WO2016060941A1 - Inhibitors of the renal outer medullary potassium channel - Google Patents

Inhibitors of the renal outer medullary potassium channel Download PDF

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Publication number
WO2016060941A1
WO2016060941A1 PCT/US2015/054805 US2015054805W WO2016060941A1 WO 2016060941 A1 WO2016060941 A1 WO 2016060941A1 US 2015054805 W US2015054805 W US 2015054805W WO 2016060941 A1 WO2016060941 A1 WO 2016060941A1
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diazaspiro
alkyl
compound
mmol
decan
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PCT/US2015/054805
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English (en)
French (fr)
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Alexander Pasternak
Barbara Pio
Harry R. Chobanian
Zhi-Cai Shi
Shuzhi DONG
Yan Guo
Shawn P. Walsh
Zhiqiang Guo
Ronald D. FERGUSON
Brian CATO
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Merck Sharp & Dohme Corp.
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Priority to EP15849961.6A priority Critical patent/EP3207030A4/en
Priority to US15/505,266 priority patent/US20170275302A1/en
Publication of WO2016060941A1 publication Critical patent/WO2016060941A1/en

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    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • C07D498/10—Spiro-condensed systems

Definitions

  • the Renal Outer Medullary Potassium (ROMK) channel Kirl . l) see e.g., Ho, K., et al., Cloning and expression of an inwardly rectifying ATP-regulated potassium channel, Nature, 1993, 362(6415): p. 31-8.1, 2; and Shuck, M.E., et al., Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel, J Biol Chem, 1994, 269(39): p.
  • TALH thick ascending loop of Henle
  • CCD cortical collecting duct
  • 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. 592-9; and Tobin, M.D., et al., Common variants in genes underlying monogenic hypertension and hypotension and blood pressure in the general population,
  • Patent application publication number WO2010/129379 published November 11, 2010 having common representative Merck Sharp & Dohme Corp., (also published as
  • R5 and R6 are independently -H, -Cl-6 alkyl, -C3-6 cycloalkyl, -CF3, -CHF2, -CH2F or
  • X is -H, -OH,-OCl-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,
  • R2 can be joined to Rl or RlOa to form a ring;
  • R3 is -H, -Ci-6 alkyl, -C3.6 cycloalkyl, -OH, -F, -OC1.3 alkyl, or -CH2OH, or R can be joined to RlOa to form a ring.
  • Patent application publication number WO2012/058116 published May 3, 2012, having common representative Merck Sharp & Dohme Corp., describes ROMK inhibitors having the generic formula: wherein R5 and R6 are independently -H, -Cl-6 alkyl or -C(0)OCl-3alkyl; and X, X 1 , Y and Y 1 are independently -H or-Cl-6alkyl; or ⁇ can be joined together with Z2 to form a fused ring system.
  • Additional published patent applications to Merck Sharp and Dohme, which describe ROMK inhibitors include: WO2013/028474; WO2013/039802; WO2013/062892;
  • the compounds of Formula I and salts thereof 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 for compounds of the formula:
  • Y is -O- or -CH 2 - ;
  • Z is a N-containing multicyclic heteroaromatic group which is optionally substituted by R 6 group, or Z is a group of the formula:
  • R is H, Ci_ 2 alkyl optionally substituted with 1-3 halogens, or -C(0)R 5 ;
  • R 1 is -OR or halogen
  • R 2 is oxo or Ci_2 alkyl optionally substituted with 1-3 F;
  • R 3 is H or CH 3 ;
  • R 4 is H or CH 3 ;
  • R 5 is CH3 or C 3 _ 6 cycloalkyl
  • R 6 is halogen, -CN, C 3 _ 6 cycloalkyl, furanyl, -S0 2 N(R 8 )(R 9 ), -OCi_ 2 alkyl which is optionally substituted with 1-5 halogens, or Ci_ 2 alkyl which is optionally substituted with or 1-5 halogens;
  • R 7 is allyl or Ci_ 2 alkyl
  • R 8 is H or CH 3 ;
  • R 9 is H or CH 3 ;
  • R 10 is H, Ci_2 alkyl, or -OCH3;
  • R 11 is H, Ci_2 alkyl, or -OCH3;
  • R 12 is H, Ci_2 alkyl or -OCH3;
  • R 13 is H, halogen, Ci_ 2 alkyl or -OCH3 ;
  • R is H, halogen, Ci_ 2 alkyl or -OCH3 ;
  • R 15 is H, halogen, Ci_ 2 alkyl or -OCH3 ;
  • R 16 is H, halogen, Ci_ 2 alkyl or -OCH3 ;
  • n 0 or 1 ;
  • n 0 or 1 ;
  • o 0, 1 or 2;
  • p is 1, 2, or 3;
  • the compounds of Formula I are inhibitors of the ROMK (Kirl .l) 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 as well as chronic kidney disease, 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 with excessive salt and water retention.
  • the invention furthermore relates to processes for preparing compounds of Formula I, and
  • compositions which comprise compounds of Formula I.
  • Embodiments of this invention comprise compounds of Formula I or pharmaceutically acceptable salts thereof.
  • R 6 -substituted N-containing multicyclic heteroaromatic group is:
  • structural elements of the compounds of this invention there are many embodiments of the structural elements of the compounds of this invention, as defined below.
  • structural elements for each substituent group can be independently substituted for one another.
  • R is H, -CH 3 ,
  • R 2 is oxo
  • R 3 is CH3.
  • R 4 is H.
  • R 5 is cyclopropyl or CH3.
  • R 6 is H, F, -S0 2 NH 2 , -CH 2 SCH 3 , CF 3 , CH 3 , C 2 H 5 , -OCH 3 , CN, cyclopropyl, or furanyl.
  • R 7 is allyl or -CH 3 . In many embodiments, R is -CH 3 .
  • R 8 is H.
  • R 9 is H.
  • R is H or CH 3 .
  • R 11 is H, CH 3 or -OCH 3 .
  • R 11 is H or -OCH 3 .
  • R 12 is H or CH 3 .
  • R 13 is H, CH 3 , or F.
  • R 14 is H, -OCH 3 or F.
  • R 15 is H, -OCH 3 or F.
  • R 16 is H, CH 3 , F or CI.
  • R 1 , R 3 , R 4 , Z and n are as defined in Formula I.
  • Another embodiment of this invention is a compound of Formula Ila, which has the structural formula:
  • R 6 is as defined in Formula I.
  • Another embodiment of this invention is a compound of Formula lib, which has the structural formula:
  • Another embodiment of this invention is a compound of Formula lie, which has the structural formula:
  • R 6 is as defined in Formula I.
  • Another embodiment of the present invention is a compound of Formula III, which has the structural formula:
  • R is as defined in Formula I.
  • Another embodiment of the present invention is a compound of Formula IV or IVa, which has the structural formula:
  • Another embodiment of the present invention is a compound of Formula V, which has the structural formula:
  • R a is H or oxo
  • R is H, halogen, Ci_ 2 alkyl, or -OCi_ 2 alkyl;
  • R is H, halogen, Ci_ 2 alkyl, or -OCi_ 2 alkyl;
  • R 15 is H, halogen, Ci_ 2 alkyl, or -OCi_ 2 alkyl; and R is H, halogen, Ci_ 2 alkyl, or -OCi_ 2 alkyl.
  • Another embodiment of the present invention is a compound of Formula VI, which has the structural formula:
  • R 10 is H or Ci_2 alkyl
  • R 11 is H, Ci_2 alkyl, or -OCi_ 2 alkyl
  • R 12 is H, Ci_2 alkyl, or -OCi_ 2 alkyl.
  • Another embodiment of the present invention is a compound of Formula VII, which has the structural formula:
  • R 4 and R 6 are as defined in Formula I.
  • Another embodiment of the present invention is a compound of Formula VIII, which has the structural formula:
  • R 4 and R 6 are as defined in Formula I.
  • Another embodiment of the present invention is a compound of Formula IX, which has the structural formula:
  • R 3 and R 4 are as defined in Formula I.
  • R 3 , R 4 and R 6 are as defined in Formula I.
  • Another embodiment of the present invention is a compound represented by Formula I, Ila, lib, lie, III, VII, VIII, X or XI or a pharmaceutically acceptable salt thereof wherein R 6 is H, -CN, halo, Ci_ 2 alkyl, Ci_ 2 alkyl-S-allyl, C3-6 cycloalkyl, furanyl, -S0 2 NH 2 , or Ci_ 2 haloalkyl, wherein Ci_ 2 haloalkyl is substituted with 1-5 halogens.
  • Another embodiment of the present invention is a compound represented by Formula I,
  • Another embodiment of the present invention is a compound represented by Formula I which is selected from any of Examples 1-95, or a pharmaceutically acceptable salt thereof.
  • Alkyl is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having , e.g., 1-12, 1-6 or 1-4 carbon atoms. Commonly used abbreviations for alkyl groups are used throughout the specification. For example the term “Cl-6 alkyl” (or
  • Alkoxy is an alkyloxy group wherein the alkyl group is as previously defined and the bond to the parent moiety is through the oxy group.
  • Non- limiting examples include -OCH 3 , -
  • Halogen means a fluorine, chlorine, bromine or iodine atom.
  • Halo means -F, -CI, -Br, or -I. A non-limiting examples includes fluorine or fluoro.
  • Haloalkyl means a halo-alkyl group in which the halo and alkyl groups are as previously defined. The bond to the parent moiety is through the alkyl group. Non-limiting examples include -CH 2 CF 3 and -CF 3 .
  • Cycloalkyl is a cyclized alkyl ring having 3-12 or 3-6 carbon atoms.
  • Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • N-containing multicyclic heteroaromatic group means a bicyclic or tricyclic fused ring system containing 9 to 14 ring members in which from 1 to 5 ring members are heteroatoms that are independently selected from the group consisting of nitrogen, sulfur or oxygen and the remainder of the ring members are carbon, provided that at least one of the ring members is nitrogen. The point of attachment to the parent moiety is through any available ring member. Further, no two adjacent ring members may be oxygen or sulfur.
  • variables depicted in a structural formula with a "floating" bond, such as R6 are permitted on any available carbon atom in the ring to which the variable is attached. If the ring is multicyclic (e.g., a bicyclic ring), then the variable may be attached to any carbon in the multicyclic (e.g., bicyclic) ring.
  • substituted shall be deemed to include multiple degrees of substitution by a named substituent. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
  • the compounds of Formula I may have one or more chiral (asymmetric) centers.
  • 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.
  • bonds to a chiral carbon are depicted as straight lines in the structural Formulas of the invention, or when a compound name is recited without an (R) or (S) chiral designation for a chiral carbon, it is understood that both the (R) and (S) configurations of each such chiral carbon, and hence each enantiomer or diastereomer and mixtures thereof, are embraced within the Formula or by the name.
  • the production of specific stereoisomers or mixtures thereof may be identified in the Examples where such stereoisomers or mixtures were obtained, but this in no way limits the inclusion of all stereoisomers and mixtures thereof from being within the scope of this invention.
  • 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.
  • the preparation of individual 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.
  • 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.
  • absolute stereochemistry may be determined by Vibrational Circular Dichroism (VCD) spectroscopy analysis.
  • VCD Vibrational Circular Dichroism
  • 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 (iH) and deuterium (3 ⁇ 4).
  • 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 as, for example but not limited to, alkali metal salts, alkaline earth metal salts or as ammonium salts.
  • alkali metal salts alkaline earth metal salts or as ammonium salts.
  • 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
  • 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. Such 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.
  • This also encompasses the use of the compounds for inhibiting ROMK in a patient comprising administering a compound of claim 1 in a therapeutically effective amount to a patient in need of diueresis, natriuresis or both.
  • the inhibition of ROMK by the compounds of Formula I can be examined, for example, in the Thallium Flux 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 Assay 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 Formula 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, such as essential hypertension (also known as primary or idiopathic hypertension) which is a form of hypertension for which no cause can be found, heart failure (which includes both acute heart failure and chronic heart failure, the latter also known as congestive heart failure) and/or other conditions associated with excessive salt and water retention.
  • hypertension such as essential hypertension (also known as primary or idiopathic hypertension) which is a form of hypertension for which no cause can be found
  • heart failure which includes both acute heart failure and chronic heart failure, the latter also known as congestive heart failure
  • other conditions associated with excessive salt and water retention
  • the compounds could also be used to treat hypertension which is associated with any of several primary diseases, such as renal, pulmonary, endocrine, and vascular diseases, including treatment of patients with medical conditions such as heart failure and/or chronic kidney disease.
  • 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 hypertension, particularly pulmonary arterial hypertension (PAH), cardiovascular disease, edematous states, 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, pre-eclampsia, cerebral edema,
  • PAH pulmonary arterial hypertension
  • cardiovascular disease edematous states
  • diabetes mellitus diabetes
  • nephropathy glomerulonephritis, nephrotic syndrome
  • acute kidney insufficiency chronic kidney insufficiency (also referred to as chronic kidney disease, or more generally as renal impairment)
  • chronic kidney disease 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, glaucoma, benign intracranial hypertension, and other conditions for which a diuretic or natriuretic or both would have therapeutic or prophylactic benefit.
  • the compounds of the invention may be administered to a patient having, or at risk of having, one or more conditions for which a diuretic or natriuretic or both would have therapeutic or prophylactic benefit such as those described herein.
  • 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
  • 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. It is expected that 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.
  • 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 prophylaxis 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.
  • the term "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
  • preventing refers 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.
  • 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.
  • 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.
  • the instant invention also encompasses a process for preparing a pharmaceutical composition comprising combining a compound of Formula I with a pharmaceutically acceptable carrier. Also encompassed is the pharmaceutical composition which is made by combining a compound of Formula I with a pharmaceutically acceptable carrier. Furthermore, a 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 about 0.1 mg to 1 g, particularly 0.1 mg to about 200 mg, more particularly from about 0.1 mg to about 100 mg, and even more particularly from about 0.1 to about 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.
  • compositions usually comprise about 0.5 to about 90 percent by weight of the active compound on a free acid/free base weight basis.
  • the compounds of Formula I inhibit ROMK. Due to this property, apart from use as pharmaceutically active compounds in human medicine and veterinary medicine, 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
  • One or more additional pharmacologically active agents may be administered in combination with a compound of Formula I.
  • the additional active agent (or agents) is intended to mean a medicinal compound that is different from the compound of Formula I, and which is a pharmaceutically active agent (or agents) that is active in the body, including pro-drugs, for example esterified forms, that convert to pharmaceutically active form after administration, and also includes free-acid, free-base and pharmaceutically acceptable salts of said additional active agents when such forms are sold commercially or are otherwise chemically possible.
  • 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).
  • additional active agent or agents including but not limited to anti-hypertensive agents, additional diuretics, anti-atherosclerotic agents such as a 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).
  • the one or more additional active agents include but are not limited to thiazide-like diuretics, e.g., hydrochlorothiazide (HCTZ or HCT); 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); dual inhibitors of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) such as omapatrilat, sampatrilat and fasidotril; angiotensin II receptor antagonists, also known as angiotensin II
  • eprosartan e.g., eprosartan mesylate (TEVETAN ® ), irbesartan (AVAPRO ® ), losartan, e.g., losartan potassium (COZAAR ® ), olmesartan, e.g, olmesartan medoximil
  • a thiazide-like diuretic such as hydrochlorothiazide (e.g., HYZAAR ® , DIOVAN HCT ® , ATACAND HCT ® ), etc.); 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; aldosterone synthase inhibitors; renin inhibitors (e.g enalkrein; RO 42-5892; A 65317; CP 80794; ES 1005; ES 8891; SQ
  • calcium channel blockers e.g., amlodipine, nifedipine, verapamil, diltiazem, felodipine, gallopamil, niludipine, nimodipine, nicardipine, bepridil, nisoldipine
  • potassium channel activators e.g., nicorandil, pinacidil, cromakalim, minoxidil, aprilkalim, loprazolam
  • sympatholitics e.g., beta-adrenergic blocking drugs (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, metoprolol, metoprolol tartate, nadolol, propranolol, sotalol, timolol); alpha adrenergic blocking drugs (e.g., doxazocin, prazocin or alpha
  • lipid lowering agents e.g., HMG-CoA reductase inhibitors such as simvastatin and lovastatin which are marketed as ZOCOR ® and MEVACOR ® in lactone pro-drug 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 reduc
  • bromocriptine combination medications such as JANUMET ® (sitagliptin with metformin), and injectable diabetes medications such as exenatide and pramlintide acetate; phosphodiesterase-5 (PDE5) inhibitors such as sildenafil (REVATIO ® , VIAGRA ® ), tadalafil (CIALIS ® ,
  • ADCIRCA ® vardenafil HC1 (LEVITRA ® ); 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, pro-drug forms (including but not limited to esters), and salts of pro-drugs of the above medicinal agents where chemically possible.
  • Trademark names of pharmaceutical drugs noted above are provided for exemplification of the marketed form of the active agent(s); such pharmaceutical drugs could be used in a separate dosage form for concurrent or sequential administration with a compound of Formula I, or the active agent(s) therein could be used in a fixed dose drug combination including a compound of Formula I.
  • N-containing multicyclic heteroaromatic group As it is used in the following schemes to illustrate chemical reactions that are used to synthesize the group Z from synthetic intermediates that contain the N-containing multicyclic heteroaromatic groups: Schemes 1-4 illustrate the chemical reactions that convert synthetic intermediates that contain N- containing multicyclic heteroaromatic groups to the groups Z.
  • N-containing multicyclic heteroaromatic groups are defined earlier in this application in the Detailed Description. The figure shown above is not meant to represent any specific chemical structure or to limit N- containing multicyclic heteroaromatic groups to bicyclic rings (some groups Z are tricyclic).
  • the compounds can be synthesized by a two stage process where the mono-protected spirobicyclic core, such as 10 (Scheme 3) or 14 (Scheme 4) is first reacted with halide 6 followed by deprotection of Boc group, and then the epoxide ring is opened as illustrated in Scheme 3 and Scheme 4.
  • 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 TM 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 C 18, 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 HPLC 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 TM single quad MS system with Electrospray Ionization, Waters 2525 Gradient Pump, Waters 2767 Injecto /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.
  • CD3CI solutions and residual CH3OH peak or TMS was used as internal reference in CD3OD solutions.
  • Chiral analytical chromatography was performed on one of CHIRALPAK ® AS, CHIRALPAK ® AD, CHIRALCEL ® OD,
  • CHIRALCEL ® IA, or CHIRALCEL ® OJ columns 250x4.6 mm) (Daicel Chemical Industries, Ltd.) with noted percentage of either ethanol in hexane (%Et/Hex) or isopropanol in heptane
  • CHIRALPAK AS Chiral preparative chromatography was conducted on one of of CHIRALPAK AS, of CHIRALPAK AD, CHIRALCEL ® OD, CHIRALCEL ® IA, CHIRALCEL ® OJ columns (20x250 mm) (Daicel Chemical Industries, Ltd.) with desired isocratic solvent systems identified on chiral analytical chromatography or by supercritical fluid (SFC) conditions.
  • SFC supercritical fluid
  • DAST diethylaminosulfur trifluoride
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • DCE 1,2-dichloroethane
  • DCM dichloromethane
  • DEA diethyl amine
  • DME dimethoxyethane
  • DIBAL-H N,N- diisopropylethylamine
  • DIEA diisopropylethylamine
  • DIPEA di-isopropylamine
  • DIP A l, -3 ⁇ 4z ' 5(diphenylphosphino)ferrocene
  • DMP Dess-Martin Periodinane
  • DMP 1,1,1 -triacetoxy- 1 , 1 -dihydro- 1 ,2-benziodoxol-3(
  • DMAP dimethylacetamide
  • DMAC dimethylacetamide
  • DPPP l,3-bis(diphenylphosphino)propane
  • HMPA hexamethylphosphoramide
  • HOBt 1-hydroxybenzotriazole hydrate
  • IP Ac isopropanol
  • KHMDS potassium bis(trimethylsilyl)amide
  • LAH lithium aluminum hydride
  • LDA lithium diisopropylamide
  • mCPBA 3-chloroperoxybenzoic acid
  • MeOH methanol
  • CH3SO2- mesyl or Ms
  • MsOH methanesulfonic acid
  • PE petroleum ether
  • Pd(PPh 3 )4 tetrakis(triphenylphosphine)palladium
  • Pd(dppf)Cl2 or PdCl2(dppf) is ⁇ , - Bis(diphenylphosphino)ferrocene]- dichloropalladium(II) which may be complexed with CH2CI2; Chloro-(2-Dicyclohexylphosphino-2',6'-diisopropoxy- 1 , 1 '-biphenyl)[2-(2- aminoethyl)phenyl]palladium(II) - methyl-t-butyl ether adduct (RuPhos precatalyst); tetra-n- butylammonium fluoride (TBAF); tetrabutylammonium tribromide (TBATB); tert- butyldimethylsilyl chloride (TBS-C1); triethylamine (TE
  • racemic or racemate (rac); starting material (SM); round-bottom flask (RB or RBF); aqueous (aq); saturated aqueous (sat'd); saturated aqueous sodium chloride solution (brine); maximum temperature (T max ) ; medium pressure liquid chromatography (MPLC); high pressure liquid chromatography (HPLC); preparative HPLC (prep-HPLC); reverse phase high pressure liquid chromatorgraphy (RP-HPLC); ionization energy (IE); flash chromatography (FC); liquid chromatography (LC); solid phase extraction (SPE); supercritical fluid
  • CELITE ® is a trademark name for diatomaceous earth
  • SOLKA FLOC 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-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), PdCi 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 RediSep® 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.
  • 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/C0 2 , flow rate 200 mL/min, 100 bar, 25°C. 500 ⁇ 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. In that case 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 round bottom flask equipped with overhead stirrer was charged NaBH 4 (87.0 g, 2.30 mol) and THF (3.0 L) and the resulting slurry was cooled to 10°C. To the slurry 3-hydroxy-2-methyl benzoic acid (175 g, 1.15 mol) was added portionwise over 20 min (T max 17°C). A stirrable slurry formed, which 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 and 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 approximately 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 about -35 to about -33°C. The reaction mixture was allowed to stir for an additional 15 min during which time the temperature decreased to -40°C.
  • MTBE (684 mL, 2 volumes) was added and the resulting 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 resulting suspension 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-hvdroxy-4-methyl-3H-isobenzofuran-l-one: 4-Bromo-3 -hydro xymethyl-2-methyl phenol (100 g, 461 mmol), CuCN (83.0 g, 921 mmol), and DMF (500 mL) were charged to a 2 L 3 -neck flask equipped with overhead stirrer, N 2 inlet, and condenser. 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 and then the reaction mixture was cooled to 95°C. 41.5 mL of 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 FLOC ® and the cake washed with 50 mL DMF.
  • the filtrate from the DMF was added to a 3 L flask containing 1 L EtOAc. A precipitate coating formed in bottom of flask.
  • the DMF/EtOAc suspension was filtered through SOLKA FLOC ® 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 MgS0 4 , filtered and evaporated.
  • Step D 4-methy 1- 1 -oxo- 1 ,3 -dihydroisobenzofuran-5 -yl trifluoromethanesulfonate : 5-Hydroxy-4-methyl-3H-isobenzofuran-l-one (46.8 g, 285 mmol) was suspended in dichloromethane (935 mL) in 2-L roundbottom flask equipped with overhead stirrer under nitrogen. Triethylamine (59.5 mL, 427 mmol) was added and the reaction mixture was cooled in an ice bath to 3.8°C.
  • Trifluoromethanesulfonic anhydride (67.4 mL, 399 mmol) was added via addition funnel over 50 min, keeping the temperature ⁇ 10°C. After stirring the reaction mixture for an additional 15 min, the reaction mixture was quenched with water (200 mL) and then stirred with DARCO ® KB (activated carbon, 25 g) for 15 min. The biphasic mixture was filtered over SOLKA FLOC ® , 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 orange-red 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.
  • the suspension was filtered, the solid washed with heptane and dried under vacuum and nitrogen, providing trifluoromethanesulfonic acid 4- methyl-l-oxo-l,3-dihydro-isobenzofuran-5-yl ester.
  • Step E 5 -( 1 -butoxy-vinyl)-4-methy l-3H-isobenzofuran- 1 -one : 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) ) were charged to a 1 L 3-neck flask and then Et 3 N (35.6 mL, 255 mmol) were added. The solution was sparged with N 2 for 20 min.
  • Step F 5-(2-bromo-acetyl)-4-methyl-3H-isobenzofuran-l-one: Crude 5-(l-butoxy-vinyl)-4- methyl-3H-isobenzofuran-l-one (55.8 g) and THF (315 mL) were added to a 1 L 3-neck flask equipped with overhead stirrer. The solution was cooled to ⁇ 5°C after which water (79 mL) was added and the solution was maintained at ⁇ 5°C. NBS (41.6 g) was then added portion- wise while maintaining T max of 19°C. The solution was then warmed to RT for 30 minutes.
  • HBr (48 %, 0.241 mL) was added and the reaction was aged at RT for approximately 1 h after which 236 mL water was then added to the batch. A water bath is used to maintain temp at 20°C. Another 315 mL of water was added (solvent composition 1 :2 THF: water) and the slurry was cooled to 15°C. The resulting solids were filtered and washed with cold 1 :2 THF:water (15°C): 150 mL displacement wash followed by 100 mL slurry wash. The solids were dried under vacuum at RT to provide 5-(2-bromo-acetyl)-4-methyl-3H-isobenzofuran-l-one.
  • Step G 4-methyl-5-[(2i?)-oxiran-2-yll-2-benzofuran-l(3H)-one: 5-(2-Bromo-acetyl)-4-methyl- 3H-isobenzofuran-l-one (48.8 g., 181 mmol) was charged to a 5 L 3 neck round bottom equipped with overhead stirrer, thermocouple, and heating mantle.
  • 2-Propanol (1.22 L ) was added, followed by 610 mL of pH 7 0.1M potassium phosphate buffer.
  • Buffer solution (610 mL) was charged to a 1.0L erlenmeyer, and 2.44 g of NADP was added to the Erlenmeyer and swirled to dissolve.
  • a reducing enzyme, KRED MIF-20 (2.44 g) (available from Codexis, Inc., 200 Penobscot Drive, Redwood City, CA 94063, www .codex s.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. The solution was aged at RT then extracted with 1.22 L IP Ac. The aqueous layer was re-extracted with 400 mL IP Ac and the combined organics were washed with 400 mL 20 % brine solution, dried over MgS0 4 , filtered and concentrated by rotary evaporation.
  • Step B 6-(oxiran-2-yl)nicotinonitrile: A solution of 6-vinylnicotinonitrile (0.742 g, 5.70 mmol) in a 2: 1 ratio of water: t-BuOH (30 mL) was treated with N-bromosuccinimide in portions over 5 minutes (1.07 g, 5.99 mmol) and stirred at 40°C for 1 h. After cooling to 5°C, the reaction was basified with dropwise addition of solution of sodium hydroxide (0.684 g in 5 ml of water, 17.1 mmol) and stirred for another 1 h. The reaction mixture was poured into water (10 ml) and extracted with EtOAc (2 X 50 mL).
  • Step A 5-bromo-2-chloro-4-methoxypyridine: To a solution of 2-chloro-4-methoxypyridine (10.0 g, 69.7 mmol) in 50 mL of sulfuric acid at 0°C was added NBS. The reaction mixture was allowed to stir and warm up to room temperature for 2 h and then heated at 60°C for 5 h. Next, the reaction mixture was cooled to room temperature, neutralized with 1 N NaOH (pH ⁇ 7), diluted with water (50 ml) and the aqueous layer was extracted with ethyl acetate (2 x 100 mL).
  • Step B 6-chloro-4-methoxynicotinonitrile: A solution of 5-bromo-2-chloro-4-methoxypyridine (5.0 g, 22.48 mmol) in DMF (80 mL) was purged nitrogen for 15 min. Next, Zn(CN) 2 (3.96 g, 33.7 mmol) and Pd(Ph 3 P) 4 (2.60 g, 2.25 mmol) were added, successively. The resulting suspension was stirred at 95°C for 12 h under nitrogen atmosphere. The reaction mixture was cooled to ambient temperature and filtered to remove inorganic solid.
  • Step C 4-methoxy-6-vinylnicotinonitrile: A 20 mL microwave tube was charged with 6-chloro- 4-methoxynicotinonitrile (200.0 mg, 1.2 mmol), bis(diphenylphosphino)ferrocene
  • the microwave tube was evacuated and filled with nitrogen (two times) and heated to 140°C. After 1 h, the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with brine and dried over Na 2 S0 4 . The extracts were concentrated and chromatographed over a column of Si0 2 eluting with 0-30% EtOAc/hexanes.
  • Step D 6-(2-bromo-l-hvdroxyethyl)-4-methoxynicotinonitrile: A solution of 4-methoxy-6- vinylnicotinonitrile (80.0 mg, 0.499 mmol) in 1,4-dioxane (8 mL) and H 2 0 (4 mL) was treated with N-bromosuccinimide (89.0 mg, 0.499 mmol, 1.0 eq). The reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was poured into H 2 0 (8 mL) and extracted with EtOAc (3 x 30 mL).
  • Step E (4-methoxy-6-(oxiran-2-yl)nicotinonitrile: A solution of 6-(2-bromo-l -hydroxyethyl)- 4-methoxynicotinonitrile (74.0 mg, 0.288 mmol) in anhydrous methanol (7 ml) with treated with sodium carbonate (61.0 mg, 0.576 mmol, 2.0 eq), and allowed to stir at room temperature overnight. The solvent was evaporated. The residue was taken up in EtOAc (30 mL) and washed with water and brine. After drying over Na 2 S0 4 , the organic layer was removed and the residue was purified over Si0 2 eluting with 10-45%) EtOAc/hexanes to yield the title compound.
  • Step B 4-formyl-2-methoxybenzonitrile: A mixture of 4-formyl-2-methoxyphenyl
  • Step C 2-methoxy-4-(oxiran-2-yl)benzonitrile: To a cool solution of NaH (0.16 g, 3.9 mmol) in THF (40 mL) was added dropwise a solution of trimethylsulfonium iodide (0.91 g, 4.5 mmol) in DMSO (20 mL). The resulting mixture was stirred at 0°C under N 2 for 20 min. The solution of 4-formyl-2-methoxybenzonitrile (0.60 g, 3.7 mmol) in THF (20 mL) was added. The resulting reaction mixture was stirred at 0°C under N 2 for 1 h, and then it was warmed gradually to room temperature and stirred at that temperature for 12 h.
  • Step A di-t-butyl 2-(2-chloro-4-cyano-5 fluorophenvDmalonate: To sodium hydride (60% in mineral oil, 3.75 g, 94 mmol) under nitrogen was added dry DMF (150 mL) and the suspension was cooled in an ice bath. Di-t-butyl malonate (8.1 g, 37.5 mmol) was added dropwise over 15 minutes via syringe with hydrogen evolution. The suspension was stirred for 30 minutes after which time 5-chloro-2,4-difluorobenzonitrile (5.0 g, 28.8 mmol) in DMF (10 mL) was added dropwise over 15 minutes and the reaction was heated to 80°C for 12 h.
  • DMF di-t-butyl 2-(2-chloro-4-cyano-5 fluorophenvDmalonate
  • the reaction was cooled to room temperature, diluted with ether and quenched with aqueous ammonium chloride. The mixture was then extracted twice with ethyl acetate and the organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified on silica gel (eluting with 2-10% ethyl acetate/hexanes) to give the title compound.
  • Step B methyl 2-(2-chloro-4-cvano-5-fluorophenyl)acetate: A solution of di-t-butyl 2-(2- chloro-4-cyano-5-fluorophenyl)malonate (9.10 g, 24.6 mmol) in 1 :2 TFA: dichloromethane (25: 50 mL) was stirred at RT for 3 hours and then concentrated in vacuo to give a solid after twice evaporating with toluene. The resulting solid was taken up in 1 : 1 methanol: dichloromethane (50 mL) and 2M trimethylsilyldiazomethane in ether was added until the yellow color persisted.
  • Step C methyl 2-(2-chloro-4-cvano-5-methoxyphenyl)acetate: A solution of methyl 2-(2- chloro-4-cyano-5-fluorophenyl)acetate (1.40 g, 6.15 mmol) in methanol (30 ml) was divided into two 20 mL microwave vials. Potassium carbonate (2 x850 mg) was added to each vial. Each was heated in a microwave at 130°C for 60 minutes, at which time HPLC/MS indicated no starting material was left, and the product was all hydrolyzed to the acid. Most of the methanol was removed in vacuo and the residue was diluted with water, acidified with 2M HCl and the mixture was extracted twice with ethyl acetate.
  • Step D 5-chloro-4-(2-hydroxyethyl)-2-methoxybenzonitrile: To a solution of methyl 2-(2- chloro-4-cyano-5-methoxyphenyl)acetate (200 mg, 0.835 mmol) in THF (5 ml) was added 2M lithium borohydride (0.835 mL, 1.67 mmol) and the reaction was stirred at RT for 16 hours. The reaction was diluted with ether and quenched into water containing 2N HC1. The mixture was extracted twice with ethyl acetate and the organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The product mixture was separated by MPLC (20- 60% ethylacetae/hexanes) to afford 5-chloro-4-(2-hydroxyethyl)-2-methoxybenzonitrile.
  • Step E 2-chloro-4-cvano-5-methoxyphenethyl methanesulfonate: A solution of 5-chloro-4-(2- hydroxyethyl)-2-methoxybenzonitrile (205 mg, 0.969 mmol), DIPEA (0.846 mL, 4.84 mmol) and pyridine (0.0780 ml, 0.969 mmol) in DCM (3 mL) was treated dropwise with mesyl chloride (0.110 mL, 1.42 mmol). The reaction was stirred for 2 hours and then diluted with DCM and washed twice with aqueous citric acid, then washed with brine, and dried over sodium sulfate and concentrated in vacuo. Purification of the residue by flash chromatography (20-50% ethyl acetate/hexanes) afforded 5-chloro-4-(2-hydroxyethyl)-2-methoxybenzonitrile.
  • Step F 5-chloro-2-methoxy-4-vinylbenzonitrile: A solution of 2-chloro-4-cyano-5- methoxyphenethyl methanesulfonate (274 mg, 0.945 mmol) in DCM (4 mL) was treated with DBU (0.712 mL, 4.73 mmol) and stirred for 3 hours at 50°C, then at RT for 12 hours. TLC
  • Step G 5-chloro-2-methoxy-4-(oxiran-2-yl)benzonitrile: A solution of 5-chloro-2-methoxy-4- vinylbenzonitrile (130 mg, 0.671 mmol) in DCM (6 mL) was treated with 85% mCPBA (226 mg, 1.10 mmol) and stirred for 5 hours at RT when another portion of mCPBA (115 mg) was added. The reaction was stirred at room temperature for another 16 hours and was then diluted with DCM and stirred with saturated sodium bicarbonate containing some sodium bisulfite.
  • Step A 4-amino-2,5-difluorobenzonitrile: 2, 4, 5-trifluorobenzonitrile (20 g, 127 mmol) was added to 15 mL of liquid NH 3 and was reacted under 1.4 MPa at 60°C for 4 hours. The reaction mixture was then cooled to room temperature, diluted with ether (200 mL), washed with brine, dried and concentrated to give 4-amino-2,5-difluorobenzonitrile.
  • Step B 4-amino-3-bromo-2,5-difluorobenzonitrile; To a solution of 4-amino-2,5- difluorobanzonitrile (19 g, 0.13 mol) in 190 mL of AcOH and 8 mL of H 2 0 was added Br 2 (6.27 mL, 0.13 mol) dropwise. The resulting mixture was stirred at room temperature for 4 hours. The mixture was then poured into water, and the white precipitates were filtered, washed with water and dried to give 4-amino-3-bromo-2,5-difluorobenzonitrile.
  • DCM/PE 1/5
  • Step D 4-Bromo-2,5-difluoro-3-methylbenzonitrile: To a solution of 4-amino-2,5-difluoro-3- methylbenzonitrile (10.5 g, 62.4 mol) in 40 mL of concentrated HBr acid and 20 mL of water was added a solution of sodium nitrite (4.72 g, 68.6 mmol) in 10 mL of water at -5 ⁇ 0°C. CuBr (18 g, 124 mol) in 40 mL concentrated HBr acid was added dropwise at room temperature. The light yellow precipitate produced was collected by filtration and was washed with concentrated hydrochloric acid and water then dried at 40-50°C by vacuum to give the title compound.
  • Step A 4-bromo-2-fluoro-5-methylaniline : To a solution of 2-fluoro-5-methylaniline (20 g, 0.16 mol) in DCM was added TBATB (118 g, 0.17 mol) at 0°C portionwise, then the mixture was stirred at rt for 1 hour, water was added and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with water and brine, dried and concentrated. The residue was purified by column chromatography to afford 4-bromo-2-fluoro-5-methylaniline.
  • Step B 4-bromo-2-fluoro-5-methylbenzonitrile: A suspension of 4-bromo-2-fluoro-5- methylaniline (15 g, 73.5 mmol) in 30 mL of concentrated HCl was added 30 mL of water and a solution of NaN0 2 (5.33 g, 77.2 mmol) in water (20 mL) was added over a 20 minute period at 0°C. This diazonium solution was then brought to pH 6 with NaHC0 3 .
  • Step A 5 -Bromo-2-(l H-tetrazol- 1-vDpyridine: To a solution of 5-bromopyridin-2-amine (5.0 g, 28.9 mmol) in acetic acid (40 ml, 699 mmol) was added (diethoxymethoxy) ethane (7.70 ml, 46.2 mmol), followed by sodium azide (2.82 g, 43.3 mmol). The mixture was heated at 80°C for 1 h, cooled to room temperature and diluted with water. Precipitate was collected by filtration and dried under high vacuum to provide the title compound.
  • Step B 5 -Ethenyl-2-(l H-tetrazol- l-yl)pyridine: To a stirring solution of 5-bromo-2-(lH- tetrazol-l-yl)pyridine (1.0 g, 4.42 mmol), in EtOH (70 mL) were added
  • Step C 5 -(Oxiran-2-yl)-2-(l H-tetrazol- 1-vDpyridine: To a solution of 5-ethenyl-2-(lH-tetrazol- l-yl)pyridine (0.664 g, 3.83 mmol) in a 2: 1 ratio of H 2 0: t-BuOH (30 mL) was added N- bromosuccinimide (0.751 g, 4.22 mmol) in portions over 5 min.
  • the mixture was heated at 40°C for 1 h, cooled to 5°C, made basic with sodium hydroxide aqueous solution (0.46 g in 5 mL of H 2 0, 11.50 mmol), stirred for another 1 h at the same temperature, and poured into H 2 0 (10 mL). The product was precipitated out as white solid. The solid was collected by filtration, washed with water, and dried in vacuum.
  • Step A tert-butyl 4-(hydroxymethyl)piperidine-l -carboxylate: The mixture of 70 g of L1AIH 4 in 1500 mL of THF was cooled to 0°C, then 180 g of 1-tert-butyl 4-methyl piperidine-1,4- dicarboxylate in THF was added dropwise. When the reaction was finished, 200 mL of ethyl acetate and solid anhydrous Na 2 S0 4 were added. Water was added until solution became clear. The mixture was filtered and the filtrates were evaporated to afford the title compound.
  • Step B fert-butyl 4-formylpiperidine-l-carboxylate: The solution of 200 mL of DMSO in CH 2 C1 2 was cooled to -78°C, and 118 mL of (COCl) 2 was added dropwise. Then 255 g of tert- butyl 4-(hydroxymethyl)piperidine-l-carboxylate was also added dropwise. The mixture was stirred for 4 hours. 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 tert- butyl 4-formylpiperidine- 1 -carboxylate.
  • Step C fert-butyl 4-(2-cvanoethyl)-4-formylpiperidine-l-carboxylate: tert-Butyl 4- formylpiperidine-1 -carboxylate was dissolved in 66 mL of acrylonitrile, and 5 g of 50% aqueous sodium hydroxide solution was added. The reaction was heated at 50°C until TLC showed it was finished. The mixture was then poured into 700 mL of ether. The organic layer was separated, and washed with brine. The crude product was purified with column chromatography to afford fert-butyl 4-(2-cyanoethyl)-4-formylpiperidine- 1 -carboxylate.
  • Step D tert-butyl 2,9-diazaspiror5.51undecane-9-carboxylate: 30 g of tert-butyl 4-(2- cyanoethyl)-4-formylpiperidine-l -carboxylate was dissolved in methanol saturated with ammonia, and 15 g of Raney Ni was added. The reaction mixture was heated to 110°C and kept at 80 atmosphere in a 2L high-pressure autoclave. After the reaction was finished, the mixture was cooled to room temperature, filtered to remove the catalyst and the filtrates were
  • tert-Butyl l-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate is commercially available from a number of vendors, for example, Shanghai AQ BioPharma Co., Ltd, catalog #ABP1882.
  • Step B tert-butyl l-oxo-2,8-diazaspiro[4.51decane-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 NH 3 H 2 0 (80 mL) was stirred at 2 MPa of hydrogen at 50°C for 18 h. The reaction mixture was filtered through a pad of CELITE ® and the filtrate was concentrated under vacuum to give a crude product, which was washed with ethyl acetate (200 mL) to give title compound.
  • 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-oxoethylidene)piperidine-l-carboxylate.
  • tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-(nitromethyl)piperidine-l-carboxylate Into a 3000-mL 4-necked round-bottom flask were placed potassium carbonate (93.2 g, 662 mmol, 0.50 equiv) and DMSO (2000 mL). The resulting solution was heated to 80°C. This was followed by the addition of tert-butyl 4-(2-ethoxy-2-oxoethylidene)piperidine-l-carboxylate
  • Step C 3-oxo-2,8-diaza-spiror4,51decane-8-carboxylic acid tert-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 rt. 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.
  • Step A tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-hydroxypiperidine-l-carboxylate: To a solution of lithium bis(trimethylsilyl)amide (120 mL, 1.0 M solution in THF, 0.12 mol) in THF (120 mL) at -78 °C was added ethyl acetate (13 mL). Next, a solution of tert-butyl 4-oxopiperidine-l- carboxylate (20 g, 0.1 mol) in THF (80 mL) was added at -78°C. After the addition, the mixture was warmed up to 0°C and stirred for another 2 h.
  • Step B 2-(l-(tert-butoxycarbonyl)-4-hvdroxypiperidin-4-yl)acetic acid: A solution of tert-butyl 4-(2-ethoxy-2-oxoethyl)-4-hydroxypiperidine-l-carboxylate (30.0 g, 0.105 mol) in methanol (130 mL) and 2N NaOH solution (100 mL, 0.2 mol) was stirred at 25°C for 1.5 h, then the mixture was evaporated and the aqueous layer was extracted with ethyl acetate.
  • the water phase was adjusted to pH 6 with 2N HC1, the aqueous layer was extracted with ethyl acetate, then the organic phase was washed with brine, dried over Na 2 S0 4 and concentrated to afford 2-(l-(tert- butoxycarbonyl)-4-hydroxypiperidin-4-yl)acetic acid.
  • Step C tert-butyl 2-oxo-l-oxa-3,8-diazaspiro[4.51decane-8-carboxylate: A mixture of 2-(l- (tert-butoxycarbonyl)-4-hydroxypiperidin-4-yl)acetic acid (22 g, 0.085 mol), DPPA (30 g, 0.11 mol), Et 3 N (150 mL) in toluene (400 mL) was stirred at 105 °C under nitrogen for 12 h.
  • Step B (R)-6-(l-hydroxy-2-(2,8-diazaspiro[4.51decan-8-yl)ethyl)-4-methoxynicotinonitrile: To a solution of (R)-tert-butyl 8-(2-(5-cyano-4-methoxypyridin-2-yl)-2-hydroxyethyl)-2,8- diazaspiro[4.5]decane-2-carboxylate (0.500 mg, 1.20 mmol) in dichloromethane (4.0 mL) at 0°C was added trifloruoacetic acid (2.0 mL). The reaction was stirred at room temperature for 30 min.
  • Step B (S)-6-(l -hydroxy-2-(2, 8-diazaspiro ⁇ 4.51 decan-8-yl)ethyl)-4-methoxynicotinonitrile
  • S -6-(l-Hydroxy-2-(2,8-diazaspiro[4.5]decan-8-yl)ethyl)-4-methoxynicotinonitrile
  • R 6-(l-hydroxy-2-(2,8- diazaspiro[4.5]decan-8-yl)ethyl)-4-methoxynicotinonitrile
  • Step A (S)-tert-butyl 8-(2-(5-cvano-4-methoxypyridin-2-yl)-2-fluoroethyl)-2,8- diazaspiro[4.51decane-2-carboxylate: To a solution of (R)-fert-butyl 8-(2-(5-cyano-4- methoxypyridin-2-yl)-2-hydroxyethyl)-2, 8-diazaspiro [4.5 ]decane-2-carboxylate
  • Step B (S)- 6-(l-fluoro-2-(2,8-diazaspiro[4.51decan-8-yl)ethyl)-4-methoxynicotinonitrile
  • Step A (S)-fert-butyl 8-(2-(5-cvano-4-methoxypyridin-2-yl)-2-methoxyethyl)-2,8- diazaspiro [4.5] decane-2-carboxy late : To a solution of (S)-tert-butyl 8-(2-(5-cyano-4- methoxypyridin-2-yl)-2-hydroxyethyl)-2, 8-diazaspiro [4.5 ]decane-2-carboxylate
  • Step B (S)-4-Methoxy-6-(l-methoxy-2-(2,8-diazaspiror4.51decan-8-yl)ethyl)nicotinonitrile:
  • the title compound was prepared in a similar fashion to that described for the synthesis of INTERMEDIATE 18 Step B starting from (S)-tert-butyl 8-(2-(5-cyano-4-methoxypyridin-2-yl)- 2-methoxyethyl)-2,8-diazaspiro[4.5]decane-2-carboxylate.
  • INTERMEDIATE 22
  • Step A (S)-tert-butyl 8-(2-(5-cyano-4-methoxypyridin-2-yl)-2-((cyclopropanecarbonyl)oxy) ethvD-2 , 8 -diazaspiro ⁇ 4.51 decane-2-carboxylate : To a solution of (S)-fert-butyl 8-(2-(5-cyano-4- methoxypyridin-2-yl)-2-hydroxyethyl)-2, 8-diazaspiro [4.5 ]decane-2-carboxylate
  • Step B (S)-l-(5-cvano-4-methoxypyridin-2-yl)-2-(2,8-diazaspiror4.51decan-8-yl)ethyl cyclopropanecarboxylate :
  • the title compound was prepared in a similar fashion to that described for the synthesis of INTERMEDIATE 18 Step B starting from (S)-ieri-butyl 8-(2-(5- cyano-4-methoxypyridin-2-yl)-2-((cyclopropanecarbonyl)oxy)ethyl)-2,8-diazaspiro[4.5]decane- 2-carboxylate.
  • Step A 2,8-diazaspiror4.51decan-l-one hydrochloride: To a solution of tert-butyl l-oxo-2,8- diazaspiro[4.5]decane-8-carboxylate (INTERMEDIATE 14) (92 g, 0.36 mol) in CH 2 C1 2 (1 L) was slowly added a 4 M HC1 solution in EtOAc (500 mL). The mixture was stirred for 8 h at rt. The mixture was concentrated under vacuum to afford the title compound.
  • tert-butyl l-oxo-2,8- diazaspiro[4.5]decane-8-carboxylate (INTERMEDIATE 14) (92 g, 0.36 mol) in CH 2 C1 2 (1 L) was slowly added a 4 M HC1 solution in EtOAc (500 mL). The mixture was stirred for 8 h at rt. The mixture was concentrated under vacuum to afford the title compound.
  • Step B (R)-8-(2-hvdroxy-2-(4-methyl-l-oxo-l,3-dihvdroisobenzofuran-5-yl)ethyl)-2,8- diazaspiro [4.5 ] decan- 1 -one : To a solution of 2,8-diazaspiro[4.5]decan-l-one hydrochloride (68 g, 0.35 mol) in ethanol (1.5 L) was added Et 3 N (55 mL). The mixture was stirred for 2 hours. Next, (R)-4-methyl-5-(oxiran-2-yl)isobenzofuran-l(3H)-one (65 g, 0.34 mol) was added.
  • Step A tert-butyl 2-(tetrazolo[l,5-blpyridazin-6-yl)-2,8-diazaspiro[4.51decane-8-carboxylate: tert-Butyl 2,8-diazaspiro[4.5]decane-8-carboxylate (0.150 g, 0.624 mmol), 6- chlorotetrazolo[l,5-P]pyridazine (0.097 g, 0.624 mmol), Huning's base (0.327 mL, 1.87 mmol) and 1,4-dioxane (2.0 mL) was charged to a microwave tube.
  • Step B 6-(2,8-diazaspiro ⁇ 4.51 decan-2-yl)tetrazolo ⁇ 1 ,5 - pipyridazine : To a solution of tert-butyl 2-(tetrazolo[l,5-P]pyridazin-6-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate (0.152 mg, 0.423 mmol) in dichloromethane (1.0 mL) at 0°C was added trifluoroacetic acid (0.5 mL). The reaction was stirred at room temperature for 30 minutes. The mixture was concentrated in vacuo.
  • 6-(2,8-Diazaspiro[4.5]decan-2-yl)-[l,2,4]triazolo[3,4-P][l,3,4]thiadiazole was prepared in a similar fashion to that described for the synthesis of INTERMEDIATE 26 starting from tert- butyl 2,8-diazaspiro[4.5]decane-8-carboxylate and 6-bromo-[l,2,4]triazolo[3,4- P][l,3,4]thiadiazole (INTERMEDIATE 27).
  • Step A tert-butyl 2-([l,2,41triazolo[4,3-alpyridin-6-yl)-2,8-diazaspiro[4.51decane-8- carboxylate: tert- utyl 2,8-diazaspiro[4.5]decane-8-carboxylate (0.200 g, 0.832 mmol), 6- bromo-[l,2,4]triazolo[4,3-a]pyridine (0.181 g, 0.915 mmol), cesium carbonate (0.407 mg, 1.25 mmol), Pd 2 (dba) 3 (0.019 mg, 0.021 mmol), Xantphos (0.036 mg, 0.062 mmol), and 1,4-dioxane (3.0 mL) were charged to a microwave tube.
  • Step A tert-butyl 3-(4-methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl)-2-oxo-l-oxa-3,8- diazaspiror4.51decane-8-carboxylate: tert-Butyl 2-oxo-l-oxa-3,8-diazaspiro[4.5]decane-8- carboxylate (INTERMEDIATE 16) (120 mg, 0.468 mmol), 4-methyl-l-oxo-l,3- dihydroisobenzofuran-5-yl trifluoromethanesulfonate (153 mg, 0.515 mmol), Pd 2 (dba) 3 (10.72 mg, 0.012 mmol), Xantphos (20.32 mg, 0.035 mmol), and Cs 2 C0 3 (229 mg, 0.702 mmol) were charged to a microwave vile.
  • Step B 3-(4-methyl-l-oxo-l ,3-dihydroisobenzofuran-5-yl)-l-oxa-3,8-diazaspiro[4.51decan-2- one:
  • the title compound was prepared in a similar fashion to that described for the synthesis of INTERMEDIATE 26 StepB from tert-butyl 3-(4-methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl)- 2-oxo-l-oxa-3,8-diazaspiro[4.5]decane-8-carboxylate.
  • 6-(2,9-Diazaspiro[5.5]undecan-2-yl)-[l,2,4]triazolo[4,3-P]pyridazine was prepared in a similar fashion to that described for the synthesis of INTERMEDIATE 26 starting from tert- butyl 2,9-diazaspiro[5.5]undecane-9-carboxylate (INTERMEDIATE 13) and 6-chloro- [l,2,4]triazolo[4,3-P]pyridazine.
  • LC/MS: [(M+l)] + 273.
  • Step A tert-butyl 2-(4-methyl-l-oxo-l,3-dihydroisobenzofuran-5-yl)-2,8- diazaspiro ⁇ 4.51 decane- 8 -carboxylate :
  • Step B 4-methyl-5-(2,8-.51decan-2-yl)isobenzofuran-l(3H)-one: tert-Butyl 2-(4-methyl-l-oxo- l,3-dihydroisobenzofuran-5-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate was dissolved in DCM and then treated with 5 mL of 4N HC1 at rt for 1 h. Excess solvent was then removed on the rotoevaporator. The residue was then re-dissolved in 4: 1 chloroform/IP A and treated with 3 mL of IN NaOH for 5 min and the then the solution was passed through a SPE column, washing with organic layer. The eluent was then concentrated to give the crude free amine which was used without further purification.
  • Absolute stereochemistry obtained from this enzymatic reaction was not identified or determined to be R or S; the stereochemistry was assigned arbitrarily as R.
  • Step A 4-bromo-N,N-diethyl-2-methylbenzamide: A solution of 4-bromo-2-methylbenzoic acid (25.0 g, 116 mmol) in DCM (400mL) was treated with oxalyl chloride (11.7mL, 134 mmol) and a catalytic amount of dry DMF (0.1 mL). The reaction was allowed to stir under nitrogen for 2 h at rt. Removal of excess solvent gave crude acid chloride which was redissolved in DCM (400 mL). The mixture was then cooled to 0°C and triethyl amine (40.5 mL, 291 mmol) was added followed by the slow addition of diethyl amine (24.3 mL, 233 mmol).
  • the reaction was then allowed to warm to rt overnight.
  • the crude mixture was then diluted with 400mL of water and extracted with DCM (3 x 500 mL). The combined organic layers were then washed with brine (200mL), dried over magnesium sulfate, filtered and then concentrated.
  • the crude material was purified via MPLC (10% EtO Ac/Hex) to afford the title compound.
  • Step B 4-bromo-N,N-diethyl-2-(2-oxopropyl)benzamide: A 2M solution of LDA (35.2 mL, 70.3 mmol) in THF (176 mL) cooled to -78°C was treated with slow addition of 4-bromo-N,N- diethyl-2-methylbenzamide (19 g, 70.3 mmol) in dry THF (176 mL). The reaction was allowed to stir at -78°C for 1 h before it was quenched with N-methoxy-N-methylacetamide (22.43 mL, 211 mmol) and allowed to slowly warm to rt.
  • Step C 4-Bromo-N,N-diethyl-2-[(2R)-2-hydroxypropyllbenzamide:
  • 135 mg glucose dehydrogenase and 270 mg NADP+ disodium was added to the glucose/buffer solution at once, a homogeneous solution was obtained after 1 min of agitation.
  • 577 mg of enzymatic reductase PI B2 was added to the reaction vessel and stirred at 500 rpm at 30°C until the enzyme is wetted (about 40 min).
  • Step D (3R)-6-Bromo-3-methyl-3,4-dihvdro-lH-isochromen-l-one: A solution of 4-bromo- N,N-diethyl-2-[(2R)-2-hydroxypropyl]benzamide (12.2 g, 38.8 mmol) dissolved in 4N HC1 in dioxane (200 mL) was stirred at room temperature and monitored by TLC. After 3 days the reaction was partitioned between EtOAc (300 mL) and water (300 mL). The aqueous phase was further extracted with EtOAc (2 x 250 mL).
  • Step B (R)-3-methyl-6-(2,8-diazaspiror4.51decan-2-yl)isochroman-l-one: The title compound (was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from (R)-tert-butyl 2-(3-methyl- 1 -oxoisochroman-6-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate.
  • Step A fert-Butyl 2-(benzo[Yl[l,2,51oxadiazol-5-yl)-2,8-diazaspiro[4.51decane-8-carboxylate: The title compound was prepared in an analogous fashion to that described for
  • Step B 5-(2,8-Diazaspiror4.51decan-2-yl)benzorYin,2,51oxadiazole: The title was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from tert-butyl 2- (benzo[y][l,2,5]oxadiazol-5-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate .
  • Step A tert-butyl 2-([l,2,51oxadiazolo[3,4-Plpyridin-6-yl)-2,8-diazaspiro[4.51decane-8- carboxylate:
  • the title compound was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step A.
  • LC/MS: [(M+l)] + 360.
  • Step B 6-(2,8-diazaspiror4.51decan-2-yl)-n,2,51oxadiazolor3,4-b1pyridine: The title compound was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from tert-butyl 2-([l,2,5]oxadiazolo[3,4-P]pyridin-6-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate.
  • a microwave vial containing tert-butyl 3-oxo-2,8-diazaspiro[4.5]decane-8- carboxylate (INTERMEDIATE 15) (200 mg, 0.786 mmol), 5-bromobenzo[Y][l,2,5]oxadiazole (156 mg, 0.786 mmol), Pd 2 (dba) 3 (144 mg, 0.157 mmol), Xantphos (182 mg, 0.315 mmol) and cesium carbonate (384 mg, 1.18 mmol) in dioxane (3.9 mL) was sealed and evacuated and purged with nitrogen before heating in microwave to 120°C for 12 min.
  • Step B 2-(benzo[Yl[l,2,51oxadiazol-5-yl)-2,8-diazaspiro[4.51decan-3-one: The title compound was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from tert-butyl 2-(benzo[y][l,2,5]oxadiazol-5-yl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate.
  • Step A fert-butyl 2-(ri,2,51oxadiazolor3,4-P1pyridin-5-yl)-3-oxo-2,8-diazaspiror4.51decane-8- carboxylate:
  • the title compound was prepared in an analogous fashion to that described for INTERMEDIATE 47.
  • LC/MS: [(M+l)] + 374.
  • Step B 2-([l,2,510xadiazolo[3,4-Plpyridin-5-yl)-2,8-diazaspiro[4.51decan-3-one: The title compound was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from the product of Step A.
  • Step A fert-butyl 2-([l,2,51oxadiazolo[3,4-Plpyridin-6-yl)-3-oxo-2,8-diazaspiro[4.51decane-8- carboxylate: The title was prepared in an analogous fashion to that described for
  • Step B 2-(ri,2,51xadiazolor3,4-P1pyridin-6-yl)-2,8-diazaspiror4.51decan-3-one: 2-([l,2,5]Oxadiazolo[3,4-P]pyridin-6-yl)-2,8-diazaspiro[4.5]decan-3-one was prepared in an analogous fashion to that described for INTERMEDIATE 42, Step B from tert-butyl 2- ([l,2,5]oxadiazolo[3,4-P]pyridin-6-yl)-3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate.
  • 1,4-dioxanes (1ml) was added. The vial was then capped and the mixture was heated at 98°C overnight. The reaction mixture was cooled to room temperature, water (1 mL) and ethyl acetate (3 mL) were added. The organic layer was then collected.
  • Step A tert-butyl 2-(ri,2,41triazolor4,3-pipyridazin-6-yl)-3-oxo-2,8-diazaspiror4.51decane-8- carboxylate: A mixture of tert-butyl 3-oxo-2,8-diazaspiro[4.5]decane-8-carboxylate
  • Step B 2-([l,2,41triazolo[4,3-Plpyridazin-6-yl)-2,8-diazaspiro[4.51decan-3-one:
  • the compound from Step A was dissolved in 3 mL of 4N HC1 in 1,4-dioxane. The mixture was stirred at rt overnight. LC-MS showed the completion of the reaction. Removal of solvent gave a solid which was filtered and washed with EtOAc. The crude 2-([l,2,4]triazolo[4,3-P]pyridazin-6-yl)- 2,8-diazaspiro[4.5]decan-3-one thus obtained was without further purification.
  • Step A fert-butyl 8-(ri,2,41triazolor4,3-pipyridazin-6-yl)-2,8-diazaspiror4.51decane-2- carboxylate: tert-Butyl 2,8-diazaspiro[4.5]decane-2-carboxylate (1.0 g, 4.16 mmol), 6-chloro- [l,2,4]triazolo[4,3-P]pyridazine (0.707 g, 4.58 mmol), and DIEA (1.45 mL, 8.32 mmol) were mixed in DMA (5 mL). The mixture was heated at 95°C overnight.
  • Step B 6-(2,8-diazaspiror4.51decan-8-yl)-n,2,41triazolor4,3-pipyridazine:
  • the crude tert-butyl 8-([l ,2,4]triazolo[4,3-P]pyridazin-6-yl)-2,8-diazaspiro[4.5]decane-2-carboxylate from Step A was treated with 5 mL of 4N HC1 in dioxane. The mixture was stirred at room temperature overnight then heated at 60°C for 3 hours until complete removal of Boc group.
  • Step A fert-Butyl 2-(imidazori,2-a1pyrazin-8-yl)-2,8-diazaspiror4.51decane-8-carboxylate: A vial containing tert-butyl 2,8-diazaspiro[4.5]decane-8-carboxylate (263 mg, 1.09 mmol), 8- chloroimidazo[l,2-a]pyrazine (168 mg, 1.09 mmol) and triethyl amine (152 uL, 1.09 mmol) in THF (5.4 mL) heated to 50°C for 4 hours. The reaction was cooled and concentrated to give the crude material, which was purified via MPLC (50-100% EtOAc/hexanes) to afford the title compound. LC-MS (357, m/z): 358 [M+l] + .
  • Step B 8 -(2 , 8 -Diazaspiro [4.51 decan-2- vDimidazo 1 ,2-alp yrazine :
  • the title compound was prepared in analogous fashion to that described for INTERMEDIATE 42, Step B from tert-butyl 2-(imidazo[l,2-a]pyrazin-8-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate.
  • Example 1 The compounds in Table 1 were prepared in an analogous fashion to Example 1 starting from INTERMEDIATE 17, 18, 20, 21, or 22 and the corresponding halide.
  • the column in Table 1 with the heading INT provides the numbers which represent the intermediates that were used in the syntheses.
  • the vial was purged with N 2 gas, sealed and heated at 85°C overnight.
  • the reaction mixture was cooled to room temperature, filtered and the filtrates were concentrated.
  • the residue was purified by semi-preparative HPLC (focused gradient 0-40% ACN over 12 minutes using 0.1%TFA as the acidic modifier). The pure fractions were combined and the solvents were removed in vacuo.
  • Potassium phosphate (111 mg, 0.523 mmol), trans-(lR,2R)-N,N'-bismethyl-l,2- cyclohexanediamine (8.24 ⁇ , 0.052 mmol), 7-bromo-[l,3]dioxolo[4,5-b]pyridine (35 mg, 0.174 mmol), (R)-8-(2-hydroxy-2-(4-methyl- 1 -oxo- 1 ,3-dihydroisobenzofuran-5-yl)ethyl)-2,8- diazaspiro[4.5]decan-l-one (INTERMEDIATE 23) (60 mg, 0.174 mmol), and copper(I) iodide (9.95 mg, 0.052 mmol) were charged to a microwave vial.
  • Example 39 The column having the heading INT provides the numbers that represent each of the two intermediates that were combined to make each exemplified compound.
  • a microwave tube was charged with 6-(2,8-diazaspiro[4.5]decan-2-yl)tetrazolo[l,5- b]pyridazine (INTERMEDIATE 26) (0.025 g, 0.096 mmol), 4-methoxy-6-(oxiran-2- yl)nicotinonitrile (INTERMEDIATE 6, isomer B) (0.017 g, 0.096 mmol) and ethanol (1.0 mL).
  • the solution was degassed and filled with nitrogen (3X), then sealed and heated in a microwave reactor to 140°C for 1 hour. The reaction was cooled to room temperature and concentrated in vacuo.
  • HEK293 cells stably expressing hROMK (hK ir l .1) were grown at 37°C in a 10% C0 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, the media was aspirated from the flask and rinsed with 10 mL calcium/magnesium- free phosphate buffered saline (PBS). 5 mL of IX trypsin (prepared in Ca/Mg Free PBS) was added to T-225 flask and the flask was returned to 37°C/C0 2 incubator for 2-3 minutes.
  • PBS calcium/magnesium- free phosphate buffered saline
  • the side of the flask was gently banged with one's hand.
  • the cells were completely titrated and then the cells were transferred to 25 mL complete media, centrifuged at 1,500 rpm for 6 min followed by resuspension in complete growth media, and the cell concentration was determined.
  • 4E6 cells/T-225 flask will attain >80% confluency in 4 days. Under ideal growth conditions and appropriate tissue culture practices, this cell line is stable for 40-45 passages.
  • 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
  • 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 was performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKirl .1 cells were 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 was replaced with the FluxORTM reagent loading buffer and incubated, protected from light, at ambient temperature (23-25°C) for 90 min. The loading buffer was replaced with assay buffer ⁇ test compound followed by 30 min incubation at ambient temperature, where the thallium/potassium stimulant was 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 ⁇ .
  • Thallium Flux Assay are shown in Table 5 below. All of the tested final product compounds in the Examples (diastereomeric mixtures and individual diastereomers) had IC50 potencies less thanl ⁇ as determined by the Thallium Flux Assay.
  • Example Number IC50 ( ⁇ ) Example Number IC50 ( ⁇ )

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