min up to 300 °C and then holds at 300 °C for 20 minutes.
  • the inlet temperatures of the GC and the MSD were held at 250 °C and 300 °C, respectively.
  • the mass spectrometer scanned from m/z 50 - 750 during the course of analysis.
  • mice were housed at a maximum of 5 per cage and kept in a specific- pathogen free (SPF) room at 20 to 24° C under a 12-h light/ 12-h dark cycle and provided with sterilized water and chow ad libitum.
  • SPF pathogen free
  • trypomastigotes of T. cruzi Y luc strain were harvested from culture supernatant and injected intraperitonealy, 10 5 trypomastigotes per mouse.
  • mice Three control groups included untreated mice, which received a vehicle, 20% Kolliphor HS 15 (also known as Solutol), and the positive control groups, which received 25 or 50 mg/kg benznidazole, all via oral gavage (p.o.), twice a day (b.i.d).
  • the infected mice were treated with test compounds at 25 mg/kg administered in 20% Kolliphor, p.o., b.i.d., for four consecutive days.
  • the luminescent signal in the mice was read upon injection of D-luciferin.
  • the absolute numbers of measured photons/s/cm 2 were averaged between all five mice in each group and compared directly between compound-treated mice and the control groups. Two tailed paired Student t test was used to assess statistical significance between luminescence values from vehicle -treated and compound-treated groups at day 7 post-infection; values are statistically significant when p ⁇ 0.05.
  • Single dose PK Single dose PK
  • CYP51 expression was induced by adding 0.25 mM isopropyl- -thiogalactopyranoside (IPTG) and ImM ⁇ -aminolevulinic acid, a precursor in heme biosynthesis, was added at that time. After induction, the growth was continued at 18°C at 180 rpm for 48 h. Cells were harvested, resuspended in 50 mM Tris, pH 8.5, 1 mM EDTA, 100 mM NaCl, 0.5 mM PMSF, 1 mM DTT and lysed using a microfluidizer.
  • IPTG isopropyl- -thiogalactopyranoside
  • ImM ⁇ -aminolevulinic acid a precursor in heme biosynthesis
  • the soluble fraction was purified by conventional Ni- NTA agarose chromatography using a linear gradient of imidazole (0 to 0.5 M) in 50 mM potassium phosphate, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM EDTA, 500 mM NaCl. After dialysis overnight against 20 mM potassium phosphate pH 7.5, 10% glycerol, 1 mM DTT, 0.5 mM EDTA, the sample was applied on MonoQ column. The flow-through fractions were applied on Mono S column and the protein was eluted in the same buffer using linear NaCl gradient (0-0.5 M). Fractions containing CYP51 were combined and concentrated using Centriprep concentrating device (Millipore). These samples were stored at -80°C and used as needed for co-crystallization and binding assays.

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Abstract

L'invention concerne des inhibiteurs d'une stérol C14-déméthylase, une nouvelle série d'inhibiteurs principaux à base de 4-aminopyridyle ciblant Trypanosoma cruzi CYP51 (TcCYP51) développés en utilisant un concept de médicament à base d'une structure ainsi que les analyses de relation structure-propriétés (SPR). Le point de départ du test de triage, LP 10 (KD < 42 nM; EC50 de 0,65 μΜ), a été optimisé pour donner les principaux potentiels qui ont une faible affinité de liaison nanomolaire pour TcCYP51 et une activité significative contre des T. cruzi amastigotes cultivés dans des myoblastes humains. Beaucoup des composés optimisés ont une stabilité de microsome améliorée et la plupart sont sélectifs contre T. cruzi CYP51 par rapport aux CYP humain 1A2, 2D6 et 3A4 (<50% d'inhibition à 1 µM). Une justification pour l'amélioration de la stabilité de microsome et la sélectivité d'inhibiteurs contre des enzymes CYP métaboliques humaines est présentée. De plus, le mode de liaison de plusieurs composés de l'invention avec l'orthologue T. brucei CYP51 (TbCYP51) a été caractérisé par l'analyse de structure par rayons X. On a montré que des composés oralement actifs et leurs complexes de cyclodextrine comme étaient efficaces contre des souris infectées par la maladie de Chagas.
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CN109232396A (zh) * 2018-11-27 2019-01-18 聊城大学 酰胺吡啶类衍生物及其用途
CN113683759A (zh) * 2021-08-31 2021-11-23 宁波聚嘉新材料科技有限公司 一种高模量热致性液晶聚芳酯薄膜及其制备方法和应用

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