WO2010074783A1 - Inhibiteurs de la phosphodiestérase et utilisations de ces derniers - Google Patents

Inhibiteurs de la phosphodiestérase et utilisations de ces derniers Download PDF

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Publication number
WO2010074783A1
WO2010074783A1 PCT/US2009/058813 US2009058813W WO2010074783A1 WO 2010074783 A1 WO2010074783 A1 WO 2010074783A1 US 2009058813 W US2009058813 W US 2009058813W WO 2010074783 A1 WO2010074783 A1 WO 2010074783A1
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atom
compound
leu
mice
gln
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PCT/US2009/058813
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English (en)
Inventor
Yan Feng
Ottavio Arancio
Shixian Deng
Donald W. Landry
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The Trustees Of Columbia University In The City Of New York
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Priority to EP09835413.7A priority Critical patent/EP2379076B1/fr
Priority to JP2011543511A priority patent/JP2012513464A/ja
Publication of WO2010074783A1 publication Critical patent/WO2010074783A1/fr
Priority to US13/167,540 priority patent/US8697875B2/en
Priority to US14/224,702 priority patent/US9422242B2/en
Priority to US15/235,736 priority patent/US9974782B2/en

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Classifications

    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3

Definitions

  • AD Alzheimer's disease
  • a ⁇ amyloid ⁇ -peptides
  • a ⁇ 42 amyloid- ⁇ -peptide 1-42
  • Phosphodiesterase 5 (PDE5) inhibitors are widely used drugs against erectile dysfunction and pulmonary hypertension. These inhibitors are believed to increase cGMP levels which enhances phosphorylation of the transcription factor and memory-affecting molecule cAMP-responsive element binding (CREB) through activation of cGMP-dependent-protein kinases.
  • CREB cAMP-responsive element binding
  • cGMP nucleotide biological second messengers
  • Intracellular receptors for these molecules include cyclic nucleotide phosphodiesterases (PDEs), cyclic nucleotide dependent protein kinases (PGK), and cyclic nucleotide-gated channels.
  • PDEs are a large family of proteins that catalyze the hydrolysis of 3',5'-cyclic nucleotides to the corresponding 5' monophosphates. There are eleven related, but biochemically distinct, human PDE gene groups.
  • PDEs are specific for hydrolysis of cAMP (such as PDE4, PDE7, and PDE8), and some are cGMP specific (such as PDE5, PDE6, and PDE9), while some PDEs have mixed specificity (such as PDEl, PDE2, PDE3, PDElO, and PDEl 1).
  • PDE 5 inhibitors are cyclic guanosine 3',5'-monophosphate type five cGMP
  • PDE inhibitors which include, but are not limited to, sildenafil, tadalafil, zaprinast, and vardenafil.
  • PDE5 inhibitors increase cGMP levels by inhibiting the degradative action of PDE5 on cGMP. No PDE inhibitor has reached the marketplace for diseases of the CNS, and no PDE5 inhibitors have been used for the treatment of AD.
  • the invention is directed to a class of quino line-containing compounds with
  • the compound is Formula V as described herein.
  • the compound is Formula V-I as described herein.
  • the compound is Formula V-Ia as described herein.
  • the compound is Formula V-IaI as described herein.
  • the compounds are those compounds depicted in compounds 1-18 as described herein.
  • the compound is compound 3 (YF012403) or compound 8 (YF016203).
  • the invention provides a method for screening compounds to treat conditions associated with accumulated amyloid-beta peptide deposits, the method comprising: (a) selecting (or identifying or screening for) a PDE5 inhibitor compound that can modulate secretase activity for at least 1 month after completion of administration of the PDE5 inhibitor compound in an animal model of amyloid-beta peptide deposit accumulation.
  • the invention provides a method for screening compounds to treat conditions associated with accumulated amyloid-beta peptide deposits, the method comprising: (a) selecting a PDE5 inhibitor compound that comprises one or both of the following features: (i) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, 1813, M816, or a combination thereof; or (ii) the 2nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH-.
  • a PDE5 inhibitor compound that comprises one or both of the following features: (i) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, 1813, M816, or a combination thereof; or (ii) the 2nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH-.
  • the invention provides a method for identifying a phosphodiesterase-binding compound to treat conditions associated with accumulated amyloid-beta peptide deposits, wherein the method comprises selecting a PDE5 inhibitor compound having one or more of the following features: (a) the IC 50 of the compound is no more than about 1000 nM; (b) the selectivity of the compound is at least a 50 fold greater potency towards PDE5 relative to PDEl, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDElO, or PDEl 1; (c) the PDE5 inhibitory activity in vitro has an IC50 no more than about 50 nM; (d) the compound penetrates the blood brain barrier; (e) the compound hydro lyzes cGMP by about 20% to about 80%; (f) the 2nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH-; or (g) the compound interacts with two
  • the phosphodiesterase in feature (g) can comprise, for example, phosphodiesterase type V (PDE5) or even another PDE.
  • feature (g) is where the compound interacts with at least all four amino acid residues F787, L804, 1813, and M816 of PDE5.
  • the compound can decrease the activity or expression of a phosphodiesterase type V (PDE5) protein
  • the above described methods further comprise testing whether the selected PDE5 inhibitor can modulate secretase activity for at least 1 month after administration in an animal model of amyloid-beta peptide deposit accumulation.
  • the secretase can be ⁇ -secretase or ⁇ -secretase.
  • the modulation can comprise a decrease in ⁇ - secretase activity or expression levels and/or an increase in ⁇ -secretase activity or expression levels.
  • the modulated secretase activity or expression of ⁇ -secretase remains decreased.
  • the modulated secretase activity or expression of ⁇ -secretase remains increased.
  • the modulated secretase activity persists more than 2 months, 3 months, 4 months, 5 months, 6 months, or 7 months after completion of the dosage period.
  • the animal model of amyloid- beta peptide deposit accumulation comprises an APP/PS1 double transgenic mouse.
  • the step of testing whether the selected PDE5 inhibitor can modulate secretase activity for at least 1 month after administration in the APP/PS1 double transgenic mouse comprises: (a) administering the selected PDE5 inhibitor to APP/PS1 double transgenic mice for a dosage period up to about 21 days; (b) testing whether the selected PDE5 inhibitor modulates secretase activity or expression in the APP/PS1 double transgenic mice immediately after completion of the dosage period as compared to a negative control; and (c) testing whether modulated secretase activity or expression in the APP/PS 1 double transgenic mice from step (b) persists more than 1 month after completion of the dosage period as compared to a negative control.
  • the selecting step of the compound based on features can involve in silico screening, molecular docking, in vivo screening, in vitro screening, or a combination thereof.
  • a dosage period of the PDE5 inhibitor compound to the animal model subject is up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, or up to about 20 days.
  • the compound has a molecular mass less than about 500 Da, a polar surface area less than about 90 A 2 , less than 8 hydrogen bonds, or a combination thereof in order to penetrate the blood brain barrier.
  • the PDE5 inhibitor compound has been first pre-screened by a method comprising: (a) providing an electronic library of test compounds; (b) providing atomic coordinates listed in Table 1 for at least 20 amino acid residues for the active site of the PDE5 protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Ca atoms, of not greater than about 2 A, in a computer readable format; (c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the PDE5 protein; (d) performing a data processing method, wherein electronic test compounds from the library are docked onto the three dimensional model of the PDE5 protein; and (e) determining which test compound fits into the active site of the three dimensional model of the PDE5 protein, thereby identifying which compound would bind to PDE5.
  • this method can further comprise: (f) synthesizing or obtaining the compound determined to dock to the active site of the PDE5 protein; (g) contacting the PDE5 protein with the compound under a condition suitable for binding; and (h) determining whether the compound modulates PDE5 protein expression or mRNA expression, or PDE5 protein activity using a diagnostic assay.
  • the PDE5 inhibitor compound comprises Formula Ia, Formula Ib, Formula Ic, Formula Id, Formula Ie, Formula Ha, Formula lib, Formula Hc, Formula Hd, Formula He, Formula Ilia, Formula IHb, Formula IHc, Formula Ilia- 1 , Formula IIIb-1, Formula IIIc-1, Formula IHd, Formula IHe, Formula IHf; Formula IVa, Formula IVb, Formula V, Formula V-I, Formula V-I -a, or Formula V-a-1 (such as either one of compounds 1-18), as described herein.
  • the PDE5 inhibitor decreases PDE5 protein or mRNA expression, or PDE5 activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or 100%.
  • the PDE5 inhibitor has an IC50 at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM.
  • methods for selecting a PDE5 inhibitor can comprise detecting whether the inhibitor can cause an increase or decease in a secondary messenger concentration.
  • the secondary messenger can comprise, for example, cyclic GMP, protein kinase G (PKG), or a combination thereof.
  • the detection can comprise an assay that measures an intracellular concentration of GTP, cyclic GMP, protein kinase G (PKG), or CREB.
  • the PDE5 inhibitor compound binds to the active site of phosphodiesterase type V (PDE5).
  • the compound has an IC50 at least about 0.1 nM, at least about
  • nM 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM.
  • the invention provides a method for increasing ⁇ -secretase protein activity or expression in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby increasing ⁇ -secretase protein activity or expression in the subject.
  • the invention provides a method for decreasing ⁇ -secretase protein activity or expression in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby decreasing ⁇ -secretase protein activity or expression in the subject.
  • the invention provides a method for reducing amyloid beta (A ⁇ ) protein deposits in a subject, the method comprising: (a) administering to the subject an effective amount of a composition comprising a PDE5 inhibitor compound, thereby decreasing A ⁇ protein deposits in the subject.
  • the subject exhibits abnormally elevated amyloid beta plaques.
  • the subject is afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy.
  • the subject is a mouse, dog, cat, horse, cow, sheep, or human.
  • the compound that is administered to the subject comprises
  • the compound is sildenafil, tadalafil, or vardenaf ⁇ l.
  • the administration comprises subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; oral or nasal delivery; or a combination thereof.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, or at least about 10 mg/kg body weight.
  • the effective amount of the administered compound is at least about 3mg/kg body weight.
  • the composition is administered at least once daily for up to 18 days, up to 19 days, up to 20 days, up to 21 days, up to 22 days, up to 23 days, up to 24 days, or up to 25 days.
  • the ⁇ -secretase protein activity or expression is increased up to 3 months post- treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment.
  • the ⁇ -secretase protein activity or expression is decreased up to 3 months post-treatment, up to 4 months post-treatment, up to 5 months post-treatment, or up to 6 months post-treatment.
  • the A ⁇ protein deposit comprises an A ⁇ 40 isomer, an A ⁇ 42 isomer, or a combination thereof.
  • PDE5 inhibitor compounds that are administered to subjects to modulate secretase activity or expression are administered infrequently due to the finding provided herein that PDE5 inhibitors can cause a long-lasting or sustained affect on secretase activity long-after administration.
  • methods of treatment are provided where subjects are administered PDE5 inhibitors for short-term periods on a regular, but infrequent basis.
  • administration can comprise a dosage regimen comprising 1 week, 2 weeks, 3 weeks, a month, or more, followed by a period of no administration that comprises 1 week, 2 weeks, 3 weeks, a month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, wherein this dosage regimen can then be repeated and varied.
  • the dosage regimen comprises a period of PDE5 inhibitor administration followed a period of no drug administration, optionally followed by further cycles.
  • the benefit of such a cyclic regimen can be, for example, to lessen the possibility of side-effects due to total drug intake-load over time.
  • FIG. IA is a graph of field input-output relationship for different stimulation intensities (5-35 V) that shows that BST is similar in 3-month-old APP/PS1 animals and WT littermates.
  • FIG. IB is a graph representing that ten minutes perfusion with sildenafil (50 nM) reverses LTP impairment in APP/PS1 mice (sildenaf ⁇ l-treated APP/PS1 mice equal to -100% of vehicle-treated WT littermates at 120 min. after tetanus, vs. -65% in vehicle- treated APP/P Sl mice; sildenaf ⁇ l-treated APP/PS1 mice: 215.08 ⁇ 11.85 % at 120 min.
  • FIG. 1C is a graph showing that sildenafil (50 nM) does not affect LTP in WT mice.
  • These experiments were interleaved with those of APP/PS1 mice.
  • FIG. 2A is a graph that shows sildenafil ameliorates cognitive dysfunction in
  • FIG. 2B is a graph demonstrating that sildenafil ameliorates cognitive dysfunction in 3-month-old APP/PS1 mice.
  • Sildenafil (3 mg/Kg, i.p.) improves spatial working memory in 3 month-old APP/PS1 mice.
  • APP/PS1 mice treated with vehicle do not learn the position of the hidden platform compared to vehicle-treated WT littermates
  • FIG. 3A is a bar graph that shows sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) ameliorates contextual fear conditioning in transgenic mice. After 24 hours, there is a reduction of freezing behavior in APP/PS1 mice compared to WT, rescued by sildenafil treatment [-97% of vehicle-treated WT mice in sildenafil-treated APP/PS1 mice, vs.
  • FIG. 3B is a graph that shows impairment of performance during radial-arm water maze testing in APP/PS1 mice is rescued by treatment with sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months).
  • Sildenafil improves the performance of APP/PS1 mice and does not affect the performance of WT mice
  • FIG. 3C is a graph depicting that the performance of APP/PS1 mice in the
  • FIG. 3D is a bar graph demonstrating that APP/PS1 mice previously treated with sildenafil search significantly more time in the target quadrant (TQ), where the platform was located during training, than do vehicle-treated APP/PS1 littermates, during the probe test.
  • Sildenafil improved the performance of the APP/PS1 mice [32.25 ⁇ 0.58% of their time given spent in TQ, or -97% of the time used by vehicle-treated WT littermates, than in other quadrants; P ⁇ 0.0001].
  • Planned comparisons confirmed that they spent significantly more time in the TQ than in the AR, in the AL, or in the OQ (P ⁇ 0.0001).
  • FIG. 4A is a graph showing that BST impairment in 6-8 month-old APP/PS 1 animals is improved by sildenaf ⁇ l-treatment (3 mg/Kg, i.p. for 3 weeks at the age of 3 months)
  • FIG. 4B is a graph demonstrating that sildenafil (3 mg/Kg, i.p. for 3 weeks at the age of 3 months) rescues the LTP impairment in 6-8 month-old APP/PS1 mice
  • APP/PS1 + sildenafil -100% of sildenafil-treated WT mice, 233.81 ⁇ 30.47 % of baseline at 120 min
  • n l slices from 6 males
  • APP/PS 1+ vehicle ⁇ 65 % of vehicle-treated WT mice, 135.56 ⁇ 22.02 % of baseline
  • FIG. 5 A are immunofluorescence photographs showing representative examples of hippocampal slices stained with a phospho-CREB antibody.
  • the slices are fixed 60 minute after either vehicle or sildenafil (50 nM) with tetanus in 3 -months old WT and APP/PS 1 animals.
  • Sildenafil re-establishes normal increase in CREB phosphorylation following tetanic stimulation in APP/PS 1 mice.
  • FIG. 5 A are immunofluorescence photographs showing representative examples of hippocampal slices stained with a phospho-CREB antibody.
  • the slices are fixed 60 minute after either vehicle or sildenafil (50 nM) with tetanus in 3 -months old WT and APP/PS 1 animals.
  • IF immunofluorescence
  • FIG. 6B are photographs of immunoblots from the brains of APP/PS1 3-month- old transgenic mice treated with sildenafil (Right Column) or vehicle (Left Column) stained for APP full length, sAPP ⁇ , sAPP ⁇ , CT83, CT99. Tubulin was used as a control.
  • Sildenafil modifies ⁇ -secretase activity in 3-4 month-old APP/PS1.
  • FIG. 7B are photographs of immunoblots from the brains of APP/PS1 mice at
  • FIG. 7D is a bar graph showing that sAPP ⁇ is increased (97 ⁇ 3.12% vs. 153.65
  • FIG. 7E is a bar graph showing that sAPP ⁇ is decreased in APP/PS 1 mice at 7-
  • FIG. 7F is a bar graph showing that no differences are observed for CTFs
  • PDE5 inhibition reverses the impairment of LTP in the CAl region of slices from 3-month-old APP/PS1 mice.
  • FIG. 8C is a graph demonstrating that IC354 (1 ⁇ M) does not reverse LTP impairment in APP/PS1 mice
  • levels of LTP IC354-treated APP/PS1 mice equal to -58% of vehicle-treated WT littermates at 120 min. after tetanus, vs. -57% in vehicle-treated APP/PS1 mice; IC354-treated APP/PS1 mice: 129.33 ⁇ 8.71 % at 120 min.
  • FIG. 9 is a bar graph that tadalafil does not ameliorate cognition in 3 -month-old
  • APP/PS1 mice Tadalafil (1 mg/Kg, i.p.) does not modify contextual fear conditioning in 3 month-old APP/PS1 mice.
  • Fear conditioning performed 24 hrs after training shows a reduction of freezing responses in APP/PS1 mice treated with vehicle compared to vehicle-treated WT littermates [freezing time in vehicle- treated APP/PS1 mice is -47% of vehicle-treated WT mice; 15.34 ⁇ 3.15% in APP/PS1, (6 males, 6 females), vs.
  • FIG. 1OA is a graph that demonstrates that four groups of mice show no difference in the time needed to find a visible platform
  • APP/PS1 mice do not show any sensory impairment at 3 months of age.
  • APP/PS1 mice do not show any /motor impairment at 7-10 months of age. These animals received daily injections of sildenafil for 3 weeks at 3 months of age.
  • FIG. 12 is a schematic showing the fused planar ring system structures in reported PDE5 inhibitors.
  • FIG. 13 are chemical structures depicting four classes of structurally related, and formally independent scaffolds (I-IV) based on structure analysis of reported PDE5 inhibitors and known Structure- Activity Relationship (SAR) data.
  • FIG. 14 is a schematic showing the synthesis of compounds comprising scaffold
  • FIG. 15 is a schematic showing the synthesis of compounds comprising scaffold
  • FIG. 16 is a schematic showing the synthesis of compounds comprising scaffold
  • FIG. 17 is a schematic showing the synthesis of compounds comprising scaffold
  • FIG. 18 is a schematic showing the synthesis of compounds comprising scaffold
  • FIG. 19 is a schematic of the NO/cGMP/CREB pathway.
  • FIG. 20 is a schematic of APP processing.
  • Administration of the PDE5 inhibitor sildenafil modifies APP process in APP/PS1 mice.
  • a decrease in sAPP ⁇ levels was detected in 3-month-old APP/PS1 mice treated with sildenafil, while an increase in CT83 and CT99 fragments was observed.
  • a persistent decrease in sAPP ⁇ levels and a persistent increase in sAPP ⁇ levels was detected at 7-10 months of age in APP/PS1 mice that were previously treated with sildenafil when 3 months old.
  • FIG. 21 is a schematic of a model depicting the action of PDE5 inhibitors on synaptic plasticity, memory, and amyloid-beta (A ⁇ ) peptide synthesis and degradation.
  • PDE5 inhibitors can increase synaptic plasticity in APP/PS1 mice; increase memory, fear conditioning and RAWM in APP/PS1 mice; increase CREB phosphorylation in APP/PS1 mice; and can decrease A ⁇ peptide levels in APP/PS1 mice.
  • FIG. 22 shows the effect of APP and PSl transgene overexpression onto active boutons in cell cultures.
  • FIG. 22A are photographs of Examples of FM 1-43 staining of active release sites before and after glutamate in WT and APP/PS1 hippocampal cultures. Scale bar, 15 ⁇ m.
  • FIG. 22B is a graph showing basal number of active boutons per unit- length-neurite was higher in cultures from Tg mice compared to WT littermates.
  • FIG. 22C is a graph demonstrating the percent increase in presynaptic active boutons 30 min after glutamate in 0 Mg++ in WT and APP/PS1 cultures. Glutamate increased active bouton number in WT but not in APP/PS1 cultures.
  • FIG. 23 represents the experimental set-up.
  • a schematic drawing of a transverse hippocampal slice is shown in the top image.
  • Schaeffer collateral fibers and CAl stratum radiatum are marked. Positions of the stimulating and recording electrodes are indicated.
  • Long-term potentiation (LTP) was induced by a theta-burst stimulation of Schaeffer collateral fibers.
  • Photograph of the interface recording chamber used for electrophysiological experiments is shown in the bottom image.
  • FIG. 24 represents a synthetic Scheme of new PDE5 inhibitors. Based on the requirement for new PDE5 inhibitors, a class of quinoline derivatives was designed. [0078] FIG. 25 depicts some synthetic scheme examples. Based on the SAR,
  • YFO 12403 cyclopropyl lead
  • YFO 16203 dimethylamino lead
  • FIG. 26 represents the IC 50 S of synthesized compounds. YFO 12403 and
  • YFO 16203 are highlighted in red.
  • FIG. 27 depicts the in vitro selectivity of PDE5 inhibitors. Two compounds,
  • YFO 12403 and YFO 16203 were picked up based on the SAR for selectivity profiling, a) Data obtained by BPS Bioscience; b) Graeme L. Card, et. al. Structure, 2004 , 12, 2233-2247; c) I Saenz de Tejada, et al., InternationalJournal of Impotence Research, 2001, 13, 282-290; d) Alain, Daugan, et. Al, Journal of Medicinal Chemistry, 2003, 46, 4533-4542.
  • FIG. 28 represents a pharmacokinetics profile.
  • One compound, YF012403, was identified based on the in vitro activity and selectivity for PK profiling as compared to sildenafil.
  • FIG. 29 represents a pharmacokinetics profile.
  • the graphs depicts a concentration/time curve of candidate YFO 12403 and sildenafil in brain tissue and plasma. The data were collected with male C57/BALB/c mice; three mice for each point.
  • FIG. 30 depicts a synthetic route for process chemistry of the dimethylamino derivative (YFO 16203).
  • FIG. 31 is a graph showing electrophysiology data.
  • YFO 12403 reverses LTP impairment in the CAl region of slices from 3-4 month-old mice treated with 200 nM oligomeric A ⁇ 42.
  • FIG. 32 depicts a synthesis scheme of Intermediate A. Dashed lines in the scheme indicate a prophetic reaction.
  • FIG. 33 depicts a synthesis scheme of Intermediate B.
  • FIG. 34 depicts a synthesis scheme of Intermediate C.
  • FIG. 35 depicts a synthesis scheme of Intermediate D.
  • FIG. 36 depicts a synthesis scheme of Formula E.
  • FIG. 37 depicts a synthesis scheme of Formula F.
  • FIG. 38 depicts the general synthesis method of scheme A.
  • FIG. 39 depicts synthesis Scheme I for compound 9a (YFO 12403; the cyclopropyl lead).
  • FIG. 40 depicts synthesis Scheme II for compound 11a (YF016203; dimethylamino lead).
  • FIG. 41 depicts synthesis Scheme III-A1 for intermediate 10a.
  • FIG. 42 are graphs that show the expression levels of PDE5 mRNA in heart, whole brain, hippocampus and cerebrum of humans.
  • the values were normalized to ⁇ -actin mRNA.
  • FIG. 42B the values shown in FIG. 42A were normalized to respective heart mRNA levels.
  • FIG. 43 shows the structures of cGMP-based molecules.
  • FIG. 44 shows the structures of ⁇ -carbolines-derived molecules.
  • FIG. 45 shows the structures of pyrazolopyridine, phthalazine and quinoline derivatives.
  • FIG. 46 shows the structures of isoquinazolinone and isoquinolinone derivatives.
  • FIG. 47 is a graph depicting PDE5 activity where 100 nM of cGMP substrate was used.
  • FIG. 49 are graphs that show the beneficial effect of YFO 12403 on A ⁇ 42- induced synaptic and cognitive dysfunction.
  • FIG. 49 A shows that YFO 12403 ameliorates the LTP deficit in A ⁇ 42-treated slices.
  • the graph represents the average of the last 5 min of recording at 60 min after the tetanus.
  • FIG. 49B shows that YFO 12403 ameliorates the contextual fear memory deficit in A ⁇ 42-infused mice.
  • FIG. 50 is a schematic showing modifications at C8 of YF012403.
  • FIG. 51 is a schematic showing modifications at C3 of YFO 12403.
  • FIG. 52 is a schematic showing modifications at C3 of YFO 12403.
  • FIG. 53 is a schematic showing modifications at C3 of YFO 12403.
  • FIG. 54 is a schematic showing modifications at other positions of YFO 12403.
  • FIG. 55 are graphs that show the acute beneficial effects of sildenafil on cognitive dysfunction of 5 month-old J20 mice during contextual fear conditioning (FC) (FIG. 55A) and RAWM (FIG. 55B) testing.
  • FIG. 56 are graphs showing that a brief perfusion of hippocampal slices with sildenafil reverses CAl-LTP impairment in 3-month-old APP/PS1 mice.
  • the graph in FIG. 56A shows that BST is similar in 3-month-old APP/PS1 animals and WT littermates.
  • FIG. 56B is a dose-response curve that shows the effect of different concentrations of sildenafil on synaptic plasticity in slices from transgenic animals.
  • 5OnM sildenafil
  • FIG. 57 are graphs that show three month old APP/PS 1 mice have normal BST associated with no changes in AMPA- and NMDA-receptor currents.
  • FIGS. 57 are graphs that show three month old APP/PS 1 mice have normal BST associated with no changes in AMPA- and NMDA-receptor currents.
  • AMPAR-mediated EPSCs were normalized to the EPSC at -90 mV.
  • NMDAR-mediated EPSCs were normalized to the NMDA response at +50 mV.
  • FIG. 57C is a comparison of AMPAR to NMDAR current ratio in the WT and APP/PS 1 pyramidal cells. The ratio was calculated by dividing the amplitude of the AMPAR current measured at -70 mV by the NMDAR current measured 50 ms after the peak at +50 mV.
  • FIG. 58 are graphs showing that sildenafil ameliorates cognitive function in 3- month-old APP/PS 1 mice.
  • FIG. 58A shows that the minimum concentration of sildenafil needed to improve contextual fear memory in APP/PS 1 mice is 3 mg/kg. A concentration of 1.5 mg/kg does not improve freezing, whereas 6 mg/kg has the same effect as 3 mg/kg [1.5 mg/kg sildenafil: (4 males, 4 females) vs.
  • FIG. 59 is a graph that shows that sildenafil does not modify cued conditioning in 3 months old mice.
  • FIG. 61 are graphs showing that tadalafil does not ameliorate cognition in 3- month-old APP/PS1 mice.
  • FIG. 61 A shows that tadalafil (1 mg/Kg, i.p.) does not modify contextual fear conditioning in 3 month-old APP/PS1 mice.
  • 61C shows that tadalafil does not improve spatial working memory in 3 month-old APP/PS 1 mice.
  • FIG. 62 shows graphs that demonstrate the minimum concentration and duration of treatment with sildenafil needed in 3-month-old APP/PS 1 mice to improve both associative and spatial memory in 6- to 8-month-old APP/PS 1 mice.
  • FIG. 63 shows graphs that demonstrate the minimum concentration and duration of treatment with sildenafil needed in 3-month-old APP/PS1 mice to improve BST and LTP as they reach 6- to 8-months of age.
  • FIG. 64 is a dose-response curve showing the effect of different concentrations of sildenafil, applied for 10 min through the bath solution, on BST and LTP in slices from 6 month old APP/PS1 animals.
  • FIG. 64 is a dose-response curve showing the effect of different concentrations of sildenafil, applied for 10 min through the bath solution, on BST and LTP in slices from 6 month old APP/PS1 animals.
  • FIG. 65 are graphs showing that sildenafil decreases A ⁇ levels in APP/PS1 mice.
  • FIG. 65B shows that daily injections of sildenafil for 3 weeks in 3-month-old APP/PS1 mice reduce A ⁇ levels in the same mice at 7-10 months of age.
  • the invention provides for a class of quino line-containing compounds which have excellent PDE5 inhibitory potency, high selectivity, reasonable pharmacokinetics and good permeability across the blood-brain-barrier (BBB). These compounds may be used to minimize the side effects for AD patients, the third most costly disease in the U.S.
  • the compounds of the invention may also be used to treat erectile dysfunction (ED), pulmonary hypertension, cardiovascular disorder, diabetes, and GI disorders.
  • the invention provides methods for identifying PDE5 inhibitors that can cause a sustained or long-term decrease in ⁇ -secretase activity or expression in a subject.
  • the invention provides methods that select for PDE5 inhibitors that can cause a decrease in ⁇ -secretase activity or expression in a subject well after administration of the PDE5 inhibitor has ended.
  • PDE5 inhibitors can be screened or selected based on their ability to cause a decrease in ⁇ -secretase activity or expression in an animal model of A ⁇ accumulation (such as APP/PS1 mice) for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more.
  • PDE5 inhibitors can first be screened or selected based on their possession of certain characteristics, such as having one or more of: an IC50 no greater than about 100 nM; a selectivity that is at least 50-fold greater for PDE5 than for other PDEs; a PDE5 inhibitory activity in vitro that has an IC 50 no greater than about 50 nM, the ability to penetrate the BBB; the ability to hydro lyze cGMP by at least about 20% (or at least about 80%); an interaction between the compound and PDE5 that comprises a second bridging ligand that is a hydroxyl group; and an interaction between the compound and PDE5 that comprises contacts with PDE5 at amino acid residues F787, L804, 1813, M816, or a combination thereof (including contacts at all four residues).
  • the candidate pool of PDE5 inhibitors to be tested in A ⁇ accumulation animal models can first be screened or selected based on "medicinal chemistry" strategies described herein (see Examples). For example, based on the structure analysis of reported PDE5 inhibitors and known SAR data (FIG. 12, four class of structurally related, but nevertheless formally independent scaffolds I-IV (see FIG. 13), are deemed as PDE5 inhibitor candidates. Compounds derived from these scaffolds can first be screened and optimized on computational models. Compounds with highest score will be synthesized and tested for potency. At this stage, the synthetic effort will be guided by the testing results of potency/selectivity.
  • PDE5 inhibitor compound does not necessarily preclude the possibility that the compound may also be able to inhibit other PDEs.
  • PDE5 inhibitor compounds display a prolonged and protective effect against synaptic dysfunction and memory loss that persists beyond the administration of the inhibitor.
  • PDE5 inhibitor compounds are desired and screened or selected for that have a prolonged inhibitory affect on ⁇ -secretase while having a prolonged enhancing effect on ⁇ -secretase.
  • methods of screening for therapeutic agents involve testing whether an agent exerts a prolonged inhibitory affect on ⁇ -secretase activity or expression and/or a prolonged stimulatory affect ⁇ -secretase activity or expression.
  • the invention is directed at identifying and using agents that interact with A ⁇ targets that lead to neuronal dysfunction.
  • the invention also provides for compounds that modulate PDE5 protein expression or activity, or that modulate activity or expression of secretases (for example, ⁇ - and ⁇ -secretase).
  • the compounds can be PDE5 inhibitors, a class of compounds that counteract the progression of neurodegenerative diseases, such as AD (Puzzo et al [12]).
  • AD therapies such as acetylcholinesterase inhibitors or NMDA antagonists
  • AD acetylcholinesterase inhibitors or NMDA antagonists
  • AD Alzheimer's disease
  • a ⁇ Alkoe, D.J. Alzheimer's disease is a synaptic failure. Science (New York, N 7298, 789-791 (2002)).
  • LTP long-term-potentiation
  • nitric oxide (NO) donors and cGMP-analogs
  • Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. JNeurosci 25, 6887-6897 (2005)).
  • NOS2 NO-synthase 2
  • APP mutated amyloid precursor protein
  • SPs are chiefly comprised of A ⁇ aggregates.
  • the major component of NFTs is the microtubule binding protein tau.
  • AD is characterized by cognitive dysfunction and begins as a synaptic disorder that involves progressively larger areas of the brain over time [I].
  • An emerging view of the processes involved in synaptic impairment shows that the subtlety and variability of the earliest amnesic symptoms, occurring in the absence of any other clinical signs of brain injury, can be due to discrete changes in the function of a single synapse, produced at least in part, by A ⁇ [5, 7, 10, H].
  • NO is a central molecule in cellular biochemical processes.
  • the gas has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity and learning and memory.
  • NO has been found to have a protective effect on A ⁇ -induced damage of the nervous system [38-40].
  • a ⁇ has been found to impair NO generation by decreasing NMDA receptor signal transduction [38], by subtracting NADPH availability to NO-synthase (NOS) [41], or by inhibiting the phosphorylation of the serine-threonine kinase Akt [42]. Moreover, i-NOS deletion enhances AD pathology in the APP mice [43]. Thus, drugs enhancing the NO-cascade have a beneficial effect against AD [44]. [00130] Despite the neuroprotective function of NO is clear and indisputable, the gas has also been viewed as a major agent of neuropathology and cell death when it is produced in high quantity.
  • PDE5 the enzyme that degrades cGMP
  • PDE5 is part of a superfamily of enzymes including 11 types/families of PDE (PDEl to PDEl 1), some of which play a critical role in memory and behavior in diverse organisms ranging from the fruit fly, Drosophila melanogaster, to humans [53].
  • PDEs are multi-domain proteins, wherein about 270 amino acids localized towards the C-terminus is highly conserved between the 11 families. This domain contains the PDEs' catalytic function. Non-homologous amino acid segments have regulatory function or confer specific binding properties.
  • PDE2, PDE5, PDE6 and PDElO contain putative GAF domains within their regulatory amino terminal portion, which have been shown to bind cGMP.
  • PDE5 a cGMP specific PDE, is found in varying concentrations in various tissues such as vascular and visceral smooth muscle, platelets, and skeletal muscle.
  • the cGMP-specific PDE is ubiquitously expressed, and can be found in several brain regions associated with cognitive function, including the hippocampus, cortex and cerebellum [17, 18].
  • PDE5 is comprised of the conserved C-terminal, zinc containing, catalytic domain, and an N-terminal regulatory domain.
  • the C -terminus of PDE5 catalyses the cleavage of cGMP, while the N terminus contains two GAF domain repeats, which each contains a cGMP-binding site (one of high affinity and the other of lower affinity).
  • PDE5 activity occurs through binding of cGMP to the high and low affinity cGMP binding sites, subsequently followed by phosphorylation, which occurs only when both sites are occupied. Inhibition of PDE5 decreases cGMP breakdown, thus allows for maintenance of cGMP levels.
  • Sildenafil for example, is a potent inhibitor of PDE5 and is the active ingredient of ViagraTM.
  • sildenafil (Viagra by Pfizer, pyrazol-[4,3-d]-pyrimidinone derivative) is reported to cross the blood brain barrier (BBB), it represents a good candidate for CNS studies. But evidence for vardenafil is indirect (Prickaerts, J., et al. Neurochem Int 45, 915- 928 (2004)), and tadalafil is unlikely to cross it. Sildenafil has an IC 50 against PDE5 of 6.0 nM and an in vivo half-life of 0.4hrs in rodents ( ⁇ 4hrs in humans) (Walker, D. K., et al.
  • NO/cGMP signaling pathway A variety of physiological processes in the nervous, cardiovascular, and immune systems are controlled by the NO/cGMP signaling pathway. For example, in smooth muscle, NO and natriuretic peptides regulate vascular tone by stimulating relaxation through cGMP. Degradation of cGMP is controlled by cyclic nucleotide PDEs, and PDE5 is the most highly expressed PDE that hydrolyzes cGMP in these cells.
  • PDE5 phosphodiesterase 5
  • 11 types of PDE some of which play a critical role in memory and behavior in diverse organisms ranging from the fruit fly, Drosophila melanogaster to humans (Davis, 1996; Barad et al, 1998; Zhang et al, 2004).
  • PDE5 phosphodiesterase 5
  • These drugs are widely used to treat erectile dysfunction and pulmonary hypertension. Thus, their side effects are known and have not precluded their use in humans.
  • PDE5 is expressed in several brain regions associated with cognitive function, such as the hippocampus, cortex and cerebellum (van Staveren, W.C., Steinbusch, H.W., Markerink-van Ittersum, M., Behrends, S. & de Vente, J. EurJNeurosci 19, 2155-2168 (2004); Van Staveren, W.C., et al. J Comp Neurol 467, 566-580 (2003)).
  • Cyclic GMP which phosphorylates the transcription factor CREB and activates cGMP dependent protein kinases (PKGs) has been implicated in the modulation of neurotransmission, LTP and memory [13-16]. Elevation of the cGMP levels through the inhibition of the cGMP-degrading enzyme phosphodiesterase-5 (PDE5), an enzyme expressed in several brain regions associated with cognitive function such as the hippocampus and cortex [17, 18], improves memory in aged rats [14] and mice [16]. Elevation of cGMP through the PDE5 inhibitor sildenafil (Viagra) also enhances selective retention and verbal recognition memory in humans [19].
  • PDE5 phosphodiesterase-5
  • the invention provides methods for identifying an agent or compound for the treatment of AD (or other A ⁇ -accumulation related conditions) that comprise selecting the agent or compound on the basis of having one or more characteristics that make the compound optimized for treating CNS diseases.
  • the characteristics can comprise: an ICsono greater than about 100 nM; a selectivity that is at least 50-fold greater for PDE5 than for other PDEs; a PDE5 inhibitory activity in vitro that has an IC50 no greater than about 50 nM, the ability to penetrate the BBB; the ability to hydrolyze cGMP by at least about 20% (or at least about 80%); an interaction between the compound and PDE5 that comprises a second bridging ligand that is a hydroxyl group; and an interaction between the compound and PDE5 that comprises contacts with PDE5 at amino acid residues F787, L804, 1813, M816, or a combination thereof.
  • the invention provides methods for identifying or designing agents or compounds for the treatment of conditions associated with A ⁇ accumulation, where computer aided-medicinal chemistry methods are used to identify and/or design agents or compounds tailored to satisfy one or more of the characteristics mentioned above and/or to suit the strengths of various bioassays described herein.
  • the invention provides for PDE5 inhibitor compounds based on four scaffold structures identified through a thorough analysis of Structure- Activity Relationship (SAR) characteristics of existing PDE5 inhibitors.
  • SAR Structure- Activity Relationship
  • the scaffold structures served and will continue to serve as leads for development of future compounds [See EXAMPLE 3].
  • Compounds based on the four scaffold structures can be screened for having one or more of the characteristics described in paragraph [0091] above, and/or for having the ability to cause a prolonged or sustained decrease in ⁇ -secretase activity or expression in an animal model of A ⁇ accumulation (such as the APP/PS1 mouse).
  • the invention provides methods for identifying compounds which can be used for treating subjects that exhibit abnormally elevated amyloid beta plaques.
  • the invention provides methods for identifying compounds which can be used for the treatment of Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy, hypertension, and erectile dysfunction.
  • the methods can comprise the identification of test compounds or agents (e.g., peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can bind to a PDE5 polypeptide molecule and/or have an inhibitory effect on the biological activity of PDE5 or its expression, and subsequently determining whether these compounds can modulate secretase activity and/or decrease A ⁇ deposits.
  • the compound is a PDE5 inhibitor.
  • modulate refers to a change in the activity or expression of a protein molecule. For example, modulation can cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of a secretase protein molecule.
  • a PDE5 inhibitor compound can be a peptide fragment of a
  • the PDE5 protein that binds to the phosphodiesterase protein.
  • the PDE5 molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NO: 1 or SEQ ID NO: 2.
  • SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the PDE5 enzyme (residues 1-875; Genbank Accession No. AAI26234):
  • SEQ ID NO: 2 is the mouse wild type amino acid sequence corresponding to the PDE5 enzyme (residues 1-865; Genbank Accession No. NP_700471):
  • Fragments include all possible amino acid lengths between and including about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids.
  • These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England).
  • the PDE5 peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared.
  • a PDE5 inhibitor compound can also be a protein, such as an antibody (monoclonal, polyclonal, humanized, and the like), or a binding fragment thereof, directed against the phosphodiesterase enzyme, PDE5.
  • An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered.
  • Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab')2, triabodies, Fc, Fab, CDRl, CDR2, CDR3, combinations of CDRs, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al, (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402).
  • Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing).
  • RNA encoding a PDE5 protein can effectively modulate the expression of the PDE5 gene from which the RNA is transcribed.
  • Inhibitors are selected from the group comprising: siRNA, interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acid, which can be RNA, DNA, or artificial nucleic acid.
  • Antisense oligonucleotides act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a PDE5 polypeptide can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al., (2006) Med. ScL Monit. ⁇ 2(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59).
  • siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell.
  • Antisense nucleotide sequences include, but are not limited to: morpho linos, 2'-O-methyl polynucleotides, DNA, RNA and the like.
  • RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA.
  • the PDE5 inhibitor compound can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.
  • these forms of nucleic acid can be single, double, triple, or quadruple stranded, (see for example Bass (2001) Nature, 411, 428 429; Elbashir et al, (2001) Nature, 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914).
  • a PDE5 inhibitor can be a small molecule that binds to a phosphodiesterase protein (for example a PDE5 protein) and disrupts its function.
  • Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized.
  • Candidate small molecules that inhibit PDE5 can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries.
  • a molecule of interest such as a PDE5 polypeptide, and the similarity of that sequence with other proteins of the same PDE family (such as PDEl, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDElO, or PDEl 1), can provide information as to the inhibitors or antagonists of the protein of interest. Identification and screening antagonists can be further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of antagonists, in addition to protein agonists.
  • the invention provides methods for screening and identifying compounds used to treat conditions associated with accumulated amyloid-beta peptide deposits, such AD.
  • the method comprises selecting a PDE5 inhibitor compound that can modulate secretase activity for at least 1 month after completion of administration of the PDE5 inhibitor compound in an animal model of amyloid-beta peptide deposit accumulation.
  • the method comprises selecting a PDE5 inhibitor compound that comprises one or both of the following features: (a) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, 1813, M816, or a combination thereof; or (b) the 2 nd bridging ligand (BL2) between the compound and a phosphodiesterase protein is OH-.
  • the method can comprise selecting a PDE5 inhibitor compound having one or more of the following features: (a) the IC 50 of the compound is no more than about 1000 nM; (b) the selectivity of the compound is at least a 50 fold greater potency towards PDE5 relative to PDEl, PDE2, PDE3, PDE4, PDE6, PDE7, PDE8, PDE9, PDElO, or PDEl 1; (c) the PDE5 inhibitory activity in vitro has an IC 50 no more than about 50 nM; (d) the compound penetrates the blood brain barrier; (e) the compound hydrolyzes cGMP by about 20% to about 80%; (f) the 2 nd bridging ligand (B L2) between the compound and a phosphodiesterase protein is OH-; or (g) the compound interacts with two or more amino acid residues of a phosphodiesterase protein, wherein the amino acid residues comprise F787, L804, 1813, M816, or a combination thereof.
  • the compound for example the PDE5 inhibitor, has an IC 50 of at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, or at least about 900 nM.
  • PDE5 inhibitory activity in vitro has an IC 50 of at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 1OnM, at least about 15nM, at least about 2OnM, at least about 25nM, at least about 3OnM, at least about 35nM, at least about 4OnM, of at least about 45nM, but no more than about 50 nM.
  • the PDE5 inhibitor compound can have a molecular mass less than about 500 Da in order to penetrate the blood brain barrier.
  • the PDE5 inhibitor compound can have a polar surface area less than about 90 A 2 and should have 8 or fewer hydrogen bonds in order to penetrate the blood brain barrier.
  • the screening and identifying of the compound can comprise in silico screening, molecular docking, in vivo screening, in vitro screening, or a combination thereof.
  • Test compounds such as PDE5 inhibitor compounds
  • PDE5 inhibitor compounds can be screened from large libraries of synthetic or natural compounds (see Wang et al., (2007) CurrMed Chem, 14(2): 133-55; Mannhold (2006) Curr Top Med Chem, 6 (10): 1031-47; and Hensen (2006) Curr Med Chem 13(4) :361-76).
  • Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds.
  • Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N. H.), and Microsource (New Milford, Conn.).
  • a rare chemical library is available from Aldrich (Milwaukee, Wis.).
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N. C), or are readily producible.
  • natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).
  • Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like.
  • Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries.
  • Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid.
  • Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts.
  • Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries, neurotransmitter libraries, and chemical libraries.
  • the libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the functional groups described herein.
  • a combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes.
  • Combinatorial libraries include a vast number of small organic compounds.
  • One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array.
  • a compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S.
  • phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. MoI. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.
  • non-peptide libraries such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91 :4708-4712), can be screened.
  • Peptoid libraries such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used.
  • Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91 :11138-11142.
  • the three dimensional geometric structure of an active site for example that of a PDE5 polypeptide can be determined by known methods in the art, such as X-ray crystallography, which can determine a complete molecular structure. Solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures can be measured with a complexed ligand, natural or artificial, which can increase the accuracy of the active site structure determined.
  • a compound that binds to a PDE5 protein can be identified via: (1) providing an electronic library of test compounds; (2) providing atomic coordinates listed in Table 1 for at least 20 amino acid residues for the active site of PDE5 (see PDB Entry No.
  • IRKP IRKP
  • the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Ca atoms, of not greater than about 2 A, in a computer readable format; (3) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the PDE5 protein; (4) performing a data processing method, wherein electronic test compounds from the library are docked onto the three dimensional model of the PDE5 protein; and determining which test compound fits into the active site of the three dimensional model of the PDE5 protein, thereby identifying which compound would bind to PDE5.
  • the method can further comprise: synthesizing or obtaining the compound determined to dock to the active site of the PDE5 protein; contacting the PDE5 protein with the compound under a condition suitable for binding; and determining whether the compound modulates PDE5 protein expression or mRNA expression, or PDE5 protein activity using a diagnostic assay.
  • ATOM 568 CB ASN A 605 49 . 681 3 . 496 65 . 145 1 . 00 28 . i C ATOM 569 CG ASN A 605 50.163 2.321 65.987 1.00 31.06 C

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Abstract

L'invention concerne un procédé de criblage de composés qui se lient à une protéine phosphodiestérase et la modulent. L'invention concerne en outre des procédés de traitement d'états associés à des accumulations de dépôts de peptide de bêta-amyloïde accumulé en administrant un composé de liaison à phosphodiestérase à un sujet.
PCT/US2009/058813 2008-12-23 2009-09-29 Inhibiteurs de la phosphodiestérase et utilisations de ces derniers WO2010074783A1 (fr)

Priority Applications (5)

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EP09835413.7A EP2379076B1 (fr) 2008-12-23 2009-09-29 Inhibiteurs de la phosphodiestérase et utilisations de ces derniers
JP2011543511A JP2012513464A (ja) 2008-12-23 2009-09-29 ホスホジエステラーゼ阻害剤及びその使用
US13/167,540 US8697875B2 (en) 2008-12-23 2011-06-23 Phosphodiesterase inhibitors and uses thereof
US14/224,702 US9422242B2 (en) 2008-12-23 2014-03-25 Phosphodiesterase inhibitors and uses thereof
US15/235,736 US9974782B2 (en) 2008-12-23 2016-08-12 Phosphodiesterase inhibitors and uses thereof

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JP2013514320A (ja) * 2009-12-16 2013-04-25 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル ７−クロロ−キノリン−４−アミン化合物、ならびにアミロイド斑の形成を含み、及び／又はａｐｐの代謝機能障害が生じる疾患の予防又は治療のためのそれらの使用
WO2013116663A1 (fr) 2012-02-01 2013-08-08 The Trustees Of Columbia University In The City Of New York Nouveaux inhibiteurs de protéases à cystéine et leurs utilisations
WO2014145485A2 (fr) 2013-03-15 2014-09-18 The Trustees Of Columbia University In The City Of New York Modulateurs de map kinase et utilisations de ceux-ci
WO2015009930A2 (fr) 2013-07-17 2015-01-22 The Trustees Of Columbia University In The City Of New York Nouveaux inhibiteurs de la phosphodiestérase et utilisations de ceux-ci
WO2020201915A3 (fr) * 2019-03-24 2020-12-17 Aribio Co., Ltd. Compositions et procédés pour la réduction de la formation de protéine bêta-amyloïde et composition à cet effet

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