WO2014015495A1 - Inhibiteurs du canal potassique médullaire externe rénal - Google Patents

Inhibiteurs du canal potassique médullaire externe rénal Download PDF

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Publication number
WO2014015495A1
WO2014015495A1 PCT/CN2012/079184 CN2012079184W WO2014015495A1 WO 2014015495 A1 WO2014015495 A1 WO 2014015495A1 CN 2012079184 W CN2012079184 W CN 2012079184W WO 2014015495 A1 WO2014015495 A1 WO 2014015495A1
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WIPO (PCT)
Prior art keywords
mmol
oxo
methyl
dihydrofuran
diazaspiro
Prior art date
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PCT/CN2012/079184
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English (en)
Inventor
Fa-Xiang Ding
Shuzhi DONG
Jessica FRIE
Xin Gu
Jinlong Jiang
Alexander Pasternak
Haifeng Tang
Zhicai Wu
Yang Yu
Takao Suzuki
Original Assignee
Merck Sharp & Dohme Corp.
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Application filed by Merck Sharp & Dohme Corp. filed Critical Merck Sharp & Dohme Corp.
Priority to PCT/CN2012/079184 priority Critical patent/WO2014015495A1/fr
Priority to ARP130102534A priority patent/AR092031A1/es
Priority to JP2015524451A priority patent/JP6166783B2/ja
Priority to MX2015001115A priority patent/MX365986B/es
Priority to US13/951,096 priority patent/US8952166B2/en
Priority to ES13745315.5T priority patent/ES2606016T3/es
Priority to BR112015001419A priority patent/BR112015001419A8/pt
Priority to PCT/US2013/052079 priority patent/WO2014018764A1/fr
Priority to KR20157001566A priority patent/KR20150036142A/ko
Priority to AU2013295722A priority patent/AU2013295722B2/en
Priority to EP13745315.5A priority patent/EP2877471B1/fr
Priority to RU2015106139A priority patent/RU2642066C2/ru
Priority to CA2876508A priority patent/CA2876508A1/fr
Priority to CN201380039265.6A priority patent/CN104540826B/zh
Priority to TW102126583A priority patent/TW201410675A/zh
Publication of WO2014015495A1 publication Critical patent/WO2014015495A1/fr
Priority to US14/569,858 priority patent/US9206198B2/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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/20—Spiro-condensed systems

Definitions

  • ROMK Renal Outer Medullary Potassium channel
  • ROMK participates in potassium recycling across the luminal membrane which is critical for the function of the Na7K + /2Cl " co-transporter, the rate-determining step for salt reuptake in this part of the nephron.
  • CCD ROMK provides a pathway for potassium secretion that is tightly coupled to sodium uptake through the amiloride- sensitive sodium channel (see Reinalter, S.C., et al, Pharmacotyping of hypokalaemic salt-losing tubular disorders, Acta Physiol Scand, 2004, 181(4): p. 513-21; and Wang, W., Renal potassium channels: recent developments, Curr Opin Nephrol Hypertens, 2004, 13(5): p. 549-55).
  • ROMK channel also referred to herein as inhibitors of ROMK or ROMK inhibitors
  • ROMK channel also referred to herein as inhibitors of ROMK or ROMK inhibitors
  • ROMK inhibitors are expected to represent novel diuretics for the treatment of hypertension and other conditions where treatment with a diuretic would be beneficial with potentially reduced liabilities (i.e., hypo- or hyperkalemia, new onset of diabetes, dyslipidemia) over the currently used clinical agents (see Lifton, R.P., A.G. Gharavi, and D.S. Geller, Molecular mechanisms of human hypertension, Cell, 2001, 104(4): p. 545-56).
  • Human genetics Ji, W., et al, Rare independent mutations in renal salt handling genes contribute to blood pressure variation, Nat Genet, 2008, 40(5): p.
  • Patent application publication number WO2010/129379 published November 1 1, 2010 having common representative Merck Sharp & Dohme Corp., (also published as
  • R5 and R 6 are independently -H, -Ci-6 alkyl, -C3-6 cycloalkyl, -CF3, -CHF2, -C3 ⁇ 4F or -CH2OH;
  • X is -H, -OH,-OCi-3alkyl, -F, oxo, NH 2 or-CH3; and
  • Xl is -H or -CH3.
  • Patent application publication number WO2012/058134 published May 3, 2012, having common representative Merck Sharp & Dohme Corp., describes ROMK inhibitors having the generic formula:
  • a and B are mono and/or bicyclic aromatic groups
  • R2 is -H, -Ci-6 alkyl, -C3.6 cycloalkyl, CF3, -CH2OH, or -CO2R, or R2 can be joined to Rl or RlOa to form a ring
  • R3 is -H, -Ci-6 alkyl, -C3-6 cycloalkyl, -OH, -F, -OCi-3 alkyl, or -CH2OH, or R3 can be joined to RlOb to form a ring.
  • R5 and R6 are independently -H, -Ci-6 alkyl or -C(0)OCi-3alkyl; and X, X 1 , Y and Y 1 are independently -H or-Ci-6alkyl; or Yl can be joined together with Z2 to form a fused ring system.
  • the compounds of Formula I of this invention are selective inhibitors of the ROMK channel and could be used for the treatment of hypertension, heart failure and other conditions where treatment with a diuretic or natriuretic would be beneficial.
  • the present invention provides compounds of Formula I
  • the compounds of Formula I are inhibitors of the ROMK (Kirl .1) channel.
  • the compounds of Formula I could be used in methods of treatment, inhibition or amelioration of one or more disease states that could benefit from inhibition of ROMK.
  • the compounds of this invention could be used in methods of treatment which comprise administering a therapeutically or prophylactically effective amount of a compound of Formula I to a patient in need of a diuretic and/or natriuretic agent. Therefore, the compounds of Formula I could be valuable pharmaceutically active compounds for the therapy, prophylaxis or both of medical conditions, including, but not limited to, cardiovascular diseases such as hypertension and heart failure, and conditions associated with excessive salt and water retention.
  • the compounds of this invention could further be used in combination with other therapeutically effective agents, including but not limited to, other drugs which are useful for the treatment of hypertension, heart failure and conditions associated withexcessive salt and water retention.
  • the invention furthermore relates to processes for preparing compounds of Formula I, and pharmaceutical compositions which comprise compounds of Formula I.
  • the present invention is directed to compounds having structural Formula I:
  • Rl is -H, -F, -OH or -OCH3;
  • n is an integer selected from zero (R3b is absent) and 1 (R3b is present);
  • n is an integer selected from 1 or 2;
  • R3 and R3b are joined together with the carbon to which they are both attached to form cyclopropyl or cyclobutyl;
  • R2 and R3a can be joined together with the carbons to which they are each attached to form (1) a phenyl ring which is fused to the pyrrolidine ring, and m is zero, or (2) a cyclopropyl ring fused to the pyrrolidine ring, and m is 1 ;
  • R5 is -H, -CI, -F, -C 1-3 alkyl, -C3-6cycloalkyl or heterocycle optionally substituted with -F, -CI or -C 1-3 alkyl;
  • R6 is -H or -C 1-3 alkyl
  • R 7 is -H or -C 1-3 alkyl optionally susbtituted with -OH, -OCH3 or 1 to 3 of -F;
  • R8 is -H, -F or -C 1-3 alkyl
  • R9 is -H, -F, -OH, -OC 1-3 alkyl, -C3 ⁇ 4OH, - H-R13 or ;
  • RlO is -H, halo, -CN, -C3-4cycloalkyl, or -Ci-3alkyl optionally substituted with 1 to 3 of-F; or R9 is -O- and is joined together with RlO to represent -CH2-CH2-O- ;
  • Rll is -H, -CH2OH, -CH2OCH3, or -Ci-3 alkyl optionally substituted with 1 to 3 of-F;
  • Rl2 is -H, -CH2OH, -CH2OCH3, or -Ci-3 alkyl optionally substituted with 1 to 3 of-F; or Rll and Rl2 are joined together to represent -CH2-CH2- , -CH2-N(CH3)-CH2- or
  • Rl3 is -H, -(CH2)o-2-C3-6cycloalkyl, -(CH2)i_2-OC3-6cycloalkyl, -(CH2)i_2-OCi-3alkyl, -(CH2)i_2-CN, -C(0)OC 1-3 alkyl, -SO2CH3 or -Ci-3alkyl optionally substituted with one three of-F; and the dashed bond (" ") represents a single, double or aromatic bond,
  • each of the variables Rl, R2, R3a R4, R5, R6, R7, R8, R9, RI O, R11, R12 a nd Rl3 and all other variables therein are as defined in Formula I, and wherein the double bond between R2 and R3 represents a non-aromatic or an aromatic double
  • Each of the above embodiments includes the pharmaceutically acceptable salts of the compounds described in the embodiment.
  • Embodiment B In a class thereof are compounds of Embodiment A, referred to as Embodiment B, wherein Rl is -H; R2 is -H at each occurrence; R3b is -H (for compounds of Formula I, II or IV); R6 is -H; R8 is -H; Rll is -H; and Rl2 is -H.
  • alkyl is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Commonly used abbreviations for alkyl groups are used throughout the specification.
  • Cycloalkyl is a cyclized alkyl ring having the indicated number of carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • Halo means -F, -CI, -Br, or -I.
  • Heterocycle is inteded to include pyridyl (all isomers), pyrazinyl, pyridazinyl or pyrimidinyl.
  • double bond refers to a covalent bond where two pairs of electrons are shared between two atoms.
  • aromaticity is likewise well- known in the art, as exemplified by benzene and phenyl which are commonly drawn as having 3 alternating double bonds, but may also be considered as having carbon-carbon bonds which are each a hybrid of a single bond and a double bond.
  • an "aromatic bond” refers to the aromatic nature of the double bond between -C(R2)- and -C(R3a). when R2 and R3a are joined together to form a phenyl ring fused to the pyrrolidinyl ring as defined in Formula I and Formula III.
  • variables depicted in a structural formula with a "floating" bond, such as R8, are permitted on any available carbon atom in the ring to which the variable is attached.
  • the present invention encompasses all stereoisomeric forms of the compounds of Formula I. Centers of asymmetry that are present in the compounds of Formula I can all independently of one another have (R) or (S) configuration.
  • the invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all ratios.
  • enantiomers are a subject of the invention in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios.
  • the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios.
  • stereoisomers can be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis.
  • customary methods for example by chromatography or crystallization
  • stereochemically uniform starting materials for the synthesis or by stereoselective synthesis optionally a
  • derivatization can be carried out before a separation of stereoisomers.
  • the separation of a mixture of stereoisomers can be carried out at an intermediate step during the synthesis of a compound of Formula I or it can be done on a final racemic product.
  • Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing a stereogenic center of known configuration.
  • compounds of this invention are capable of tautomerization, all individual tautomers as well as mixtures thereof are included in the scope of this invention.
  • the present invention includes all such isomers, as well as salts, solvates (which includes hydrates) and solvated salts of such racemates, enantiomers, diastereomers and tautomers and mixtures thereof.
  • the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
  • the present invention is meant to include all suitable isotopic variations of the compounds of Formula I.
  • different isotopic forms of hydrogen (H) include protium (lH) and deuterium (2H).
  • Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
  • Isotopically-enriched compounds within Formula I can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and/or intermediates.
  • the invention also includes the corresponding pharmaceutically acceptable salts.
  • the compounds of Formula I which contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts.
  • such salts include but are not limited to sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids.
  • Compounds of Formula I which contain one or more basic groups i.e.
  • the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). Salts can be obtained from the compounds of Formula I by customary methods which are known to the person skilled in the art, for example by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts.
  • the present invention also includes all salts of the compounds of Formula I which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
  • compounds of the present invention may exist in amorphous form and/or one or more crystalline forms, and as such all amorphous and crystalline forms and mixtures thereof of the compounds of Formula I are intended to be included within the scope of the present invention.
  • some of the compounds of the instant invention may form solvates with water (i.e., a hydrate) or common organic solvents.
  • solvates and hydrates, particularly the pharmaceutically acceptable solvates and hydrates, of the instant compounds are likewise encompassed within the scope of this invention, along with un-solvated and anhydrous forms.
  • esters can optionally be made by esterification of an available carboxylic acid group or by formation of an ester on an available hydroxy group in a compound.
  • labile amides can be made.
  • Pharmaceutically acceptable esters or amides of the compounds of this invention may be prepared to act as pro-drugs which can be hydrolyzed back to an acid (or -COO- depending on the pH of the fluid or tissue where conversion takes place) or hydroxy form particularly in vivo and as such are encompassed within the scope of this invention.
  • Examples of pharmaceutically acceptable pro-drug modifications include, but are not limited to, -Ci-6alkyl esters and -Ci-6alkyl substituted with phenyl esters.
  • the compounds within the generic structural formulas, embodiments and specific compounds described and claimed herein encompass salts, all possible stereoisomers and tautomers, physical forms (e.g., amorphous and crystalline forms), solvate and hydrate forms thereof and any combination of these forms, as well as the salts thereof, pro-drug forms thereof, and salts of pro-drug forms thereof, where such forms are possible unless specified otherwise.
  • the compounds of Formula I according to the invention are inhibitors of ROMK, and therefore could be used as diuretic and/or natriuretic agents.
  • ROMK inhibitors may be used to help to increase urination and increase urine volume and also to prevent or reduce reabsorption of sodium in the kidneys leading to increased excretion of sodium and water. Therefore, the compounds could be used for treatment or prophylaxis or both of disorders that benefit from increased excretion of water and sodium from the body. Accordingly, the compounds of this invention could be used in a method for inhibiting ROMK comprising administering a compound of Formula I in a ROMK-inhibitory effective amount to a patient in need thereof.
  • the inhibition of ROMK by the compounds of Formula I can be examined, for example, in the Thallium Flux Assay and/or Electrophysiology Assay described below.
  • this invention also relates to the use of the compounds of Formula I or salts thereof to validate in vitro assays, for example but not limited to the Thallium Flux and Electrophysiology Assays described herein.
  • the compounds of this invention could be used in a method for causing diuresis, natriuresis or both, comprising administering a compound of Formula I in a therapeutically effective amount to a patient in need thereof. Therefore, the compounds of Formual I of this invention could be used in methods for treatment of, prevention of or reduction of risk for developing medical conditions that benefit from increased excretion of water and sodium, such as but not limited to one or more of hypertension, heart failure (both acute and chronic, the latter also known as congestive heart failure) and/or other conditions associated with excessive salt and water retention.
  • the compounds of Formula I could be used in methods for treatment of, prevention of or reduction of risk for developing one or more disorders such as pulmonary arterial hypertension (PAH), cardiovascular disease, diabetes mellitus, diabetes insipidus, post-operative volume overload,, endothelial dysfunction, diastolic dysfunction, systolic dysfunction, stable and unstable angina pectoris, thromboses, restenosis, myocardial infarction, stroke, cardiac insufficiency, pulmonary hypertonia, atherosclerosis, hepatic cirrhosis, ascitis, preeclampsia, cerebral edema, nephropathy, glomerulonephritis, nephrotic syndrome, acute and chronic kidney insufficiency (also referred to as chronic kidney disease, or more generally as renal impairment), acute tubular necrosis, hypercalcemia, idiopathic edema, Dent's disease, Meniere's disease, edematous states, glaucoma, benign intra
  • the compounds of Formula I may potentially have reduced liabilities (for example, hypo- or hyperkalemia, new onset of diabetes, dyslipidemia, etc.) over currently used clinical agents. Also the compounds may have reduced risk for diuretic tolerance, which can be a problem with long-term use of loop diuretics.
  • compounds that are ROMK inhibitors can be identified as those compounds which, when tested, have an IC50 of 5 ⁇ or less, preferably 1 ⁇ or less, and more preferably
  • Electrophysiology Assay These assays are described in more detail further below.
  • the dosage amount of the compound to be administered depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, weight and individual responsiveness of the human or animal to be treated, on the efficacy and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to compounds of Formula I. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically effective or prophylactically effective dosage amount needed to prevent, counter, or arrest the progress of the condition.
  • the compound will be administered chronically on a daily basis for a length of time appropriate to treat or prevent the medical condition relevant to the patient, including a course of therapy lasting days, months, years or the life of the patient.
  • a daily dose of approximately 0.001 to 100 mg/kg, preferably 0.001 to 30 mg/kg, in particular 0.001 to 10 mg/kg (in each case mg per kg of bodyweight) is appropriate for administration to an adult weighing approximately 75 kg in order to obtain the desired results.
  • the daily dose is preferably administered in a single dose or can be divided into several, for example two, three or four individual doses, and may be, for example but not limited to, 0.1 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 20 mg, 40 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, etc., on a daily basis. In some cases, depending on the potency of the compound or the individual response, it may be necessary to deviate upwards or downwards from the given daily dose. Furthermore, the compound may be formulated for immediate or modified release such as extended or controlled release.
  • patient includes animals, preferably mammals and especially humans, who use the instant active agents for the prohylaxis or treatment of a medical condition.
  • Administering of the drug to the patient includes both self-administration and administration to the patient by another person.
  • the patient may be in need of treatment for an existing disease or medical condition, or may desire prophylactic treatment to prevent or reduce the risk for developing said disease or medical condition or developing long-term complications from a disease or medical condition.
  • therapeutically effective amount is intended to mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • a prophylactically effective amount is intended to mean that amount of a
  • prophylactic and derivatives of these terms as used herein refer to administering a compound to a patient before the onset of clinical symptoms of a condition not yet present in the patient. It is understood that a specific daily dosage amount can simultaneously be both a therapeutically effective amount, e.g., for treatment of hypertension, and a prophylactically effective amount, e.g., for prevention or reduction of risk of myocardial infarction or prevention or reduction of risk for complications related to hypertension.
  • the ROMK inhibitors may be administered via any suitable route of administration such as, for example, orally, parenterally, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes subcutaneous injections, intravenous (IV), intramuscular, intrasternal injection or infusion techniques.
  • Oral formulations are preferred for treatment of chronic indications such as hypertension or chronic heart failure, particularly solid oral dosage units such as pills, tablets or capsules, and more particularly tablets. IV dosing is preferred for acute treatment, for example for the treatment of acute heart failure.
  • compositions comprised of a compound of Formula I and a pharmaceutically acceptable carrier which is comprised of one or more excipients or additives.
  • An excipient or additive is an inert substance used to formulate the active drug ingredient.
  • the pharmaceutical compositions of this invention containing the active ingredient may be in forms such as pills, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • the excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, mannitol, calcium phosphate or sodium phosphate;
  • granulating and disintegrating agents for example, corn starch, or alginic acid
  • binding agents for example starch, gelatin or acacia
  • lubricating agents for example, magnesium stearate, stearic acid or talc.
  • compositions may also contain other customary additives, for example but not limited to, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • customary additives for example but not limited to, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
  • Oral immediate-release and time-controlled release dosage forms may be employed, as well as enterically coated oral dosage forms. Tablets may be uncoated or they may be coated by known techniques for aesthetic purposes, to mask taste or for other reasons. Coatings can also be used to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or miscible solvents such as propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or miscible solvents such as propylene glycol, PEGs and ethanol
  • an oil medium for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose.
  • therapeutically effective amount of a compound of this invention can be used for the preparation of a medicament useful for inhibiting ROMK, for causing diuresis and/or natriuresis, and/or for treating, preventing or reducing the risk for any of the medical conditions described herein, in dosage amounts described herein.
  • the amount of active compound of Formula I and/or its pharmaceutically acceptable salts in the pharmaceutical composition may be, for example but not limited to, from from 0.1 mg to 1 g, particularly 0.1 mg to 200 mg, more particularly from 0.1 mg to 100 mg, and even more particularly from 0.1 to 50 mg, per dose on a free acid/free base weight basis, but depending on the type of the pharmaceutical composition, potency of the active ingredient and/or the medical condition being treated, it could also be lower or higher.
  • Pharmaceutical compositions usually comprise 0.5 to 90 percent by weight of the active compound on a free acid/free base weight basis.
  • the compounds of Formula I inhibit ROMK.
  • They can also be employed as a scientific tool or as aid for biochemical investigations in which such an effect on ROMK is intended, and also for diagnostic purposes, for example in the in vitro diagnosis of cell samples or tissue samples.
  • the compounds of Formula I can also be employed as intermediates for the preparation of other pharmaceutically active compounds.
  • One or more additional pharmacologically active agents may be administered in
  • the additional active agent is intended to mean a a compound that is different from the compound of Formula I, and which is a
  • any suitable additional active agent or agents including but not limited to anti-hypertensive agents, additional diuretics, anti- atherosclerotic agents such as a lipid modifying compound, anti-diabetic agents and/or anti- obesity agents may be used in any combination with the compound of Formula I in a single dosage formulation (a fixed dose drug combination), or may be administered to the patient in one or more separate dosage formulations which allows for concurrent or sequential administration of the active agents (co-administration of the separate active agents).
  • angiotensin converting enzyme inhibitors e.g, alacepril, benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril); angiotensin II receptor antagonists also known as angiotensin receptor blockers or ARBs (e.g., losartan i.e., COZAAR®, valsartan, candesartan, olmesartan, telmesartan, eprosartan, irbesartan, azilsartan and any of these drugs used in combination with thiazide like diure
  • angiotensin II receptor antagonists also known as
  • potassium sparing diuretics such as amiloride HC1, spironolactone, epleranone, triamterene, each with or without HCTZ; carbonic anhydrase inhibitors, such as acetazolamide; neutral
  • endopeptidase inhibitors e.g., thiorphan and phosphoramidon
  • aldosterone antagonists e.g., thiorphan and phosphoramidon
  • aldosterone synthase inhibitors renin inhibitors (e.g. urea derivatives of di- and tri-peptides (See U.S. Pat. No. 5, 116,835), amino acids and derivatives (U.S. Patents 5,095, 119 and 5, 104,869), amino acid chains linked by non-peptidic bonds (U.S. Patent 5, 114,937), di- and tri-peptide derivatives (U.S. Patent 5, 106,835), peptidyl amino diols (U.S. Patents 5,063,208 and 4,845,079) and peptidyl beta-aminoacyl aminodiol carbamates (U.S.
  • renin inhibitors e.g. urea derivatives of di- and tri-peptides (See U.S. Pat. No. 5, 116,835), amino acids and derivatives (U.S. Patents 5,095, 119 and 5, 104,869), amino acid chains linked by non-peptidic bonds (U.
  • Patent 5,089,471) also, a variety of other peptide analogs as disclosed in the following U.S. Patents 5,071,837; 5,064,965; 5,063,207; 5,036,054; 5,036,053; 5,034,512 and 4,894,437, and small molecule renin inhibitors (including diol sulfonamides and sulfinyls (U.S. Patent 5,098,924), N-morpholino derivatives (U.S. Patent 5,055,466), N-heterocyclic alcohols (U.S. Patent 4,885,292) and pyrolimidazolones (U.S. Patent 5,075,451); also, pepstatin derivatives (U.S.
  • Patent 4,980,283 and fluoro- and chloro-derivatives of statone-containing peptides U.S. Patent 5,066,643; enalkrein; RO 42-5892; A 65317; CP 80794; ES 1005; ES 8891; SQ 34017; aliskiren (2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2- methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)-phenyl]- octanamid hemifumarate) SPP600, SPP630 and SPP635); endothelin receptor antagonists;
  • vasodilators e.g. nitroprusside
  • calcium channel blockers e.g., amlodipine, nifedipine, verapamil, diltiazem, , felodipine, gallopamil, niludipine, nimodipine, nicardipine
  • potassium channel activators e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam
  • sympatholitics e.g., acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, metoprolol, metoprolol tartate, nadolol, propranolol, sotalol, timolol
  • alpha adrenergic blocking drugs e.g., doxazocin, prazocin or alpha methyldopa
  • central alpha adrenergic agonists e.g. doxazocin, prazocin or alpha methyldopa
  • peripheral vasodilators e.g. hydralazine
  • nitrates or nitric oxide donating compounds e.g.
  • lipid lowering agents e.g., HMG-CoA reductase inhibitors such as simvastatin and lovastatin which are marketed as ZOCOR® and MEVACOR® in lactone prodrug form and function as inhibitors after administration, and pharmaceutically acceptable salts of dihydroxy open ring acid HMG-CoA reductase inhibitors such as atorvastatin (particularly the calcium salt sold in LIPITOR®), rosuvastatin (particularly the calcium salt sold in CRESTOR®), pravastatin (particularly the sodium salt sold in PRAVACHOL®), and fluvastatin (particularly the sodium salt sold in LESCOL®); a cholesterol absorption inhibitor such as ezetimibe (ZETIA®), and ezetimibe in combination with any other lipid lowering agents such as the HMG-CoA reductase inhibitors noted above and particularly with simvastatin (VYTORIN®) or with atorvastatin calcium; niacin in
  • medications such as exenatide and pramlintide acetate; or with other drugs beneficial for the prevention or the treatment of the above-mentioned diseases including but not limited to diazoxide; and including the free-acid, free-base, and pharmaceutically acceptable salt forms of the above active agents where chemically possible.
  • reaction can be carried out with conventional heating, or by heating using a microwave apparatus.
  • solvents can be used in this reaction, for example, ethanol and 2-propanol.
  • Spirocyclic amines may be free bases, or they may be salts, in which case a base such as triethylamine or N;N-diisopropylethylamine may be added.
  • Alhehydes or ketones 3 may be used in reductive alkylation reactions of spirocyclic amines 2 to afford ROMK inhibitors of the formula IB by using various reductive amination conditions (for example using sodium cyanoborohydride, sodium triacetoxy borohydride, or titanium tetra- isopropoxide, followed by sodium borohydride or sodium cyanoborohydride).
  • the epoxides 1 (and single enatiomers (R)-l and (S)-l) can be prepared following the method detailed in Scheme 3.
  • Treatment of 4 (where X is chloride, bromide, iodide, or trifluoromethane sulfonate) with commercially available potassium vinyl trifluoroborate (Molander, G.; Luciana, A. Journal of Organic Chemistry, 2005, 70(10), 3950-3956) under palladium catalyzed coupling conditions with an appropriate phosphine ligand gives rise to styrene 5
  • styrenes 5 can be converted to the corresponding epoxides 1 under various epoxidation conditions, for example, with m-CPBA (Fringuelli, F. et al. Organic Preparations and Procedures International, 1989, 21(6), 757-761).
  • m-CPBA Frringuelli, F. et al. Organic Preparations and Procedures International, 1989, 21(6), 757-761).
  • the racemic epoxide 1 can be resolved under chiral HPLC chromatography conditions to afford its enantiomers, which can be used in place of 1 according to Scheme 1.
  • enantiopure epoxides (R)-l or (S)-l can be prepared as shown in Scheme 4.
  • Treatment of 4 (where X is bromide, iodide, or trifluoromethane sulfonate) with commercial available vinyl butylether 6 under palladium catalyzed conditions with a suitable ligand (for example Pd(OAc) 2 , DPPP) can provide the enol ethers 7.
  • Enol ethers may be prepared using other methods known to the chemist. Treatment of the resulting enol ethers 7 with BS or other similar reagents affords the corresponding bromomethyl ketones 8.
  • Aldehydes 3A may be prepared in numerous ways, with two approaches described in Scheme 5. Treatment of 4 (where X is bromide, iodide, or trifluoromethane sulfonate) with bromo(l,3-dioxolan-2-ylmethyl)zinc in the presence of an appropriate palladium catalyst and ligand, such as palladium(II) acetate and tri-t-butylphosphine-BF4 complex, provides the corresponding aryl l,3-dioxolan-2-ylmethyl derivative 9 . Then the aldehydes 3 A may be obtained by treatment with HCl in the presence of water and an organic solvent. Alternatively, reaction of 4 (where X is bromide, iodide, or trifluoromethane sulfonate) with
  • allyltributylstannane in the presence of palladium catalyst affords the allyl product 10.
  • Spirocyclic aminofuranones 2 can be prepared as described in Scheme 6.
  • Spirocyclic diamines or amino lactams (where R 11 and R 12 together represent a carbonyl group) 11, protected as appropriate (Greene, T.; Wuts, P. G. M. protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York, NY 1991)
  • furanone triflates or bromides 12 using a palladium catalyst and ligand, for example palladium acetate and 4,5-Bis(diphenylphosphino)-9,9- dimethylxanthene.
  • spirocyclic diamines or amino lactams 11 described herein are commercially available; others can prepared as described in the experimental section below.
  • 4- Bromofuran-2(5H)-one is commercially available; other furanones 12 can be prepared as described in the examples below.
  • Intermediates 13 are converted to spirocyclic aminofuranones 2 by removal of the protective group, for example, tert-butoxycarbonyl can be removed with TFA or HC1.
  • Spirocyclic amino lactams 11 A can be prepared in numerous ways, including those described in Scheme 7.
  • Commercially available aminoesters 14 can be alkylated with
  • the subject compounds may be prepared by modification of the procedures disclosed in the Examples as appropriate. Starting materials are commercially available or made by known procedures or as illustrated. The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.
  • reactions sensitive to moisture or air were performed under nitrogen or argon using anhydrous solvents and reagents.
  • the progress of reactions was determined by either analytical thin layer chromatography (TLC) usually performed with E. Merck pre-coated TLC plates, silica gel 60F-254, layer thickness 0.25 mm or liquid chromatography-mass spectrometry (LC-MS).
  • TLC analytical thin layer chromatography
  • LC-MS liquid chromatography-mass spectrometry
  • the analytical LC-MS system used consisted of a Waters ZQ platform with electrospray ionization in positive ion detection mode with an Agilent 1100 series HPLC with autosampler.
  • the column was usually a Water Xterra MS C18, 3.0 x 50 mm, 5 ⁇ .
  • the flow rate was 1 mL/min, and the injection volume was 10 /L.
  • UV detection was in the range 210-400 nm.
  • the mobile phase consisted of solvent A (water plus 0.06% TFA) and solvent B (acetonitrile plus 0.05% TFA) with a gradient of 100% solvent A for 0.7 min changing to 100% solvent B over 3.75 min, maintained for 1.1 min, then reverting to 100% solvent A over 0.2 min.
  • Preparative FIPLC purifications were usually performed using a mass spectrometry directed system. Usually they were performed on a Waters Chromatography Workstation configured with LC-MS System Consisting of: Waters ZQ single quad MS system with Electrospray Ionization, Waters 2525 Gradient Pump, Waters 2767 Injector / Collector, Waters 996 PDA Detector, the MS Conditions of: 150-750 amu, Positive Electrospray, Collection Triggered by MS, and a Waters Sunfire C-18 5 micron, 30 mm (id) x 100 mm column.
  • the mobile phases consisted of mixtures of acetonitrile (10-100%) in water containing 0.1%TFA. Flow rates were maintained at 50 mL/min, the injection volume was 1800 /L, and the UV detection range was 210-400 nm. Mobile phase gradients were optimized for the individual compounds.
  • chromatographic conditions such as the column used, the condition of the column, and the solvent system and instruments used.
  • EtOAc ethyl acetate
  • Cbz benzyloxycarbonyl
  • dibenzylideneacetone dba
  • 11- chloroethylchloroformate ACE-Cl
  • Ph phenyl
  • DCM dichloromethane
  • SM starting material
  • ether or Et 2 0 trifluoroacetic acid
  • TAA triethylamine
  • TAA 1,8- Diazabicyclo[5.4.0]undec-7-ene
  • DBU N,N-diisopropylethylamine
  • DIEA Hunig's base, DIPEA
  • DMS dimethylsulfide
  • DMF 4-dimethylaminopyridine
  • DMA dimethylacetamide
  • DM AC 3- chloroperoxybenzoic acid
  • mCPBA nicotinamide adenine dinucleotide phosphate
  • PE petroleum ether
  • LAH lithium aluminum hydride
  • DIP A di-isopropylamine
  • Pd(dppf)Cl2 or PdCl2(dppf) is l, l '-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) which may be complexed with CH2CI2, hexamethylphosphoramide (HMPA), isopropyl acetate (IP Ac); N-methylmorpholine-N-oxide (NMO); tetrakis(triphenylphosphine)palladium (Pd(PPh 3 ) 4 );
  • tetrafluoroborate (XtalFluor-E); 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos); N,N,A ⁇ -Tetramethylethylenediamine (TMEDA); round-bottom flask (RB or RBF), aqueous (aq); saturated aqueous (sat'd), saturated aqueous sodium chloride solution (brine); medium pressure liquid chromatography (MPLC), high pressure liquid chromatography (HPLC), flash chromatography (FC); liquid chromatography (LC), supercritical fluid chromatography (SFC); thin layer chromatography (TLC), mass spectrum (ms or MS); liquid chromatography-mass spectrometry (LC-MS or LC/MS), column volume (CV), room temperature (rt, r.t.
  • MPLC medium pressure liquid chromatography
  • HPLC high pressure liquid chromatography
  • FC flash chromatography
  • TLC thin layer chromatography
  • MS liquid chromat
  • Celite is a trademark name for diatomaceous earth
  • Solka Floe is a trademark name for powdered cellulose.
  • X or x may be used to express the number of times an action was repeated (e.g., washed with 2 x 200 mL IN HC1), or to convey a dimension (e.g., the dimension of a column is 30 x 250mm).
  • Step A 5-(l -Dioxolan-2-ylmethyl)-2-benzofuran-l(3H)-one: A three-neck 5L round bottomed flask equipped with a stir bar, firestone valve, thermocouple, condenser and heating mantle was charged with tri-t-butyl phosphonium tetrafluoroborate (500 mg, 1.72 mmol), palladium (II) acetate (250 mg, 1.1 mmol) and 5-bromo-2-benzofuran-l(3H)-one (100 g, 470 mmol). DMF (1.88 L) was added to the flask, and the mixture was degassed three times by alternating vacuum and nitrogen purge.
  • tri-t-butyl phosphonium tetrafluoroborate 500 mg, 1.72 mmol
  • palladium (II) acetate 250 mg, 1.1 mmol
  • bromo(l,3-dioxolan-2-ylmethyl)zinc solution (1.03 L, 516 mmol) was added via canula and the mixture was again degassed three times. The mixture was then heated at 85 °C for 5 h. Analysis by HPLC-MS indicated the reaction was not complete. The mixture was stirred at 85 °C for 5 more h. The mixture was then allowed to return to room temperature for overnight. 2-methylTHF (2L) and brine were added, and the mixture was stirred for 5 min. The layers were separated and the aqueous layer was extracted again with 2- methylTHF. The organic layers were combined, washed three times with brine (4L each), dried over MgS0 4 , filtered, and concentrated.
  • Step B (l-Oxo-1.3-dihydro-2-benzofuran-5-yl)acetaldehyde: 5-(l,3-Dioxolan-2-ylmethyl)-2- benzofuran-l(3H)-one (61 g, 280 mmol) was combined with water (2.2 L) in a 5 L round bottomed flask equipped with a Claisen adapter, thermocouple, stir bar and nitrogen bubbler. Aqueous HCl solution (2M, 1.14 L, 2.29 mol) was added and the resulting mixture was heated at 40 °C for 8 h. Then the mixture was stirred overnight at room temperature. The mixture was extracted three times with 2 L of ethyl acetate.
  • Step B 5 -bromo-4-methyl-2-benzofuran- 1 (3H)-one : To a flask charged with (3-bromo-2- methylphenyl)methanol (6.0 g, 30 mmol) was added a 1M TFA solution of Thallium
  • Trifluoroacetate (16.2 g, 29.8 mmol). The mixture was stirred at RT overnight. Analysis by TLC showed no starting material remaining. The solvent was removed under vacuum, and the residue was pumped under high vacuum for 30 min to ensure complete removal of TFA. To the residue was then added Palladium(II) Chloride (529 mg, 2.98 mmol), Lithium Chloride (2.53 g, 59.7 mmol), Magnesium Oxide (2.41 g, 59.7 mmol), and MeOH (150 mL). The reaction was flushed with CO twice, and kept under CO at room temperature. Analysis by LC showed a big product spot within 2 hours. To this solution was added ethyl acetate to precipitate the salts.
  • Step A 4-Methyl-5-prop-2-en-l-yl-2-benzofuran-l(3H)-one: To a flask charged with 5-bromo- 4-methyl-2-benzofuran-l(3H)-one (320 mg, 1.409 mmol) and a stir bar was added allyl tri-n- butyltin (0.655 mL, 2.11 mmol), Pd(PPh 3 ) 4 (244 mg, 0.211 mmol), lithium chloride (179 mg, 4.23 mmol), and toluene (15 mL). The reaction was purged with nitrogen 2 times then was heated at reflux for 4 hours. The product was separated by silica gel chromatography to give 4-methyl-5- prop-2-en- 1 -yl-2-benzofuran- 1 (3H)-one.
  • Step B (4-Methyl- 1 -oxo- 1.3-dihydro-2-benzofuran-5-yl)acetaldehyde: A solution of the above olefin (220 mg, 1.2 mmol) in MeOH (20 mL) was cooled to -78 °C. To this solution was bubbled ozone until the reaction turned blue. Nitrogen was bubbled through the reaction to drive off excess ozone, followed by addition of DMS (0.870 mL, 11.7 mmol). The reaction was allowed to warm up to RT. The crude product was purified by flash chromatography to afford the title compound.
  • Step A 5-ethenyl-4-methyl-2-benzofuran-l(3H)-one: 5-Bromo-4-methyl-2-benzofuran- 1 (3H)- one (598 mg, 4.47 mmol), potassium vinyl trifluoroborate (507 mg, 2.23 mmmol), PdCl 2 (dppf)- CH 2 Cl 2 Adduct (182 mg, 0.223 mmmol) , and TEA (0.622 mL, 4.47 mmol) were added to 10 mL ethanol in a 20 mL microwave tube. The tube was sealed and degassed, then heated to 140 °C for 20 min. Analysis by LC-MS showed product peak.
  • the reaction mixture was diluted with ethyl acetate, washed with brine twice, dried and evaporated to dryness.
  • the crude product was purified by MPLC chromatography using a 120g Redi-sep column and 0-80% ETOAC/Hexane solvent system to yield 5-ethenyl-4-methyl-2-benzofuran-l(3H)-one.
  • Step B 4-methyl-5-oxiran-2-yl-2-benzofuran-l(3H)-one: 5-ethenyl-4-methyl-2-benzofuran- l(3H)-one (1.46 g, 8.38 mmol) was added to DCM (25 mL) at 0 °C then mCPBA (2.89 g, 16.8 mmol) was added and the mixture was stirred at RT overnight. The reaction mixture was washed once each with saturated aqueous Na 2 S 2 0 3 , NaHC0 3 , and brine. The organic layer was dried over Na 2 S0 4 , filtered, and evaporated to dryness. The crude material was purified by MPLC
  • Racemic 4-methyl-5-oxiran-2-yl-2-benzofuran-l(3H)-one was resolved on a ChiralPak® AD-H column (5x25cm)under supercritical fluid chromatography (SFC) conditions on a Berger MGIII preparative SFC instrument.
  • the racemate was diluted to 50 mg/mL in 1 : 1 DCM:MeOH.
  • the separation was accomplished using 10% EtOH/C02, flow rate 200 mL/min, 100 bar, 25 °C. 500ul Injections were spaced every 2.12 mins.
  • the resolution could also be achieved using a mobile phase of 8%MeOH / 98% C0 2 with a flow rate of lOOmL/min.
  • the sample was prepared by dissolving in methanol, 20mg/mL, and using a 1 mL volume per injection. After separation, the fractions were dried off via rotary evaporator at bath temperature 40°C.
  • Step A 3-hydroxymethyl-2-methyl phenol: To a 5L 3 neck RB equipped with overhead stirrer was charged NaBH4 (87.0 g, 2.30 mol) and THF (3.0 L) and the resulting slurry was cooled to 10 °C. To the slurry was then added 3-hydroxy-2-methyl benzoic acid (175 g, 1.15 mol) portionwise over 20 min (Tmax 17 °C). A stirrable slurry formed, and was aged for an additional 45 min at 10-15 °C after which BF 3 -OEt 2 (321 mL, 2.53 mol) was added slowly over 1.5 hours. The slurry was aged at 10°C-15°C for 2 h then assayed for reaction completion (98.5 % conversion).
  • the slurry was cooled to ⁇ 10 °C and quenched with 931 mL MeOH slowly over 1.5 h (gas evolution). The resulting slurry was aged overnight at RT. The batch was cooled to ⁇ 10 °C then quenched with 1 N HC1 (1.5 L) to get a homogeneous solution (pH solution ⁇ 1), which was aged for 30 min and then the organic solvents were removed by rotary evaporation to approximately 1.8 L of total reaction volume (bath temperature was set to 50 °C; internal temp of concentrate after rotary evaporation was ⁇ 40 °C). The slurry was held at 45 °C for 30 min then cooled slowly to 15 °C.
  • Step B 4-Bromo-3-hydroxymethyl-2-methyl phenol: 3-Hydroxymethyl-2-methyl phenol (113.9 g, 824.0 mmol) was dissolved in a mixture of acetonitrile (850 mL) and trifluoroacetic acid (750.0 mL, 9,735 mmol) in a 3 -neck 5-L flask under nitrogen. The reaction mixture was cooled to -33 °C. N-bromosuccinimide (141 g, 791 mmol) was added over 15 minutes, with the temperature during addition in the range of -35 to -33 °C. The reaction mixture was allowed to stir for an additional 15 min during which time the temperature decreased to -40 °C.
  • the cooling bath was removed, and potassium carbonate (741.0 g, 5,358 mmol) diluted with water to a total of 1.0 L was added. Off-gassing was observed, and the temperature increased to 25 °C. MTBE (1.5 L) was added, and the reaction mixture was transferred to a separatory funnel. The layers were separated. The aqueous layer was diluted with water (500 mL) and extracted with MTBE (1 L) + EtOAc (500 mL), and then MTBE (500 mL) + EtOAc (250 mL). The combined organic layers were washed with water (240 mL) and dried over sodium sulfate.
  • potassium carbonate 741.0 g, 5,358 mmol
  • the sodium sulfate was removed by filtration, washed with additional MTBE and concentrated under reduced pressure.
  • MTBE (684 mL, 2 volumes) was added, and the suspension was heated to 40 °C to produce a homogeneous solution. The solution was allowed to cool to room temperature.
  • Six volumes of heptane were added, and the supension was stirred overnight.
  • the suspension was filtered, and the crystals were washed with 4: 1 heptane: MTBE (500 mL), followed by heptane (500 mL).
  • the solid was dried under vacuum, providing 4-bromo-3-hydroxymethyl-2-methyl phenol.
  • Step C 5-Hydroxy-4-methyl-3H-isobenzofuran-l-one: To a 2 L 3 neck flask equipped with overhead stirrer, N2 inlet, and condenser were charged 4-bromo-3-hydroxymethyl-2-methyl phenol (100 g, 461 mmol), CuCN (83.0 g, 921 mmol), and DMF (500 mL). The solution was sparged with N 2 for 15 min then heated to 145 °C to obtain a homogeneous solution. The solution was aged at 145 °C for 2h, then the reaction mixture was cooled to 95 °C. 41.5 mL water was added (sparged with N 2 ), and the reaction aged for 20 h.
  • the reaction was cooled to RT then the solids filtered through solka flok and the cake washed with 50 mL DMF.
  • To a 3 L flask containing 1 L EtOAc was added the DMF filtrate. A precipitate coating formed in bottom of flask.
  • the DMF/EtOAc suspension was filtered through solka flok and the cake was washed with 250 mL EtOAc.
  • the resulting filtrate was washed with 5 % brine solution (3x500 mL).
  • the aqueous layers were extracted with 500 mL EtOAc and the combined organics were dried over MgS04, fitered and evaporated.
  • Step D 4-methyl-l-oxo-1.3-dihydroisobenzofuran-5-yl trifluoromethanesulfonate
  • the biphasic mixture was filtered over Solka floe, washing with additional dichloromethane, and transferred to a separatory funnel, whereupon it was diluted with additional water (300 mL). The layers were separated, and the organic layer was washed with water (500 mL) and 10% brine (200 mL).
  • the dichloromethane solution was dried over sodium sulfate, filtered and evaporated. The solid was adsorbed onto silica gel (27.5 g) and eluted through a pad of silica gel (271 g) with 25% ethyl acetate/hexanes. The resulting solution was concentrated under vacuum with the product crystallizing during concentration.
  • Step E 5-(l-Butoxy-vinyl)-4-methyl-3H-isobenzofuran-l-one: To a 1 L 3 -neck was charged trifluoromethanesulfonic acid 4-methyl-l-oxo-l,3-dihydro-isobenzofuran-5-yl ester (63.0 g, 213 mmol), DMF (315 mL), butyl vinyl ether (138 mL, 1063 mmol) )then Et 3 N (35.6 mL, 255 mmol). The solution was sparged with N 2 for 20 min.
  • KRED MIF-20 (available from Codexis, Inc., 200 Penobscot Drive, Redwood City, CA 94063, www.codexis.com. tel. 1- 650-421-8100) was added to the erlenmeyer flask and the mixture was swirled to dissolve the solids. The resulting solution was added to the 5 L round bottom, which was then heated to 28 °C and aged for 6 hours, at which point the reaction was cooled to RT and triethylamine (50.2 mL, 360 mmol) was added. The resulting solution was aged at 40 °C for 1 h. The light slurry solution was cooled to RT, after which 122 g NaCl was added.
  • Step A (R)-5-(2 -Hydroxy- 1 -methoxyethyl)-4-methylisobenzofuran- 1 (3H)-one: To a solution of ( ⁇ -4-methyl-5-(oxiran-2-yl)isobenzofuran-l(3H)-one (2.00 g, 10.5 mmol) in MeOH (20 mL) was added p-toluenesulfonic acid monohydrate (0.100 g, 0.526 mmol). After heating at 80°C for 48 h, the reaction mixture was cooled to RT and then concentrated. The crude product was purified by column chromatography eluting with 0- 45% EtOAc/Hexane.
  • Step A ( ⁇ -4-Methyl-5-(prop-l-en-l-yl)isobenzofiiTan-l(3H)-one: To Pd(dppf)Cl 2 (0.220 g, 0.338 mmol), K 3 P0 4 (6.75 mL, 1 M in water, 6.75 mmol) in THF (22 mL) was added potassium ( J E)-trifluoro(prop-l-en-l-yl)borate (0.749 g, 5.06 mmol) and 4-methyl-l-oxo-l,3- dihydroisobenzofuran-5-yl trifluoromethanesulfonate (INTERMEDIATE 4B, Method 2, step D, 1.0 g, 3.38 mmol) .
  • Step B 5-(1.2-Dihydroxypropyl)-4-methylisobenzofuran-l(3H)-one: To (£)-4-methyl-5-(prop-l- en-l-yl)isobenzofuran-l(3H)-one (300 mg, 1.59 mmol) in acetonitrile/water (10/1, 18 mL) was added NMO (243 mg, 2.07 mmol) and potassium osmate(VI) dihydrate (29.4 mg, 0.080 mmol) at 0 °C. The reaction mixture was allowed to warm to rt and stirred at rt for 2 h. TLC showed the reaction completed. The reaction mixture was filtered through a pad of silica gel, rinsed with 10% MeOH/DCM. The crude product was purified with column chromatography (0-10%
  • Step C 5-(2-hydroxyethyl)-6-iodo-2-benzofuran-l(3H)-one: To a cooled (0 °C) solution of 5- (2-hydroxyethyl)-2-benzofuran-l(3H)-one (9.00 g, 50.6 mmol) in 100 mL of TfOH was added NIS (12.5 g, 55.6 mmol), then the mixture was stirred at 0 °C for 2 hrs and then poured into ice- water (500 mL). The solution was extracted three times with 500 mL of EtOAc and the combined organic layers were washed with saturated NaHC0 3 and brine, dried over anhydrous sodium sulfate, filtered and concentrated.
  • Step D 5-(2-hydroxyethyl)-6-methyl-2-benzofuran-l(3H)-one: To a flask charged with 5-(2- hydroxyethyl)-6-iodo-2-benzofuran-l(3H)-one (6.00 g, 19.7 mmol) and a stir bar was added Pd 2 (dba) 3 (452 mg,0.493 mmol), PPh 3 (1 g, 4 mmol) and MP (50 mL). The mixture was purged with N 2 and heated to 50 °C for 10 min, followed by addition of Cul (375 mg, 1.97 mmol).
  • Step E 2-(6-methyl-l-oxo-l .3-dihydro-2-benzofuran-5-yl)ethyl methanesulfonate: To a solution of 5-(2-hydroxyethyl)-6-methyl-2-benzofuran-l(3H)-one (1.20 g, 6.25 mmol) and TEA (2.5 g, 25 mmol) in DCM (100 mL) was added MsCl (1.40 g, 12.5 mmol) at 0 °C. The mixture was stirred at ambient temperature overnight, then was washed with water and brine. The organic layer was dried and concentrated to dryness. The collected title compound was used for the next step without any purification.
  • Step F 5-ethenyl-6-methyl-2-benzofuran-l(3H)-one: To a mixture of 2-(6-methyl-l-oxo-l,3- dihydro-2-benzofuran-5-yl)ethyl methanesulfonate (2.00 g, 7.41 mmol) and TEA (5 mL) in DCM (50 mL) was added DBU (5 mL) slowly at 0 °C. The mixture was stirred at r.t. overnight, and then was diluted with 50 mL of DCM, washed with 2 N HC1 in three times and brine. The organic layer was dried and concentrated to dryness. The residue was purified by prep-TLC to give 5-ethenyl-6-methyl-2-benzofuran- 1 (3H)-one.
  • Step G 6-methyl-5-oxiran-2-yl-2-benzofuran-l(3H)-one: To a solution of 5-ethenyl-6-methyl-2- benzofuran-l(3H)-one (1.00 g, 5.75 mmol) in 50 mL of DCM was slowly added Mcpba (3.50 g, 17.4 mmol) in 50 mL of DCM at 0 °C. The mixture was warmed to room temperature, and stirred for 2 days. The mixture was washed with aqueous Na 2 S0 3 until KI indicator paper didn't change color. The organic layer was washed with brine and then concentrated. The residue was purified via silica column to give title compound. _LC-MS M+l (calc. 191, found 191).
  • Step A 5-bromo-4-iodo-2-benzofuran-l(3H]-one: To a solution of 5-bromo-2-benzofuran- l(3H)-one (5.00 g, 23.5 mmol) at 0 °C in TfOH (100 mL) was added NIS (5.55 g, 24.6 mmol).
  • reaction mixture was then poured slowly into ice-water (1 L) with stirring. To the solution was then added EtOAc (500 mL) and subsequently stirred for 10 min. The mixture was filtered and the organic layer separated. The aqueous layer was
  • Step B 5 -bromo-4-prop-2-en- 1 -yl-2-benzofuran- 1 (3H)-one : A mixture of 5-bromo-4-iodo-2- benzofuran-l(3H]-one (2.42 g, 7.13 mmol), allyltributyltin (2.36 g, 7.13 mmol), LiCl (1.50 g,
  • Step C 5-bromo-4-(2-hydroxyethyl)-2-benzofuran-l(3H)-one: To a solution of 5-bromo-4- prop-2-en-l-yl-2-benzofuran-l(3H)-one (1.27 g, 5.02 mmol) in MeOH (50 mL) and DCM (50 mL) was bubbled 0 3 at -78 °C until the solution turned blue; excess ozone was removed on high vacuum. After the solution's color changed into colorless, NaBH 4 (0.8 g, 20 mmol) was added to the reaction mixture and subsequently stirred at room temperature for 30 min; LC and TLC
  • Step D 5-ethenyl-4-(2-hydroxyethyl)-2-benzofuran-l(3H)-one: A mixture of 5-bromo-4-(2- hydroxyethyl)-2-benzofuran-l(3H)-one (0.460 g, 1.78 mmol), tributyl(vinyl)tin (0.676 g, 2.13 mmol), LiCl (0.224 g, 5.33 mmol) and Pd (PPh 3 )4 (0.10 g, 0.087 mmol) in toluene (50 mL) was heated at 100-110 °C under N 2 overnight; TLC indicated that reaction had gone to completion and to the solution was poured EtOAc (100 mL) and washed with brine, water, dried over
  • Step E 4-(2-hydroxyethyl)-5-oxiran-2-yl-2-benzofuran-l(3H)-one: 5-Ethenyl-4-(2-hydroxyethyl)-2- L benzofuran-l(3H)-one (1.2 g, 5.9 mmol) was added to a flask containing a stir bar. To the flask was then added dichloromethane (20 mL).
  • Reaction mixture was treated with dichloromethane and washed with NaCl, water and dried over Na 2 S0 4 , filtered and concentrated to -j dryness, absorbed into silica gel and was then subjected for purification over silica gel; the title compouni was isolated with the solvent system of hexanes/EtOAc (1/0.5).
  • Step A ethyl 4-bromo-2-methyl-3-oxobutanoate: To the solution of ethyl 2-methyl-3- oxobutanoate (5.05 g, 35.0 mmol) in water (10 mL) at 0 °C was added bromine (1.805 mL, 35.0 mmol) dropwise over 2h. The resulting solution was stirred at rt for 16h. The reaction mixture was extracted with ethyl acetate, the organic phase was dried over sodium sulfate, and
  • Step B 4-hydroxy-3-methylfuran-2(5H)-one: Ethyl 4-bromo-2-methyl-3-oxobutanoate (7.81 g,
  • Step C 4-methyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To the solution of 4- hydroxy-3-methylfuran-2(5H)-one (400 mg, 3.51 mmol) in dichloromethane (10 mL) at -78 °C was added 2,6-lutidine (0.612 mL, 5.26 mmol) and triflic anhydride (0.711 mL, 4.21 mmol)
  • Step A fert-Butyl 4-(hydroxymethyl)piperdinecarboxylate: The mixture of 70 g of LiAlH 4 in 1500 mL of THF was cooled to 0 °C, then 180 g of 1-tert-butyl 4-methyl piperidine-l,4-dicarboxylate in THF was added dropwise. When the reaction was finished, 200 mL of ethyl acetate and solid anhydrous Na 2 S0 4 . Water was added until solution became clear. The mixture was filtered and the filtrate was evaporated to afford title compound.
  • Step B fert-Butyl 4-formypiperidinecarboxylate: The solution of 200 mL of DMSO in CH 2 C1 2 cooled to -78 °C, 118 mL of (COCl) 2 was added drop-wise. Then 255 g of tert-butyl 4- (hydroxymethyl)piperdinecarboxylate was also added drop-wise. The mixture was stirred for 4 h. After the reaction was finished, 638 mL of Et 3 N was added at -78 °C. The organic layer was washed by brine, dried and purified by column chromatography to afford title compound.
  • Step C fert-Butyl 4-formyl-4-propylpiperidinecarboxylate: tert-Butyl 4- formypiperidinecarboxylat was dissolved in 66 mL of acrylonitrile, and 5 g of 50% aq. Sodium hydroxide solution was added. Heated to 50 °C, the reaction was finished (TLC). The mixture was then poured into 700 mL of ether. Washed with brine and purified with column
  • Step D fert-Butyl 3.8-diazaspiro[5.5]undecane-3-carboxylate: The 30 g of tert-butyl 4-formyl-4- propylpiperidinecarboxylate was dissolved in methanol of saturated ammonia, and 15 g of Raney Ni was added. The reaction mixture was heated to 110 °C and allowed to 80 atmospheres in 2 L of high-pressure autoclave. The mixture was filtered to remove the catalyst and the filtrate was concentrated to give residue which was purified by column chromatography to afford tert-butyl
  • the title compound is commercially available from a number of vendors, for example, Shanghai AQ BioPharma Co., Ltd, catalog #ABP1882. Alternatively, it may be prepared in various ways, including the procedure described below.
  • Step A 1-fert-Butyl 4-methyl 4-(cyanomethyl)piperidine-1.4-dicarboxylate: To a solution of commercially available 1-tert-butyl 4-methyl piperidine-l,4-dicarboxylate (200 g, 0.82 mol) in anhydrous THF (2 L) was added LDA (2M in THF, 575 mL, 1.15 mol) drop-wise at -65 °C under N 2 . The mixture was stirred at -65 °C for 1.5 h. To the mixture was added bromoacetonitrile (148 g, 1.23 mol) in anhydrous THF (500 mL) at -65 °C.
  • Step B fert-Butyl l-oxo-2.8-diazaspiro[4.5]decane-8-carboxylate: A suspension of 1-tert-butyl 4-methyl 4-(cyanomethyl)piperidine-l,4-dicarboxylate (70.0 g, 247.9 mmol) and Raney Ni (60 g) in MeOH (1500 mL) and ⁇ 3 ⁇ 2 0 (80 mL) was stirred at 2 MPa of hydrogen pressure at 50 °C for 18 h. The reaction mixture was filtered through a pad of celite and the filtrate was
  • Step A fert-butyl 4-(2-ethoxy-2-oxoethylidene)piperidine-l-carboxylate: Into a 10-L 4-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a suspension of NaH (74.0 g, 2.16 mol 1.05 equiv, 70%) in tetrahydrofuran (2000 mL) at 0°C, then added dropwise ethyl 2-(diethoxyphosphoryl)acetate (514 g, 2.06 mol, 1.05 equiv, 98%) with stirring at 0°C.
  • Step B tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)piperidine-l-carboxylate: Into a 3000- mL 4-necked round-bottom flask were potassium carbonate (93.2 g, 662 mmol, 0.50 equiv) and DMSO (2000 mL). The resulting solution was heated to 80°C.
  • Step C 3-oxo-2.8-diaza-spiro[4.5]decane-8-carboxylic acid fert-butylester: A mixture of tert- butyl 4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)piperidine-l-carboxylate (330 g, 990 mmol, 1.00 equiv, 99%) and Ni (40 g, 0.15 equiv) in ethanol (1200 mL) was stirred for 24 h under a hydrogen atmosphere at room temperature. The solid was filtered out. The filtrate was concentrated under vacuum. The crude product was purified by re-crystallization from ether to afford the title compound.
  • Boc-piperidine-4-carboxylic acid methyl ester (2.00 g, 8.22 mmol) in THF (40 mL) was cooled to -78 °C . Under nitrogen, a 2.0 M THF solution of LDA (6.17 mL, 12.3 mmol) was added dropwise. The reaction mixture was stirred at -78 °C for 30 minutes before a solution of 3- bromo-2-methylpropene (1.60 g, 11.9 mmol) in THF (2 mL) was added. After the reaction was stirred for 1 hour at this temperature, a sample was taken for LC-MS analysis and it showed that the reaction was completed.
  • Step B 1 -fert-Butyl 4-methyl 4-(2-oxopropyl)piperidine- 1.4-dicarboxylate: To a solution of 1- tert-butyl 4-methyl 4-(2-methylallyl)piperidine-l,4-dicarboxylate (2.2 g, 7.4 mmol) in
  • Step C fert-Butyl 3-methyl-l-oxo-2.8-diazaspiro[4.5]decane-8-carboxylate: 1-tert-Butyl 4- methyl 4-(2-oxopropyl)piperidine-l,4-dicarboxylate (1.15 g, 3.84 mmol) in methanol (25 mL) was treated with ammonium acetate(3.85 g, 49.9 mmol) , sodium cyanoborohydride (0.681 g, 10.83 mmol) and magnesium sulfate (2.54 g, 21.1 mmol). The mixture was heated at 80 °C in a sealed tube for 12 hours.
  • Step A fert-Butyl 2-(5-oxo-2.5-dihydrofuran-3-yl)-2.9-diazaspiro[5.5]undecane-9-carboxylate
  • Hunig's Base 275 ⁇ , 1.57 mmol
  • tert-butyl 3,8-diazaspiro[5,5]undecane-3- carboxylate 200 mg, 0.786 mmol.
  • the reaction mixture was stirred at 76 °C overnight, concentrated and purified by column chromatography (0-10% MeOH in DCM) to afford the title compound.
  • LC/MS: [(M+l)] + 337
  • Step B 4-(2.9-Diazaspiro[5.5]undecan-2-yl)furan-2(5H)-one: To tert-butyl 2-(5-oxo-2,5- dihydrofuran-3-yl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (266 mg, 0.792 mmol) in DCM (2 mL) was added TFA (2 mL, 26.0 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h, then concentrated under reduced pressure, then placed on the high vac. The residue was dissolved in MeOH and loaded onto a 2 g Bond Elut SCX column (pre-rinsed with MeOH).
  • Step A fert-butyl l-oxo-2-(5-oxo-2.5-dihydrofuran-3-yl)-2.8-diazaspiro[4.5]decane-8- carboxylate: The mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (1.83 g, 7.20 mmol), commercially available 4-bromofuran-2(5H)-one (1.41 g, 8.63 mmol), xantphos (0.416 g, 0.720 mmol), water (0.389 mL, 21.6 mmol) , and potassium carbonate (1.989 g, 14.39 mmol) in toluene (50 mL) was degassed with nitrogen followed by addition of palladium acetate (0.081 g, 0.36 mmol).
  • Step B 2-(5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To the solution of tert- butyl l-oxo-2-(5-oxo-2,5-dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate (5.70 g, 16.9 mmol) in methylene chloride (10 mL) was added trifluoroacetic acid (26.1 mL, 339 mmol) and the resulting solution was stirred at rt for lh.
  • Step A fert-Butyl 2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane-8- carboxylate: To a mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate
  • Step B 2-(4-Methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a mixture of of tert-butyl 2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8-diazaspiro[4.5]decane-8- carboxylate (57.0 g, 163 mmol) in EtOAc (180 mL) was added saturated HCl(g)/ EtOAc (712 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 3 h.
  • Step A fert-Butyl l-oxo-2-(5-oxo-2.5-dihydrofuran-3-yl)-2.9-diazaspiro[5.5]undecane-9- carboxylate: To a microwave vial was charged commercially available tert-butyl l-oxo-2,9- diazaspiro[5.5]undecane-9-carboxylate (Shanghai AQ BioPharma Co., Ltd, catalog # ABP3640, 100 mg, 0.373 mmol), 4-bromofuran-2(5H)-one (72.9 mg, 0.447 mmol), Pd 2 (dba) 3 (17.06 mg, 0.019 mmol), Xantphos (32.3 mg, 0.056 mmol), and cesium carbonate (182 mg, 0.559 mmol).
  • Step B 2-(5 -Oxo-2.5 -dihydrofuran-3 -yl)-2.9-diazaspiro [5.5 jundecan- 1 -one : tert-Butyl l-oxo-2- (5-oxo-2,5-dihydrofuran-3-yl)-2,9-diazaspiro[5.5]undecane-9-carboxylate (100 mg, 0.285 mmol) in DCM (2 mL) was treated with TFA (660 ⁇ , 8.56 mmol) at 0 °C to give TFA salt.
  • Step A fert-Butyl 2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.9-diazaspiro[5.5]undecane- 9-carboxylate: To a microwave vial was charged commercially available tert-butyl l-oxo-2,9- diazaspiro[5.5]undecane-9-carboxylate (Shanghai AQ BioPharma Co., Ltd, catalog # ABP3640, 100 mg, 0.373 mmol), 4-methyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (110 mg, 0.447 mmol), Pd 2 (dba) 3 (17 mg, 0.019 mmol), Xantphos (32 mg, 0.056 mmol), and cesium carbonate (182 mg, 0.559 mmol).
  • the title compound was prepared from 3-oxo-2,8-diaza-spiro[4,5]decane-8-carboxylic acid tert- butyl ester and 4-methyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate in two steps in an analogous fashion to that described for 2-(4-Methyl-5-oxo-2,5-dihydrofuran-3-yl)-2,9- diazaspiro[5.5]undecan- 1 -one above.
  • Step A fert-Butyl 3-methyl-l-oxo-2-(5-oxo-2.5-dihydrofuran-3-yl)-2.8-diazaspiro[4.5]decane-8- carboxylate: A mixture of tert-butyl 3-methyl-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (505 mg, 1.88 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (109 mg, 0.188 mmol), palladium(II) acetate (21 mg, 0.094 mmol), potassium carbonate (520 mg, 3.76 mmol), water (102 ⁇ , 5.65 mmol) and commercially available 4-bromofuran-2-one (368 mg, 2.26 mmol) in toluene (13 mL) was degassed and then heated at 60 °C for 16 hours.
  • Step B 3 -methyl-2-(5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : tert-Butyl 3- methyl-l-oxo-2-(5-oxo-2,5-dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate (90 mg, 0.257 mmol) was dissolved in dichloromethane (2 mL) and treated with TFA (1 mL). After stirring at room temperature for 1.5 hours, the reaction mixture was concentrated to remove excess of the reagent and co-evaporated with dichloromethane three times. LC-MS (IE, m/z) : 251 (M+l) + .
  • Step A ethyl 4-bromo-2-ethyl-3-oxobutanoate: To a solution of ethyl 2-ethyl-3-oxobutanoate (5.17 g, 32.7 mmol) in water (10 mL) at 0°C was added bromine (1.684 mL, 32.7 mmol) dropwise over 2h. The resulting solution was stirred at rt for 16 h. The mixture was extracted with ethyl acetate (100 mL) and the organic phase was dried over sodium sulfate, concentrated to give ethyl 4-bromo-2-ethyl-3-oxobutanoate.
  • Step B 3-ethyl-4-hydroxyfuran-2(5H)-one: A mixture of ethyl 4-bromo-2-ethyl-3-oxobutanoate and hydrogen bromide (48%, 0.037 mL, 0.327 mmol) was heated at 100 °C for 20 h. After cooling to rt, the solid was collected by filtration followed by diethyl ether washing to give 3- ethyl-4-hydroxyfuran-2(5H)-one. LC/MS: (M+l) + : 129.05.
  • Step C 4-ethyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To a solution of 3-ethyl- 4-hydroxyfuran-2(5H)-one (400 mg, 3.12 mmol) in dichloromethane (10 mL) at -78 °C was added 2,6-lutidine (0.545 mL, 4.68 mmol) and triflic anhydride (0.633 mL, 3.75 mmol) dropwise. The reaction solution was stirred at -78 °C for lh before warmed to rt for 2h.
  • Step D fert-butyl 2-(4-ethyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane-8- carboxylate: A mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (200 mg, 0.786 mmol), 4-ethyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (246 mg, 0.944 mmol), xantphos (45.5, 0.079 mmol), palladium (II) acetate (8.8 mg, 0.039 mmol), water (0.043 mL, 2.4 mmol), and potassium carbonate (217 mg, 1.57 mmol) in toluene (20 mL) was heated at 60 °C for 16 h.
  • Step E 2-(4-ethyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of tert-butyl 2-(4-ethyl-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (0.34 g, 0.93 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (2.16 mL, 28 mmol) and the resulting solution was stirred at rt for lh.
  • Step A ethyl 4-bromo-2-isopropyl-3-oxobutanoate: To a solution of ethyl 2-acetyl-3- methylbutanoate (5.10 g, 29.6 mmol) in water (10 mL) at 0 °C was added bromine (1.53 mL, 29.6 mmol) dropwise over 2h. Chloroform (30 mL) was added and the resulting solution was stirred at rt for 16h. ethyl acetate (300 mL) was adde to the reaction solution and the organic phase was dried over sodium sulfate, concentrated to give the title compound. 1 HNMR (500 MHz,
  • Step B 4-hydroxy-3-isopropylfuran-2(5H)-one: A mixture of ethyl 4-bromo-2-isopropyl-3- oxobutanoate (7.1 g, 28 mmol) and hydrogen bromide (48%, 0.032 mL, 0.28 mmol) was heated at 100 °C for 8h. After cooling to rt, the solid was collected by filtration followed by diethyl ether washing to give 4-hydroxy-3-isopropylfuran-2(5H)-one. LC/MS: (M+l) + : 143.09.
  • Step C 4-isopropyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To a solution of 4- hydroxy-3-isopropylfuran-2(5H)-one (400 mg, 2.81 mmol) in methylene chloride (10 mL) at -78 °C was added 2,6-lutidine (0.492 mL, 4.22 mmol) and triflic anhydride (0.570 mL, 3.38 mmol) dropwise, the reaction temperature was maintained at -78 °C for lh before warmed to rt for 2h. The mixture was partitioned between methylene chloride and 1 N hydrogen chloride. The organic phase was washed with 1 N hydrogen chloride, diluted sodium bicarbonate, dried over sodium sulfate, concentrated to give the title compound. LC/MS: (M+l) + : 275.07.
  • Step D fert-butyl 2-(4-isopropyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane- 8-carboxylate: A mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (200 mg, 0.786 mmol), 4-isopropyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (259 mg, 0.944 mmol), Xantphos (46 mg, 0.079 mmol), palladium (II) acetate (8.8 mg, 0.039 mmol), water (0.043 mL, 2.4 mmol), and potassium carbonate (217 mg, 1.57 mmol) in toluene (20 mL) was heated at 66 °C for 16h. After filtration
  • Step E 2-(4-isopropyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of tert-butyl 2-(4-isopropyl-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8- diazaspiro[4.5]decane-8-carboxylate (195 mg, 0.515 mmol) in methylene chloride was added trifluoroacetic acid at rt and the resulting solution was stirred at rt for lh.
  • the reaction mixture was diluted with DCM (20 mL), washed with water (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate and filtered. The filtrates were concentrated and the crude product was purified by column chromatography (ISCO 40 g silica gel column), eluting with 50-100% ethyl acetate/hexane gradient to give the title compound.
  • Step B fert-Butyl 2-(4-cyclopropyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8- diazaspiro [4.5 ] decane- 8 -carboxylate : In a microwave vial, tert-butyl 2-(4-bromo-5-oxo-2,5- dihydrofuran-3-yl)-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (90 mg, 0.22 mmol) was dissolved in toluene (2 mL) and water (0.2 mL).
  • Step C 2-(4-cyclopropyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one :
  • the title compound can be prepared in a similar fashion to that described for 2-(4-isopropyl-5-oxo-2,5- dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one above using TFA.
  • Step A ethyl 4-bromo-3-oxopentanoate: To a solution of ethyl 3-oxopentanoate (5.00 g, 34.7 mmol) in chloroform (27 mL) at 0 °C was added bromine (1.79 mL, 34.7 mmol) in chloroform (10 mL) drop-wise. The resulting solution was stirred at rt for 16 h. The solution was washed with water, dried over sodium sulfate, concentrated to give ethyl 4-bromo-3-oxopentanoate.
  • Step B 4-hydroxy-5-methylfuran-2(5H)-one: Ethyl 4-bromo-3-oxopentanoate (7.49 g, 33.6 mmol) was treated with potassium hydroxide (5.03 g, 90 mmol) in water (36 mL) at 0 °C. The resulting mixture was vigorously stirred at 0°C for 4h. The reaction mixture was extracted with methylene chloride (twice with 100 mL). The alkaline phase was acidified to pH ⁇ 1 by 6 N hydrogen chloride. The acidic phase was extracted with methylene chloride (3x100 mL). The latter combined organic phase was dried over sodium sulfate, concentrated to give title compound.
  • Step C 2-methyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To a solution of 4- hydroxy-5-methylfuran-2(5H)-one in methylene chloride (10 mL) at -78 °C was added 2,6- lutidine (0.612 mL, 5.26 mmol) and triflic anhydride (0.711 mL, 4.21 mmol) drop-wise.
  • Step D fert-butyl 2-(2-methyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane-8- carboxylate: A mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (200 mg, 0.786 mmol), 2-methyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (232 mg, 0.944 mmol), xantphos (45.5 mg, 0.079 mmol), palladium (II) acetate (8.83 mg, 0.039 mmol), water
  • Step E 2-(2-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of tert-butyl 2-(2-methyl-5 -oxo-2, 5 -dihydrofuran-3 -yl)- 1 -oxo-2, 8-diazaspiro [4.5] decane-8- carboxylate (127 mg, 0.362 mmol) in methylene chloride (1 mL) was added trifluoroacetic acid (1.396 mL, 18.12 mmol), the resulting solution was stirred at rt for lh. After concentration, the residue was treated with methylene chloride (1 mL) and hydrogen chloride (1 mL, 4 N in dioxane).
  • Step A ethyl 4-bromo-2-methyl-3-oxopentanoate: To a solution of ethyl 2-methyl-3- oxopentanoate (5.0 g, 34.7 mmol) in chloroform at 0 °C was added bromine (1.79 mL, 34.7 mmol) in chloroform (10 mL) drop-wise. The resulting solution was stirred at rt for 16 h. The solution was washed with water, dried over sodium sulfate, concentrated to give title compound.
  • Step B 4-hydroxy-3.5-dimethylfuran-2(5H)-one: To ethyl 4-bromo-2-methyl-3-oxopentanoate (7.49 g, 31.6 mmol) was added cold potassium hydroxide (4.7 g, 84 mmol) in water (36 mL) at 0 °C, the resulting mixture was vigorously stirred at 0 °C for 4h. The reaction mixture was extracted with methylene chloride (2x100 mL), the alkaline phase was acidified to Ph 1 by 6 N hydrogen chloride followed by extraction with methylene chloride (3 times 100 mL).
  • Step C 2.4-dimethyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To a solution of 4- hydroxy-3,5-dimethylfuran-2(5H)-one (400 mg, 3.12 mmol) in methylene chloride (10 mL) at -78 °C was added 2,6-lutidine (0.545 mL, 4.68 mmol) and triflic anhydride (0.633 mL, 3.75 mmol) drop-wise. The reaction temperature was maintained at -78 °C for lh before warming to rt for 2h.
  • Step D fert-butyl 2-(2.4-dimethyl-5-oxo-2.5-dihvdrofuran-3-ylVl-oxo-2.8- diazaspiro [4.5 ] decane- 8 -carboxylate : The mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-
  • Step E 2-(2.4-dimethyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of tert-butyl 2-(2,4-dimethyl-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8- diazaspiro[4.5]decane-8-carboxylate (195 mg, 0.535 mmol) in methylene chloride (1 mL) was added trifiuoroacetic acid (2.06 mL, 26.8 mmol) and the resulting solution was stirred at rt for lh.
  • Step A fert-butyl 2-(4-chloro-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane-8- carboxylate: To a solution of tert-butyl l-oxo-2-(5-oxo-2,5-dihydrofuran-3-yl)-2,8- diazaspiro[4.5]decane-8-carboxylate (2.1 g, 6.2 mmol) in chloroform (50 mL) was added NCS (1.00 g, 7.49 mmol) at rt and the resulting solution was heated at 60 °C overnight. After removing the volatiles the residue was purified on silica gel column using EtOAc/hexane as eluting solvents to give the title compound. LC/MS: (M+l) + : 371.11, 372.99.
  • Step B 2-(4-chloro-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of tert-butyl 2-(4-chloro-5 -oxo-2, 5 -dihydrofuran-3 -yl)- 1 -oxo-2, 8-diazaspiro [4.5] decane-8- carboxylate (2.26 g, 6.09 mmol) in methylene chloride (10 mL) was added trifluoroacetic acid (9.39 mL, 122 mmol) and the resulting solution was stirred at rt for lh, after removing the volatile, the residue was participated between methylene chloride (100 mL) and IN sodium hydroxide (100 mL).
  • Step A 3-bromo-4-ethoxy-3-fluoro-4-hydroxydihydrofuran-2(3H)-one: To the solution of 4- hydroxyfuran-2(5H)-one (2.25 g, 22.5 mmol) in ethanol (20 mL) was added NBS (4.00 g, 22.5 mmol), the resulting solution was stirred at rt for 40 min. Then l-chloromethyl-4-fluoro-l,4- diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (7.97 g, 22.5 mmol) was added and the resulting mixture was stirred at rt overnight.
  • Step B 3-fluoro-4-hydroxyfuran-2(5H)-one: To the solution of 3-bromo-4-ethoxy-3-fluoro-4- hydroxydihydrofuran-2(3H)-one (4.39 g, 18.1 mmol) in tetrahydrofuran (20 mL) was added tri-n- butyltin hydride (9.39 mL, 35.0 mmol) at 0 °C under N2. The resulting solution was stirred at rt overnight. After removing the volatile, the residue was stirred in 30 mL 50% acetic acid and 30 mL hexane at rt for 30 min.
  • the acidic phase was washed with hexane (3x30 mL) before concentration.
  • the reside was dissolved in sodium carbonate (50 mL, 2N), extracted with 40% EtOAc/hexane (4x50 mL), the alkaline phase was acidified to pH ⁇ 1 by 1 N hydrogen chloride.
  • the acidic phase was then extracted with ethyl acetate (8 times 60 mL).
  • the combined organic phase was dried over sodium sulfate, concentrated to give 3-fluoro-4-hydroxyfuran-2(5H)-one.
  • Step C 4-fluoro-2-methyl-5-oxo-2.5-dihydrofuran-3-yl trifluoromethanesulfonate: To a solution of 3-fluoro-4-hydroxyfuran-2(5H)-one (400 mg, 3.39 mmol) in methylene chloride (10 mL) at - 78°C was added 2,6-lutidine (0.592 mL, 5.08 mmol) and trifiic anhydride (0.687 mL, 4.07 mmol) drop-wise, the reaction temperature was maintained at -78°C for 1 h before warmed to rt for 2h.
  • Step D fert-butyl 2-(4-fluoro-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8-diazaspiro[4.5]decane-8- carboxylate: A mixture of tert-butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (150 mg, 0.590 mmol), 4-fluoro-2-methyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (148 mg, 0.590 mmol), xantphos (34.1 mg, 0.059 mmol), water (0.032 mL, 1.77 mmol) in toluene (20 mL) was degassed by nitrogen followed by addition of palladium acetate (6.6 mg, 0.029 mmol).
  • Step E 2-(4-fluoro-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To the solution of tert-butyl 2-(4-fluoro-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8-diazaspiro[4.5]decane- 8-carboxylate (109 mg, 0.308 mmol) in methylene chloride (1 mL) was added trifluoroacetic acid (1.896 mL, 24.61 mmol) and the resulting solution was stirred at rt for lh.
  • Step B nR.3r.5,Sy8-fert-butyl 3-methyl 3-(cyanomethyl)-8-azabicyclo[3.2.1]octane-3.8- dicarboxylate: To a solution of ( ⁇ R,3r,5S)-8-tert-buty ⁇ 3-methyl 8-azabicyclo[3.2.1]octane-3,8- dicarboxylate (5.00 g, 18.6 mmol) in THF (100 mL) was added LDA (13.9 mL, 27.8 mmol) at - 78 °C.
  • Step C nR.3r.5,Sy8-fert-butyl 3-methyl 3-(2-aminoethyl)-8-azabicyclo[3.2.1]octane-3.8- dicarboxylate: To a solution of ( ⁇ R,3r,5S)-8-tert-buty ⁇ 3-methyl 3-(cyanomethyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (4.0 g, 12.97 mmol) in ethanol (20 mL) and AcOH (20 mL) was added platinum(IV) oxide (0.295 g, 1.30 mmol). The mixture was hydrogenated on a shaker (45 psi hydrogen ) for 24 hr. The catalyst was filtered off through a celite pad, and the filtrate was concentrated. The crude material was used in the next step without further
  • Step D (lR3r,5S)-tert-buty ⁇ 2'-oxo-8-azaspiro[bicyclo [3.2.1]octane-3.3'-pyrrolidine]-8- carboxylate: A mixture of (lR,3r,5,S)-8-tert-butyl 3-methyl 3-(2-aminoethyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (4.2 g, 13.4 mmol) and potassium carbonate (9.29 g, 67.2 mmol) in MeOH (50 mL) was heated at 60 °C for 1 hr. The mixture was concentrated, and DCM (50 mL) was added.
  • Step E dR.3r.5,Syfert-butyl r-(4-methyl-5-oxo-2.5-dihvdrofuran-3-vn-2'-oxo-8- azaspiro [bicyclo [3.2.1 ] octane-3.3 '-pyrrolidine] -8-carboxylate : A mixture of 4-methyl-5-oxo-2,5- dihydrofuran-3-yl trifluoromethanesulfonate (1.861 g, 7.56 mmol), (lR,3r,5,S)-tert-butyl 2'-oxo-8- azaspiro[bicyclo[3.2.1]octane-3,3'-pyrrolidine]-8-carboxylate (1.63 g, 5.81 mmol), palladium(II) acetate (0.065 g, 0.291 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxant
  • Step F (lRJr.5,5 - -(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-8-azaspiro[bicyclo[3.2. ljoctane- 3.3 '-pyrrolidin] -2'-one : A solution of (lR,3r,5,S)-tert-butyl l'-(4-methyl-5-oxo-2,5-dihydrofuran- 3-yl)-2'-oxo-8-azaspiro[bicyclo [3.2.1]octane-3,3'-pyrrolidine]-8-carboxylate in DCM (50 mL) and TFA (10 mL) was stirred at rt for lh.
  • Step A (lR.5S.,Z)-tert-butyl 3-(l-cyano-2-methoxy-2-oxoethylidene)-8-azabicyclo[3.2.1]octane- 8-carboxylate: Methyl 2-cyanoacetate (3.63 g, 36.6 mmol), (lR,5S)-tert-butyl 3-oxo-8- azabicyclo[3.2.1]octane-8-carboxylate (5.5 g, 24.41 mmol), ammonium acetate (2.51 mL, 36.6 mmol), acetic acid (5.59 mL, 98 mmol) and toluene (100 mL) were placed in a 500-mL round- bottomed flask attached to a Dean- Stark constant water separator which was connected to a reflux condenser.
  • the reaction was quenched with saturated ammonium acetate aqueous, and the mixture was diluted with EtOAc (100 mL). The organic layer was separated, and the aqueous was extracted with EtOAc (100 mL). The combined organic layers were washed with bring, dried over MgS0 4 and concentrated. The crude material was purified by column (eluted with 0-50% EtOAc, then 50% EtOAc in hexane).
  • Step C nR.3s.5,Syfert-butyl 3-(cyanomethyl)-3-vinyl-8-azabicyclo[3.2.1]octane-8-carboxylate: A suspension of (lR,3r,5,S)-tert-butyl 3-(l-cyano-2-methoxy-2-oxoethyl)-3-vinyl-8- azabicyclo[3.2.1]octane-8-carboxylate (7.2 g, 21.53 mmol) and sodium chloride (1.258 g, 21.53 mmol) in DMSO (40 mL) and water (4 mL) was heated in an 160 °C oil bath for 2 h, then cooled down to RT.
  • Step D (lR3s.5S)-tert-buty ⁇ 3-(cyanomethyl)-3-formyl-8-azabicyclo[3.2.1]octane-8-carboxylate: To a suspension of ( ⁇ R,3s,5S)-tert-buty ⁇ 3-(cyanomethyl)-3-vinyl-8-azabicyclo[3.2. l]octane-8- carboxylate (4.00 g, 14.5 mmol, water (15 mL) and sodium periodate (12.4 g, 57.9 mmol) in dioxane (45 mL) was added osmium tetroxide (0.184 g, 0.724 mmol).
  • Step E (lR.3s.5,5 r )-8-(fert-butoxycarbonyl)-3-(cyanomethyl)-8-azabicyclo[3.2. l]octane-3- carboxylic acid: To a solution of ( ⁇ R,3s,5S)-tert-buty ⁇ 3-(cyanomethyl)-3-formyl-8- azabicyclo[3.2. l]octane-8-carboxylate (5.10 g, 18.3 mmol) in t-BuOH/H20 (2: 1) was added sodium dihydrogenphosphate hydrate (7.59 g, 55.0 mmol) and 2-methylbut-2-ene (9.7 mL, 92 mmol).
  • Step F nR.3s.5,Sy8-fert-butyl 3-methyl 3-(cyanomethyl)-8-azabicyclo[3.2.1]octane-3.8- dicarboxylate: To a solution of (lR,3s,5,S)-8-(tert-butoxycarbonyl)-3-(cyanomethyl)-8- azabicyclo[3.2.1]octane-3-carboxylic acid (5.20 g, 17.7 mmol) in a mixture solvent of MeOH (30 mL) and DCM (30 mL) was slowly added (trimethylsilyl)diazomethane (13.3 mL, 26.5 mmol) at rt. The mixture was stirred at the same temperature for 0.5 h.
  • Step G nR.3s.5,Sy8-fert-butyl 3-methyl 3-(2-aminoethyl)-8-azabicyclo[3.2.1]octane-3.8- dicarboxylate: A mixture of ( ⁇ R,3s,5S)-8-tert-buty ⁇ 3-methyl 3-(cyanomethyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (3.5 g, 11.4 mmol) and platinum(iv) oxide (0.258 g, 1.14 mmol) in ethanol (20 mL) and AcOH (20 mL), was hydrogenated on a shaker (45 psi hydrogen ) at rt for 48 hr. The catalyst was filtered off through a celite pad. The filtration was concentrated. LCMS 313.25 (+1), 257.25 (+1-56).
  • Step H (lR3s,5S)-tert-buty ⁇ 2'-oxo-8-azaspiro[bicyclo[3.2.1]octane-3.3'-pyrrolidine]-8- carboxylate: A mixture of (lR,3s,5S)-8-tert-buty ⁇ 3-methyl 3-(2-aminoethyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (3.50 g, 11.2 mmol) and potassium carbonate (7.74 g, 56.0 mmol) in MeOH (50 mL) was heated at 60 °C for 1 hr. The mixture was concentrated, and DCM (50 mL) was added.
  • Step I (lR.3s.5.S)-fer ⁇ butyl l'-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-2'-oxo-8- azaspiro [bicyclo [3.2.1 ] octane-3.3 '-pyrrolidine] -8-carboxylate : A mixture of (lR,3s,5,S)-tert-butyl 2'-oxo-8-azaspiro[bicyclo[3.2.1]octane-3,3'-pyrrolidine]-8-carboxylate (2.00 g, 7.13 mmol), 4- methyl-5-oxo-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (1.93 g, 7.85 mmol),
  • Step B (lR.3r.5,S)-8-fer ⁇ butyl 3-methyl 3-(2-methylallylV8-azabicvclo[3.2.11octane-3.8- dicarboxylate: To a solution of diisopropylamine (5.94 mL, 41.7 mmol) in tetrahydrofuran (5 mL) at 0 °C was added n-butyllithium (16.7 mL, 41.7 mmol) drop-wise, the resulting solution was stirred at 0°C for 0.5h.
  • Step C nR.3r.5,Sy8-fert-butyl 3-methyl 3-(2-oxopropyl)-8-azabicyclo[3.2.1]octane-3.8- dicarboxylate: To a solution of (lR,3r,5,S)-8-tert-butyl 3-methyl 3-(2-methylallyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (7.99 g, 24.7 mmol) in dioxane (100 mL) and water (50 mL) was added sodium periodate (10.6 g, 49.4 mmol) and osmium tetroxide (0.126 g, 0.494 mmol).
  • Step D (lR3r,5S)-tert-buty ⁇ 5'-methyl-2'-oxo-8-azaspiro[bicyclo[3.2. l]octane-3.3 '-pyrrolidine] - 8-carboxylate: To the solution of (lR,3r,5,S)-8-tert-butyl 3-methyl 3-(2-oxopropyl)-8- azabicyclo[3.2.1]octane-3,8-dicarboxylate (7.90 g, 24.3 mmol) in methanol (50 mL) was added magnesium sulfate (5.84 g, 48.6 mmol), ammonium acetate (3.74 g, 48.6 mmol), and sodium cyanoborohydride (3.05 g, 48.6 mmol).
  • Step E (lR.3r.5,S)-fer ⁇ butyl 5'-methyl-2'-oxo-l'-(5-oxo-2.5-dihydrofuran-3-yl)-8- azaspiro[bicyclo[3.2.1]octane-3.3'-pyrrolidine]-8-carboxylate
  • A and (lR3r,5S)-tert-buty ⁇ 5'- methyl-2'-oxo- -(5-oxo-2.5-dihydrofuran-3-yl)-8-azaspiro[bicyclo [3.2.1]octane-3.3'- pyrrolidine]-8-carboxylate
  • B To a solution of (lR,3r,5,S)-tert-butyl 5'-methyl-2'-oxo-8- azaspiro[bicyclo [3.2.1]octane-3,3'-pyrrolidine]-8-carboxylate (2.41 g, 8.19
  • Step F ( IR, 3 r.5S)-5 '-methyl- 1 '-(5 -oxo-2.5 -dihydrofuran-3 -yl)-8-azaspiro [bicyclo [3.2.1 ] octane- 3.3'-pyrrolidin
  • Step B dR.5,Sy9-fert-butyl 7-methyl 3-methyl-3.9-diazabicvclo[3.3.11non-6-ene-7.9- dicarboxylate: To a solution of (lR,5,S)-tert-butyl 3-methyl-7-(((trifluoromethyl)sulfonyl)oxy)- 3,9-diazabicyclo[3.3.1]non-6-ene-9-carboxylate (14 g, 36 mmol) and diisopropylethylamine (9.47 mL, 54.3 mmol) in methanol (100 mL) and DMF (100 mL) was triphenylphosphine (0.95 g, 3.62 mmol) and palladium (II) acetate (0.407 g, 1.81 mmol).
  • Step C nR.5,Sy9-fert-butyl 7-methyl 3-methyl-3.9-diazabicyclo[3.3.1]nonane-7.9-dicarboxylate: To a solution of (lR,5,S)-9-tert-butyl 7-methyl 3-methyl-3,9-diazabicyclo[3.3.1]non-6-ene-7,9- dicarboxylate (4.69 g, 15.8 mmol) in methanol (50 mL) was added palladium on carbon (10%, 1.684 g, 1.583 mmol) and the resulting mixture was subjected to hydrogenation under 40 psi for three days. After filtration through celite under nitrogen the filtrate was concentrated to give title compound. LC/MS: (M+l) + : 299.1.
  • Step D ( ⁇ R5S sY9-tert-butv ⁇ 7-methyl 7-(cvanomethylV3-methyl-3.9- diazabicyclo[3.3.1]nonane-7.9-dicarboxylate: To a solution of diisopropylamine (3.28 mL, 23.02 mmol) in tetrahydrofuran ( 5 mL) was added n-butyllithium (11.51 mL, 23.02) dropwise at 0 °C, and the resulting solution was stirred at 0 °C for 0.5 h.
  • Step E (lR.5£7s)-9-fer ⁇ butyl 7-methyl 7-(2-aminoethyl)-3-methyl-3.9- diazabicyclo[3.3.1]nonane-7.9-dicarboxylate: To a solution of ( ⁇ R,5S,7s)-9-tert-buty ⁇ 7-methyl 7- (cyanomethyl)-3-methyl-3,9-diazabicyclo[3.3.1]nonane-7,9-dicarboxylate (4.39 g, 13.0 mmol) in methanol (30 mL) was added platinum (IV) oxide (0.207 g, 0.911 mmol) and the resulting mixture was hydrogenated at 40 psi for 16 h. After filtration through celite under nitrogen, the filtrate was concentrated to give the title compound. LC/MS: (M+l) + : 342.2.
  • Step F (lR3's,5S)-tert-buty ⁇ 3-methyl-2'-oxo-3.9-diazaspiro[bicyclo[3.3.1]nonane-7.3'- pyrrolidine] -9-carboxylate: To a solution of ( ⁇ R,5S,7s)-9-tert-buty ⁇ 7-methyl 7-(2-aminoethyl)-3- methyl-3,9-diazabicyclo[3.3.1]nonane-7,9-dicarboxylate (4.44 g, 13.0 mmol) in methanol (100 mL) was added potassium carbonate (10.8 g, 78 mmol), and the resulting solution was heated at reflux for 8h.
  • Step G ( ⁇ R3's.5S)-tert-butv ⁇ 3-methyl-r-(4-methyl-5-oxo-2.5-dihvdrofuran-3-vn-2'-oxo-3.9- diazaspiro [bicyclo [3.3.1 ]nonane-7.3 '-pyrrolidine] -9-carboxylate : A mixture of ( ⁇ R,3's,5S)-tert- butyl 3-methyl-2'-oxo-3,9-diazaspiro[bicyclo[3.3.
  • Step H ( ⁇ R.3's.5S)-3 -methyl- 1 '-(4-methyl-5 - oxo-2.5 -dihvdrofuran-3 -vD-3.9- diazaspiro [bicyclo [3.3.1 ]nonane-7.3 '-pyrrolidin] -2'-one : To the solution of ( ⁇ R,3's,5S)-tert-buty ⁇ 3 -methyl- 1 '-(4-methyl-5 -oxo-2, 5 -dihydrofuran-3 -yl)-2'-oxo-3 , 9-diazaspiro [bicyclo [3.3.1 Jnonane- 7, 3 '-pyrrolidine] -9-carboxylate (2.63 g, 6.49 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (10 mL, 130 mmol) and the resulting solution was stirred at rt for lh. After
  • Step A Ethyl l-benzyl-4-(cyanomethyl)-3-oxopiperidine-4-carboxylate: To a flask charged with ethyl l-benzyl-3-oxopiperidine-4-carboxylate (1.0 g, 3.8 mmol) and a stir bar was added K2CO3 (1.06 g, 7.6 mmol), Bromoacetonitrile (0.92 g, 7.6 mmoO, and acetone (15 mL). The reaction was allowed to stir at RT for 2 hours. LC showed slow reaction. It was then heated to 45 °C for 3 hours. LC showed complete reaction at that point. The reaction was quenched with NH4CI, extracted with EtOAc, dried over Na2S04, filtered and concentrated. The crude product was purified by MPLC to furnish title compound. LCMS: mlz 301 (M+H) + .
  • Step B 8-Benzyl-6-hydroxy-2.8-diazaspiro[4.5]decan-l-one: To a flask charged with Ethyl 1- benzyl-4-(cyanomethyl)-3-oxopiperidine-4-carboxylate (900 mg, 3.0 mmol) and a stir bar was added Platinum Oxide (100 mg, 0.44 mmol), MeOH (20 mL) and Acetic acid (20 mL). The mixture was allowed to stir vigorously under an atmosphere of hydrogen for 24 hours. LC indicated complete reaction at that point. The catalyst was removed by filtration through a pad of celite, and the filtrate was concentrated under reduced pressure.
  • Step C 8-Benzyl-6-hydroxy-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decani-one: To a flask charged with 8-Benzyl-6-hydroxy-2,8-diazaspiro[4.5]decan-l-one (520 mg, 2.0 mmol) and a stir bar was added palladium acetate (22 mg, 0.10 mmol), K2CO3 (550 mg, 4.00 mmol), Xantphos (120 mg, 0.20 mmol), 4-Methyl-5-oxo-2,5-dihydrofuran-3-yl
  • Step D 6-Hydroxy-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one
  • 8-Benzyl-6-hydroxy-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)-2,8- diazaspiro[4.5]decan-l-one 150 mg, 0.42 mmol
  • MeOH mL
  • the mixture was allowed to stir under an atmosphere of hydrogen for 16 hours.
  • LC indicated complete reaction.
  • the catalyst was filtered off, and the crude material was used without further purification.
  • Step A 8-benzyl-6-fluoro-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 - one: To a flask charged with DCM (5 mL) and a stir bar was added DAST (0.092 mL, 0.69 mmol) at -78 °C, which was followed by addition of 8-Benzyl-6-hydroxy-2-(4-methyl-5-oxo-2,5- dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one (165 mg, 0.46 mmol) in DCM.
  • Step B-l 6-Fluoro-2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-2.8-diazaspiro[4.5]decan-l-one
  • Isomer B To a solution of 8-benzyl-6-fluoro-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)-2,8- diazaspiro[4.5]decan-l-one (Isomer B) (100 mg, 0.28 mmol) in DCE (2 mL) was added ACE-C1 (0.15 mL, 1.4 mmol). The mixture was heated to 80 °C for 3 hours.
  • Step B-2 6-Fluoro-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one (Isomer A): The intermediate was prepared following the same method as Isomer B (above) from 8-benzyl-6-fluoro-2-(4-methyl-5 -oxo-2, 5 -dihydrofuran-3 -yl)-2, 8-diazaspiro [4.5] decan- 1 -one (Isomer A). LCMS: mlz 269 (M+H) + .
  • Step A 1-fert-butyl 4-methyl 4-(2-((fert-butoxycarbonyl)amino)-l -hydroxyethyDpiperidine- 1.4- dicarboxylate: To a solution of LDA (prepared by adding «-butyllithium (20.0 mL, 49.3 mmol) to diisopropylamine (5.16 mg, 51.0 mmol) in THF (40 mL) at 0 °C, stir for 30 min) was added 1- fert-butyl 4-methyl piperidine-l,4-dicarboxylate (4.00 g, 16.4 mmol) in TMEDA (15 mL, 99 mmol) drop-wise via syringe pump at -78 °C for 10 min.
  • LDA prepared by adding «-butyllithium (20.0 mL, 49.3 mmol) to diisopropylamine (5.16 mg, 51.0 mmol) in THF (40 mL) at 0 °C
  • Step B fert-Butyl 4-hydroxy-l-oxo-2.8-diazaspiro[4.5]decane-8-carboxylate: To the solution of
  • Step C fert-Butyl 4-hydroxy-2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8- diazaspiro [4.5 ] decane- 8 -carboxylate : To a round bottom flask was charged tert-butyl 4- hydroxy-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (400 mg, 1.48 mmol), 4-methyl-5-oxo- 2, 5 -dihydrofuran-3 -yl trifluoromethanesulfonate (546 mg, 2.22 mmol), Pd 2 (dba) 3 (33.9 mg, 0.037 mmol), Xantphos (64.2 mg, 0.111 mmol), and cesium carbonate (964 mg, 2.96 mmol). The flask was equipped with a condenser, vacuumed and back filled with N2 and filled with Dioxane (6 mL
  • Step D 4-hydroxy-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one, derived from faster eluting isomer (A) and slower eluting isomer (B):
  • the title compounds were prepared from isomers A and B of tert-Butyl 4-hydroxy-2-(4-methyl-5-oxo-2,5-dihydrofuran-3- yl)-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate with using TFA in a similar fashion as described previously for INTERMEDIATE 19.
  • Step A fert-Butyl 2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-1.4-dioxo-2.8- diazaspiro [4.5 ] decane- 8 -carboxylate : _To tert-butyl 4-hydroxy-2-(4-methyl-5-oxo-2,5- dihydrofuran-3-yl)-l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate (200 mg, 0.546 mmol) in DCM (2.8 mL) was added sodium bicarbonate (68.8 mg, 0.819 mmol) and Dess-Martin periodinane (347 mg, 0.819 mmol).
  • Step B 2-(4-Methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decane- 1.4-dione :
  • the title compound was prepared from tert-Butyl 2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)-l,4-dioxo- 2,8-diazaspiro[4.5]decane-8-carboxylate in a similar fashion as described for INTERMEDIATE 19.
  • LC/MS: [(M+l)] + 265
  • Step A fert-Butyl 4-methoxy-2-(4-methyl-5-oxo-2.5-dihydrofuran-3-yl)-l-oxo-2.8- diazaspiro[4.5]decane-8-carboxylate (faster and slower eluting isomers A and B.
  • Step B 4-methoxy-2-(4-methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one (derived from faster and slower eluting precursor isomers.
  • Step B 2-(4-Methyl-5 - oxo-2.5 -dihvdrofuran-3 -yl)-2.8-diazaspiro [4.5] dec-3 -en- 1 -one :
  • the title compound was prepared from tert-Butyl 2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)-l-oxo-2,8- diazaspiro[4.5]dec-3-ene-8-carboxylate using TFA in an analogous fashion to that described for making INTERMEDIATE 19, Step B.
  • LC/MS: [(M+l)] + 249.
  • Step A fert-Butyl 4-hydroxy-l-oxo-2-(5-oxo-2.5-dihydrofuran-3-yl)-2.8-diazaspiro[4.5]decane- 8-carboxylate: To a round bottom flask was charged tert-butyl 4-hydroxy-l-oxo-2,8- diazaspiro[4.5]decane-8-carboxylate (300 mg, 1.11 mmol), 4-bromofuran-2(5H)-one (271 mg, 1.66 mmol), Pd(Oac) 2 (24.9 mg, 0.111 mmol), Xantphos (96 mg, 0.166 mmol), and K 2 C0 3 (307 mg, 2.22 mmol). The flask was sealed, vacuumed and back filled with N 2 and filled with dioxane
  • Step B fert-Butyl l-oxo-2-(5-oxo-2.5-dihydrofuran-3-yl)-2.8-diazaspiro[4.5]dec-3-ene-8- carboxylate: To a solution of tert-butyl 4-hydroxy-l-oxo-2-(5-oxo-2,5-dihydrofuran-3-yl)-2,8- diazaspiro[4.5]decane-8-carboxylate (210 mg, 0.596 mmol) in DCM (6 mL) at 0 °C was added DBU (269 ⁇ , 1.79 mmol), and XtalFluor-E (409 mg, 1.79 mmol).
  • Step C 2-(5 -Oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] dec-3 -en- 1 -one :
  • the title compound was prepared from tert-Butyl l-oxo-2-(5-oxo-2,5-dihydrofuran-3-yl)-2,8-diazaspiro[4.5]dec-3- ene-8-carboxylate using TFA in an analogous fashion to that described for INTERMEDIATE 19,
  • Step A (,5 r )-8-(2-Hydroxy-2-(4-methyl- 1 -oxo- 1 J-dihydroisobenzofuran-5-yl)ethyl)-2-(4-methyl- 5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a suspension of (,S)-4-methyl-5- (oxiran-2-yl) isobenzofuran-l(3H)-one (200 mg, 1.05 mmol) and 2-(4-methyl-5-oxo-2, 5- dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one (300 mg, 1.05 mmol) in 10 mL ofEtOH was added DIPEA (271 mg, 2.10 mmol).
  • Step B 8-[(2RV2-fluoro-2-(4-methyl-l-oxo-1.3-dihvdro-2-benzofuran-5-vnethyl]-2-(4-methyl-5- oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of (,S)-8-(2-hydroxy-2- (4-methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl)ethyl)-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)- 2,8-diazaspiro[4.5]decan-l-one (170 mg, 0.39 mmol) in 5 mL of DCM was added Et 3 N 3HF (10 drops) and DAST (5 drops) at -78 °C.
  • Step B except starting from 8-[(2R)-2-hydroxy-2-(4-methyl- 1 -oxo- 1 ,3-dihydro-2-benzofuran-5-yl)ethyl]-2-(4-methyl-5- oxo-2,5-dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one (Step B) except starting from 8-[(2R)-2-hydroxy-2-(4-methyl- 1 -oxo- 1 ,3-dihydro-2-benzofuran-5-yl)ethyl]-2-(4-methyl-5- oxo-2,5-dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one.
  • Step A (S)-8-(2-chloro-2-(4-methyl-l-oxo-l .3-dihvdroisobenzofuran-5-vnethylV2-(4-methyl-5- oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of (R)-8-(2-hydroxy-2- (4-methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl)ethyl)-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)- 2,8-diazaspiro[4.5]decan-l-one (1.1 g, 2.5 mmol) and methanesulfonyl chloride (0.584 mL, 7.49 mmol) in DCM (30 mL) was added triethylamine (1.22 mL, 8.74 mmol
  • Step B 8-[(2R)-2-methoxy-2-(4-methyl- 1 -oxo- 1.3-dihvdro-2-benzofuran-5-vnethyl]-2-(4-methyl- 5 -oxo-2.5 -dihydrofuran-3 -yl)- 1 -oxo-2-aza-8-azoniaspiro [4.5] decane : One small piece of sodium metal in methanol (20 mL) was stirred until the sodium disappeared.
  • the reaction mixture was concentrated and purified by column chromatography (0-10% MeOH/DCM) to separate the syn and anti isomers.
  • the resulting syn and anti isomers were then respectively purified by SFC-HPLC, using the following conditions: chiralpak AD, 30x250mm, 65% MeOH+0.2% DEA, 70mL/min to give four single isomers of the title compound. Absolute stereochemistry has not been assigned with certainty for each isomer at this time. They are designated as follows: 53 A: faster eluting from MeOH column chromatography, faster eluting from SFC-HPLC; LC/MS:
  • Step A 8-(2-(4-methyl-l-oxo-l J-dihydroisobenzofuran-5-yl)-2-oxoethyl)-2-(4-methyl-5-oxo- 2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one
  • Step B 8-(2-(cyclopropylamino)-2-(4-methyl- 1 -oxo- 1.3-dihydroisobenzofuran-5-yl)ethyl)-2-(4- methyl-5 -oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one
  • Step A .(,S)-8-(2-aado-2-(4-methy.- ⁇
  • Step B ( t S r )-8-(2-amino-2-(4-methyl- 1 -oxo- 1.3-dihvdroisobenzofuran-5-vnethylV2-(4-methyl-5- oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of (,S)-8-(2-azido-2-(4- methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl) ethyl)-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)- 2,8-diazaspiro[4.5]decan-l-one (200 mg, 0.43 mmol) in a mixed solvent of tetrahydrofuran and water (v : v, 12 : 1, 20 mL) was added triphenylphosphin
  • Step A (RV8-(2-azido-2-(4-methyl- 1 -oxo- 1.3-dihvdroisobenzofuran-5-vnethylV2-(4-methyl-5- oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a suspension of 8-[(2S)-2- hydroxy-2-(4-methyl-l-oxo-l,3-dihydro-2-benzofuran-5-yl)ethyl]-2-(4-methyl-5-oxo-2,5- dihydrofuran-3-yl)-2,8-diazaspiro[4.5]decan-l-one (Example 8, 500 mg, 1.13 mmol)
  • Step C (R)-8-(2-amino-2-(4-methyl-l-oxo-l J-dihydroisobenzofuran-5-yl)ethyl)-2-(4-methyl-5- oxo-2.5 -dihydrofuran-3 -yl)-2.8-diazaspiro [4.5] decan- 1 -one : To a solution of (R)-8-(2-azido-2-(4- methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl) ethyl)-2-(4-methyl-5-oxo-2,5-dihydrofuran-3-yl)- 2,8-diazaspiro[4.5]decan-l-one (250 mg, 0.54 mmol) in a mixed solvent of tetrahydrofuran and water (v : v, 12 : 1, 20 mL) was added triphenylphosphine (283 mg, 1.
  • Commercially available spiro[indoline-3,4'-piperidin]-2-one hydrochloride 500 mg, 2.10 mmol
  • (,S)-4-methyl-5-(oxiran-2-yl)isobenzofuran-l(3H)-one (398 mg, 2.10 mmol) and DIEA (439 ⁇ , 2.51 mmol) in ethanol (7 mL) and heated at 80 °C for 3 h.
  • Step B 1 '-
  • Thallium Flux Assay was performed on each of the final product compounds in the Examples unless otherwise noted in an Example.
  • HEK293 cells stably expressing hROMK (hK ir l . l) were grown at 37°C in a 10%CO 2 humidified incubator in complete growth media: Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, Penicillin/Streptomycin/Glutamine, G418 and FBS. At >80% confluency, aspirate the media from the flask and rinse with 10 mL
  • Probenecid (Component D) - Lyophilized sample is kept at -20°C. Water soluble, 100X after solubilization in 1 mL water. Store at 4°C.
  • Probenecid/ Assay Buffer 100 mL of IX FluxORTM Assay Buffer; 1 mL of reconstituted component D; Store at 4°C
  • Loading Buffer (per microplate): 10 ⁇ 1000X FluxORTM Reagent; 100 ⁇ component C; 10 mL Probenecid/ Assay Buffer
  • Compound Buffer (per microplate): 20 mL Probenecid/ Assay Buffer; 0.3 mM ouabain (10 mM ouabain in water can be stored in amber bottle/aluminum foil at room temperature); Test compound
  • IX FluxORTMChloride-Free Buffer Prepare IX working solution in water. Can be stored at room temperature • Stimulant Buffer (prepared at 5X final concentration in IX FluxORTMChloride-Free Buffer): 7.5 mM Thallium sulfate and 0.75 mM Potassium sulfate (to give a final assay concentration of 3 mM Thallium/ 0.3 mM Potassium). Store at 4°C when not in use. If kept sterile, this solution is good for months.
  • Assay protocol- The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. FIEK-hKirl . l cells are seeded in Poly-D-Lysine microplates and kept in a 37°C-10%CO 2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxORTM reagent loading buffer and incubated, protected from light, at ambient temperature (23-25°C) for 90 min. The loading buffer is replaced with assay buffer ⁇ test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate.
  • control compound is included to support that the assay is giving consistent results compared to previous measurements, although the control is not required to obtain the results for the test compounds.
  • the control can be any compound of Formula I of the present invention, preferably with an IC50 potency of less than 1 ⁇ in this assay.
  • the control could be another compound (outside the scope of Formula I) that has an IC50 potency in this assay of less than 1 ⁇ .
  • test pulse consisting of a 100 ms step to 0 mV from a holding potential of -70 mV, followed by a 100 ms voltage ramp from -70 mV to +70 mV, was applied before and after a 6 min compound incubation period.
  • Test compounds were prepared by diluting DMSO stock solutions into the bath solution at 3x the final concentration and placed in the instrument in 96-well polypropylene plates. Current amplitudes were measured using the IonWorks software.
  • control compound is included to support that the assay is giving consistent results compared to previous measurements, although the control is not required to obtain the results for the test compounds.
  • the control can be any compound of Formula I of the present invention, preferably with an IC50 potency of less than 1 ⁇ in this assay.
  • the control could be another compound (outside the scope of Formula I) that has an IC50 potency in this assay of less than 1 ⁇ .
  • the spontaneously hypertensive rat exhibits age-dependent hypertension that does not require administration of exogenous agents to elevate blood pressure nor does it require the use of a high salt diet to elevate blood pressure. Thus it resembles human essential hypertension and provides an opportunity to assess the dose-dependence of novel agents for their ability to lower blood pressure.
  • HCTZ 25 mg/kg/day, PO
  • the blood pressure lowering efficacy of compounds of the present invention compared to vehicle control was evaluated following a single oral gavage each day for a typical duration of three to fourteen days. Data were collected as hourly averages, and changes in blood pressure were calculated by subtracting vehicle control baseline data on an hourly basis.
  • Example numbers 5, 7, 9 and 43 were evaluated at PO, QD doses at one or more doses within the range of 0.3 to 10 mg/kg and resulted in typical reductions in daily (24 h) mean systolic blood pressure ranging from 6 mmHg to 24 mmHg at the doses used by the last day of the studies.
  • the Spontaneously Hypertensive Rat Assay is well known and often used in the art as an experimental model simulating human hypertension (see, e.g., Lerman, L.O., et al, J Lab Clin Med, 2005;146: 160-173).

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Abstract

La présente invention concerne des composés de la formule I et des sels de qualité pharmaceutique de ceux-ci, lesdits composés étant des inhibiteurs du canal ROMK (Kir1.1). Les composés peuvent être utilisés en tant qu'agents diurétiques et/ou natriurétiques et pour la thérapie et la prophylaxie d'états médicaux, comprenant des maladies cardiovasculaires telles que l'hypertension, l'insuffisance cardiaque et des états associés à une rétention excessive de sel et d'eau.
PCT/CN2012/079184 2012-07-26 2012-07-26 Inhibiteurs du canal potassique médullaire externe rénal WO2014015495A1 (fr)

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PCT/CN2012/079184 WO2014015495A1 (fr) 2012-07-26 2012-07-26 Inhibiteurs du canal potassique médullaire externe rénal
ARP130102534A AR092031A1 (es) 2012-07-26 2013-07-16 Inhibidores del canal de potasio medular externo renal
PCT/US2013/052079 WO2014018764A1 (fr) 2012-07-26 2013-07-25 Dérivés de pipéridine spiro-fusionnée à utiliser comme inhibiteurs du canal potassique médullaire externe rénal
AU2013295722A AU2013295722B2 (en) 2012-07-26 2013-07-25 Spiro - fused piperidine derivatives for use as inhibitors of the renal outer medullary potassium channel
US13/951,096 US8952166B2 (en) 2012-07-26 2013-07-25 Inhibitors of the renal outer medullary potassium channel
ES13745315.5T ES2606016T3 (es) 2012-07-26 2013-07-25 Derivados de piperidina condensados en espiro para su uso como inhibidores del canal de potasio medular externo renal
BR112015001419A BR112015001419A8 (pt) 2012-07-26 2013-07-25 composto, composição farmacêutica, e, uso de um composto ou de um sal farmaceuticamente aceitável do mesmo
JP2015524451A JP6166783B2 (ja) 2012-07-26 2013-07-25 腎髄質外層カリウムチャンネルの阻害薬として使用されるスピロ縮合ピペリジン誘導体
KR20157001566A KR20150036142A (ko) 2012-07-26 2013-07-25 신장 외수질 칼륨 채널의 억제제로서 사용하기 위한 스피로 - 융합된 피페리딘 유도체
MX2015001115A MX365986B (es) 2012-07-26 2013-07-25 Derivativos de piperidina fusionados con espiro para usarse como inhibidores del canal de potasio medular externo renal.
EP13745315.5A EP2877471B1 (fr) 2012-07-26 2013-07-25 Dérivés de pipéridine spiro-fusionnée à utiliser comme inhibiteurs du canal potassique médullaire externe rénal
RU2015106139A RU2642066C2 (ru) 2012-07-26 2013-07-25 Спиро-конденсированные производные пиперидина для применения в качестве ингибиторов калиевого канала наружного медуллярного слоя
CA2876508A CA2876508A1 (fr) 2012-07-26 2013-07-25 Derives de piperidine spiro-fusionnee a utiliser comme inhibiteurs du canal potassique medullaire externe renal
CN201380039265.6A CN104540826B (zh) 2012-07-26 2013-07-25 用作肾外髓钾通道的抑制剂的螺稠合的哌啶衍生物
TW102126583A TW201410675A (zh) 2012-07-26 2013-07-25 腎外髓質鉀離子通道抑制劑
US14/569,858 US9206198B2 (en) 2012-07-26 2014-12-15 Inhibitors of the renal outer medullary potassium channel

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103864673A (zh) * 2014-03-04 2014-06-18 雅本化学股份有限公司 手性-1-叔丁氧羰基-3-羟基哌啶的制备以及手性翻转的方法
US8999991B2 (en) 2011-10-25 2015-04-07 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US8999990B2 (en) 2011-10-25 2015-04-07 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US9108947B2 (en) 2011-10-31 2015-08-18 Merck Sharp & Dohme Corp. Inhibitors of the Renal Outer Medullary Potassium channel
US9139585B2 (en) 2011-10-31 2015-09-22 Merck Sharp & Dohme Corp. Inhibitors of the Renal Outer Medullary Potassium channel
US9206199B2 (en) 2011-12-16 2015-12-08 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
WO2016065602A1 (fr) * 2014-10-31 2016-05-06 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
WO2016127358A1 (fr) * 2015-02-12 2016-08-18 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
US9493474B2 (en) 2011-10-31 2016-11-15 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US9527830B2 (en) 2011-09-16 2016-12-27 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
WO2017184662A1 (fr) 2016-04-20 2017-10-26 Bristol-Myers Squibb Company Composés hétérocycliques bicycliques substitués
WO2018093569A1 (fr) 2016-11-03 2018-05-24 Bristol-Myers Squibb Company Dérivés hétérocycliques bicycliques substitués utiles en tant qu'inhibiteurs de canal romk
WO2018222795A1 (fr) 2017-06-01 2018-12-06 Bristol-Myers Squibb Company Composés contenant de l'azote substitué
US10364234B2 (en) 2014-12-08 2019-07-30 Jiangsu Hengrui Medicine Co., Ltd. Pyridinecarboxamide derivatives, preparation method thereof and pharmaceutical uses thereof
US10513518B2 (en) 2015-01-29 2019-12-24 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010129379A1 (fr) * 2009-05-06 2010-11-11 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
WO2012058116A1 (fr) * 2010-10-27 2012-05-03 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique de la médullaire externe rénale
WO2012058134A1 (fr) * 2010-10-29 2012-05-03 Merck Sharp & Dohme Corp. Inhibiteurs du canal à potassium de la medulla externe du rein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010129379A1 (fr) * 2009-05-06 2010-11-11 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
WO2012058116A1 (fr) * 2010-10-27 2012-05-03 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique de la médullaire externe rénale
WO2012058134A1 (fr) * 2010-10-29 2012-05-03 Merck Sharp & Dohme Corp. Inhibiteurs du canal à potassium de la medulla externe du rein

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US9527830B2 (en) 2011-09-16 2016-12-27 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US8999991B2 (en) 2011-10-25 2015-04-07 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US8999990B2 (en) 2011-10-25 2015-04-07 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US9493474B2 (en) 2011-10-31 2016-11-15 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US9108947B2 (en) 2011-10-31 2015-08-18 Merck Sharp & Dohme Corp. Inhibitors of the Renal Outer Medullary Potassium channel
US9139585B2 (en) 2011-10-31 2015-09-22 Merck Sharp & Dohme Corp. Inhibitors of the Renal Outer Medullary Potassium channel
US9206199B2 (en) 2011-12-16 2015-12-08 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
CN103864673B (zh) * 2014-03-04 2016-03-23 雅本化学股份有限公司 手性-1-叔丁氧羰基-3-羟基哌啶的制备以及手性翻转的方法
CN103864673A (zh) * 2014-03-04 2014-06-18 雅本化学股份有限公司 手性-1-叔丁氧羰基-3-羟基哌啶的制备以及手性翻转的方法
WO2016065602A1 (fr) * 2014-10-31 2016-05-06 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
US9850245B2 (en) 2014-10-31 2017-12-26 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US10759788B2 (en) 2014-12-08 2020-09-01 Jiangsu Hengrui Medicine Co., Ltd. Pharmaceutical uses of pyridine carboxamide derivatives
US10442794B2 (en) 2014-12-08 2019-10-15 Jiangsu Hengrui Medicine Co., Ltd. Processes for preparing pyridine carboxamide derivatives
US10364234B2 (en) 2014-12-08 2019-07-30 Jiangsu Hengrui Medicine Co., Ltd. Pyridinecarboxamide derivatives, preparation method thereof and pharmaceutical uses thereof
US10513518B2 (en) 2015-01-29 2019-12-24 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
US10160751B2 (en) 2015-02-12 2018-12-25 Merck Sharp & Dohme Corp. Inhibitors of the renal outer medullary potassium channel
WO2016127358A1 (fr) * 2015-02-12 2016-08-18 Merck Sharp & Dohme Corp. Inhibiteurs du canal potassique médullaire externe rénal
WO2017184662A1 (fr) 2016-04-20 2017-10-26 Bristol-Myers Squibb Company Composés hétérocycliques bicycliques substitués
US10851108B2 (en) 2016-04-20 2020-12-01 Bristol-Myers Squibb Company Substituted bicyclic heterocyclic compounds
WO2018093569A1 (fr) 2016-11-03 2018-05-24 Bristol-Myers Squibb Company Dérivés hétérocycliques bicycliques substitués utiles en tant qu'inhibiteurs de canal romk
WO2018222795A1 (fr) 2017-06-01 2018-12-06 Bristol-Myers Squibb Company Composés contenant de l'azote substitué
EP3929194A1 (fr) 2017-06-01 2021-12-29 Bristol-Myers Squibb Company Composés contenant de l'azote substitué
USRE49700E1 (en) 2017-06-01 2023-10-17 Bristol-Myers Squibb Company Substituted nitrogen containing compounds

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