US20120302562A1 - 4h-thieno[3,2-c]chromene-based inhibitors of notum pectinacetylesterase and methods of their use - Google Patents

4h-thieno[3,2-c]chromene-based inhibitors of notum pectinacetylesterase and methods of their use Download PDF

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US20120302562A1
US20120302562A1 US13/478,529 US201213478529A US2012302562A1 US 20120302562 A1 US20120302562 A1 US 20120302562A1 US 201213478529 A US201213478529 A US 201213478529A US 2012302562 A1 US2012302562 A1 US 2012302562A1
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Prior art keywords
thieno
carboxylic acid
thiophene
mmol
dihydronaphtho
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Inventor
Joseph Barbosa
Kenneth Gordon Carson
Michael Walter GARDYAN
Jason Patrick Healy
Qiang Han
Ross Mabon
Praveen PABBA
James TARVER, JR.
Kristen M. Terranova
Ashok Tunoori
Xiaolian Xu
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Lexicon Pharmaceuticals Inc
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Lexicon Pharmaceuticals Inc
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Priority to US13/478,529 priority Critical patent/US20120302562A1/en
Assigned to LEXICON PHARMACEUTICALS, INC. reassignment LEXICON PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TUNOORI, ASHOK, PABBA, PRAVEEN, HEALY, JASON PATRICK, TARVER, JAMES, JR., BARBOSA, JOSEPH, CARSON, KENNETH GORDON, GARDYAN, MICHAEL WALTER, HAN, QIANG, MABON, ROSS, TERRANOVA, Kristen M.
Assigned to LEXICON PHARMACEUTICALS, INC. reassignment LEXICON PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: XU, XIAOLIAN
Publication of US20120302562A1 publication Critical patent/US20120302562A1/en
Priority to US14/284,106 priority patent/US9624238B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes

Definitions

  • This invention relates small molecule inhibitors of Notum Pectinacetylesterase, compositions comprising them, and methods of their use.
  • Bone health depends on the coordinated activities of bone forming osteoblasts and bone resorbing osteoclasts. “Bone turnover reflects a balance between these anabolic and catabolic cellular functions and ensures that the mature skeleton can repair itself when damaged and sustain its endocrine function by release of minerals such as calcium and phosphorous into the circulation.” Allen, J. G. et al., J. Med. Chem., 53 (Jun. 10, 2010), pp. 4332-4353, 4332. Many disease states alter this balance, resulting in increased or decreased bone mass or changes in bone quality. Gradual loss of bone mineral density is known as osteopenia; severe loss of bone is known as osteoporosis. Id.
  • the current standard of care for the treatment and prevention of osteoporosis utilizes the bisphosphonate class of oral, small molecule antiresportives. Id. at 4333. Zoledronic acid, raloxifene, calcium, and vitamin D supplements are also typically used in the osteoporosis treatment. Id. While antiresporptive agents can help prevent bone loss, anabolic agents “are capable of increasing bone mass to a greater degree . . . and also have the capacity to improve bone quality and increase bone strength.” Guo, H., et al., J. Med. Chem., 53 (Feb. 25, 2010), pp. 1819-1829, 1819. In the United States, human PTH is the only FDA-approved anabolic agent.
  • WNT pathway Another of these pathways is the WNT pathway, which is implicated in a variety of developmental and regenerative processes. Allen at 4340.
  • the pathway is complex, however, and much about it and about how its components affect bone remains unclear. For example, it has been suggested that LRP-5, mutations of which are associated with increased bone mass in humans, and ⁇ -catenin, through which canonical WNT signaling occurs, “may not be linked directly via WNT signaling to the control of bone mass.” Id.
  • This invention encompasses compounds of the formula:
  • A is aryl or a 5- or 6-membered heterocycle
  • X is C(R 4 ) 2 , C(R 4 ), O, S, S(O), or S(O) 2
  • each R 1 is independently R 1A or alkyl or heteroalkyl optionally substituted with one or more R 1A
  • each R 1A is independently alkoxyl, amido, amino, carbamide, carboxyl, cyano, halo, hydroxyl, nitro, sulfanyl, sulfinyl, sulfonyl, or thio
  • n is 0-4
  • R 2 is —C(O)R 2A or 5- or 6-membered cycloalkyl or heterocycle optionally substituted with one or more R 2B
  • R 2A is —OR 2C , —N(R 2C ) 2 , —C(R 2C ) 2 NO 2 , —C(R 2C ) 2 OR 2C
  • This invention also encompasses pharmaceutical compositions comprising the compounds disclosed herein, and methods of their use.
  • This invention further encompasses methods of inhibiting NOTUM, methods of stimulating endocortical bone formation, and methods of treating, managing, and preventing diseases and disorders associated with bone loss, such as osteoporosis.
  • FIG. 1 provides a graphical representation of differences between the cortical thicknesses of various bone sites in NOTUM homozygous knockout mice (“HOM”) and those in their wildtype littermates (“WT”).
  • HOM NOTUM homozygous knockout mice
  • WT wildtype littermates
  • FIG. 2 provides a graphical representation of an increase in cortical bone thicknesses observed in both NOTUM homozygous and heterozygous (“HET”) knockout mice as compared to their wildtype littermates.
  • FIG. 3 provides a graphical representation of results obtained from femur breaking strength and spine compression tests performed on the bones of male NOTUM homozygous and heterozygous knockout mice and their wildtype littermates.
  • FIG. 4 provides a graphical representation of results obtained from femur breaking strength and spine compression tests performed on the bones of female NOTUM homozygous and heterozygous knockout mice and their wildtype littermates
  • FIG. 5 shows the effects of administering 6,9-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid on the midshaft femur cortical thickness of mice.
  • FIG. 6 shows the effects of administering 4H-thieno[3,2-c]thiochromene-2-carboxylic acid 5,5-dioxide on the midshaft femur cortical thickness of mice.
  • FIG. 7 shows the effects of administering 6,8-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid on the midshaft femur cortical thickness of mice.
  • This invention is based, in part, on the discovery that inhibition of NOTUM can affect endocortical bone formation.
  • Particular aspects of the invention are based on studies of mice lacking a functional NOTUM gene (“knockout mice”), on the discovery of compounds that inhibit NOTUM, and on the discovery that such compounds can be used to stimulate cortical bone formation in mice and rats.
  • alkenyl means a C 2-20 (e.g., C 2-10 , C 2-4 ) straight chained, branched or cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • Representative alkenyl moieties include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl and 3-decenyl.
  • alkyl means a C 1-20 (e.g., C 1-10 , C 1-4 ) straight chained, branched or cyclic hydrocarbon. C 1-4 alkyl moieties are referred to as “lower alkyl.” Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.
  • Cycloalkyl moieties may be monocyclic or multicyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Additional examples of alkyl moieties have linear, branched and/or cyclic portions (e.g., 1-ethyl-4-methylcyclohexyl).
  • alkyl includes saturated hydrocarbons as well as alkenyl and alkynyl moieties.
  • alkoxy means an —O-alkyl group.
  • alkoxy groups include —OCH 3 , —OCH 2 CH 3 , —O(CH 2 ) 2 CH 3 , —O(CH 2 ) 3 CH 3 , —O(CH 2 ) 4 CH 3 , and —O(CH 2 ) 5 CH 3 .
  • alkynyl means a C 2-20 (e.g., C 2-10 , C 2-4 ) straight chain, branched or cyclic hydrocarbon containing at least one carbon-carbon triple bond.
  • Representative alkynyl moieties include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyn
  • aryl means a C 6-12 aromatic or partially aromatic ring or ring system composed of carbon and hydrogen atoms.
  • An aryl moiety may comprise multiple rings bound or fused together.
  • Examples of aryl moieties include anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, and tolyl.
  • halogen and “halo” encompass fluorine, chlorine, bromine, and iodine.
  • heteroalkyl refers to an alkyl moiety (e.g., linear, branched or cyclic) in which at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S).
  • heteroaryl means an aryl moiety wherein at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S).
  • heteroatom e.g., N, O or S.
  • examples include acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and tri
  • heterocycle refers to 5-12-membered (e.g., 5, 6-membered) monocyclic or polycyclic ring or ring system comprised of carbon, hydrogen and at least one heteroatom (e.g., N, O or S).
  • a heterocycle may comprise multiple (i.e., two or more) rings fused or bound together.
  • Heterocycles include heteroaryls.
  • Examples include benzo[1,3]dioxolyl, 2,3-dihydro-benzo[1,4]dioxinyl, cinnolinyl, furanyl, hydantoinyl, morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl, pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl and valerolactamyl.
  • hydrogen or “H” encompasses both hydrogen and deuterium.
  • a generic (e.g., markush) chemical structure comprising hydrogen atoms is to be interpreted as encompassing their deuterated equivalents.
  • the terms “manage,” “managing” and “management” encompass preventing the recurrence of the specified disease or disorder, or of one or more of its symptoms, in a patient who has already suffered from the disease or disorder, and/or lengthening the time that a patient who has suffered from the disease or disorder remains in remission.
  • the terms encompass modulating the threshold, development and/or duration of the disease or disorder, or changing the way that a patient responds to the disease or disorder.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid.
  • inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethe
  • Non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids.
  • Examples of specific salts thus include hydrochloride and mesylate salts.
  • Others are well-known in the art. See, e.g., Remington's Pharmaceutical Sciences, 18 th ed. (Mack Publishing, Easton Pa.: 1990) and Remington: The Science and Practice of Pharmacy, 19 th ed. (Mack Publishing, Easton Pa.: 1995).
  • the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a patient begins to suffer from the specified disease or disorder, which inhibits or reduces the severity of the disease or disorder, or of one or more of its symptoms.
  • the terms encompass prophylaxis.
  • a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or condition, or one or more symptoms associated with the disease or condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • substituted when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with an atom, chemical moiety or functional group such as, but not limited to, alcohol, aldehylde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (—OC(O)alkyl), amide (—C(O)NH-alkyl- or -alkylNHC(O)alkyl), amidinyl (—C(NH)NH-alkyl or —C(NR)NH 2 ), amine (primary, secondary and tertiary such as alkylamino, arylamino, arylalkylamino), aroyl, aryl,
  • the term substituted refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with alcohol, alkoxy, alkyl (e.g., methyl, ethyl, propyl, t-butyl), amide (—C(O)NH-alkyl- or -alkylNHC(O)alkyl), amidinyl (—C(NH)NH-alkyl or —C(NR)NH 2 ), amine (primary, secondary and tertiary such as alkylamino, arylamino, arylalkylamino), aryl, carbamoyl (—NHC(O)O-alkyl- or —OC(O)NH-alkyl), carbamyl (e.g., CONH 2 , as well as CONH-alkyl, CONH-aryl), halo, haloalkyl (e.g., —CCl 3 , —CF
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition.
  • a therapeutically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease or condition.
  • the term “therapeutically effective amount” encompasses an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a patient is suffering from the specified disease or disorder, which reduces the severity of the disease or disorder, or one or more of its symptoms, or retards or slows the progression of the disease or disorder.
  • the term “include” has the same meaning as “include, but are not limited to” and the term “includes” has the same meaning as “includes, but is not limited to.” Similarly, the term “such as” has the same meaning as the term “such as, but not limited to.”
  • one or more adjectives immediately preceding a series of nouns is to be construed as applying to each of the nouns.
  • the phrase “optionally substituted alky, aryl, or heteroaryl” has the same meaning as “optionally substituted alky, optionally substituted aryl, or optionally substituted heteroaryl.”
  • a chemical moiety that forms part of a larger compound may be described herein using a name commonly accorded it when it exists as a single molecule or a name commonly accorded its radical.
  • the terms “pyridine” and “pyridyl” are accorded the same meaning when used to describe a moiety attached to other chemical moieties.
  • the two phrases “XOH, wherein X is pyridyl” and “XOH, wherein X is pyridine” are accorded the same meaning, and encompass the compounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.
  • stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or the portion of the structure is to be interpreted as encompassing all stereoisomers of it.
  • names of compounds having one or more chiral centers that do not specify the stereochemistry of those centers encompass pure stereoisomers and mixtures thereof.
  • any atom shown in a drawing with unsatisfied valences is assumed to be attached to enough hydrogen atoms to satisfy the valences.
  • chemical bonds depicted with one solid line parallel to one dashed line encompass both single and double (e.g., aromatic) bonds, if valences permit.
  • This invention encompasses tautomers and solvates (e.g., hydrates) of the compounds disclosed herein.
  • This invention encompasses compounds, and methods of using compounds, of the formula:
  • A is aryl or a 5- or 6-membered heterocycle
  • X is C(R 4 ) 2 , C(R 4 ), O, S, S(O), or S(O) 2
  • each R 1 is independently R 1A or alkyl or heteroalkyl optionally substituted with one or more R 1A
  • each R 1A is independently alkoxyl, amido, amino, carbamide, carboxyl, cyano, halo, hydroxyl, nitro, sulfanyl, sulfinyl, sulfonyl, or thio
  • n is 0-4
  • R 2 is —C(O)R 2A or a 5- or 6-membered cycloalkyl or heterocycle optionally substituted with one or more R 2B
  • R 2A is —OR 2C , —N(R 2C ) 2 , —C(R 2C ) 2 NO 2 , —C(R 2C ) 2
  • X and Z are each independently CH, CH 2 , N, NH, O, or S.
  • Y is S and Z is N or NH.
  • Y is CH and Z is 0.
  • Y is S and Z is CH.
  • X is C(R 4 ) 2 , C(R 4 ), O, S, S(O), or S(O) 2 ; each R 1 is independently R 1A or alkyl or heteroalkyl optionally substituted with one or more R 1A ; each R 1A is independently alkoxyl, amido, amino, carbamide, carboxyl, cyano, halo, hydroxyl, nitro, sulfanyl, sulfinyl, sulfonyl, or thio; n is 0-4; R 2 is —C(O)R 2A or a 5- or 6-membered cycloalkyl or heterocycle optionally substituted with one or more R 2B ; R 2A is —OR 2C , —N(R 2C ) 2 , —C(R 2C ) 2 NO 2 , —C(R 2C ) 2 OR 2C , —C(R 2C ) 2 CN
  • R 2 when X is CH 2 , n is 0, m is 0, and R 2 is —C(O)R 2A , R 2A is not —OR 2C or —N(R 2C ) 2 .
  • R 2 when X is 0, n is 0, m is 0, and R 2 is —C(O)R 2A , R 2A is not —O-alkyl.
  • R 1 when X is 0, n is 1, R 1 is fluoro, m is 0, and R 2 is —C(O)R 2A , R 2A is not —O-alkyl.
  • R 3 when X is 0, n is 0, R 3 is not methyl.
  • X is C(R 4 ) 2 . In some, at least one R 4 is H.
  • the bond between X and C 4 is a single bond.
  • X is 0. In some, X is S, S(O), or S(O) 2 .
  • R 1 is R 1A .
  • R 1A is halo.
  • R 1A is cyano.
  • R 1A is nitro.
  • R 2 is C(O)OH. In some, R 2 is optionally substituted 5- or 6-membered heterocycle.
  • n is 2. In some, n is 3.
  • m is 0 (i.e., one or two hydrogen atoms are bound to the C 4 carbon, depending on whether there is a double bond between it and X).
  • n 1 and R 3 is methyl.
  • thieno[3,2-c]chromene carboxylic acids dihydronaptho[1,2-b]thiophene-2-carboxylic acids
  • thieno[3,2-c]thiochromene-2-carboxylic acid derivatives were synthesized from the corresponding substituted chroman-4-ones, dihydronaphthalen-1(2H)-ones and thiochroman-4-ones, as shown in Scheme 1.
  • ketones were treated with phosphorus oxychloride and dimethylformamide under Vislmeier-Hack conditions to generate the ⁇ , ⁇ -unsaturated ⁇ -chloroaldehydes (B). Subsequent treatment with ethyl mercaptoacetate and sodium ethoxide effected cyclization to generate substituted thiophenes (C). Saponification of the esters with aqueous base yielded a range of carboxylic acids (D). These acids could be further functionalized to amides, heterocycles or ketoacids, as demonstrated herein.
  • This invention encompasses methods of inhibiting NOTUM, which comprise contacting NOTUM with a compound of the invention (i.e., a compound disclosed herein).
  • This invention also encompasses a method of upregulating the WNT pathway, which comprises contacting NOTUM with a compound of the invention.
  • This invention encompasses a method of stimulating endocortical bone formation in a patient, which comprises administering to a patient in need thereof an effective amount of a compound of the invention. It also encompasses a method of increasing cortical bone thickness, comprising administering to a patient in need thereof an effective amount of a compound of the invention.
  • This invention encompasses a method of treating, managing, or preventing a disease or disorder associated with bone loss, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention.
  • diseases and disorders include osteoporosis (e.g., postmenopausal osteoporosis, steroid- or glucocorticoid-induced osteoporosis), osteopenia, and Paget's disease.
  • Also encompassed by the invention is a method of accelerating or facilitating bone healing in a patient, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention.
  • This invention also encompasses a method of treating, managing, or preventing bone fractures, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention.
  • Particular bone fractures are associated with metastatic bone disease, i.e., cancer that has metastasized to bone.
  • metastatic bone disease i.e., cancer that has metastasized to bone.
  • cancers that can metastasize to bone include prostate, breast, lung, thyroid, and kidney cancer.
  • This invention also encompasses a method of treating, managing, or preventing bone loss associated with, or caused by, a disease or disorder, which comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention.
  • diseases and disorders include celiac disease, Crohns Disease, Cushing's syndrome, hyperparathyroidism, inflammatory bowel disease, and ulcerative colitis.
  • Examples of patients that may benefit from methods of this invention include men and women aged 55 years or older, post-menopausal women, and patients suffering from renal insufficiency.
  • Compounds of the invention can be administered in combination (e.g., at the same or at different times) with other drugs known to be useful in the treatment, management, or prevention of diseases or conditions affecting the bone.
  • drugs known to be useful in the treatment, management, or prevention of diseases or conditions affecting the bone.
  • examples include: androgen receptor modulators; bisphosphonates; calcitonin; calcium sensing receptor antagonists; cathepsin K inhibitors; estrogen and estrogen receptor modulators; integrin binders, antibodies, and receptor antagonists; parathyroid hormone (PTH) and analogues and mimics thereof; and Vitamin D and synthetic Vitamin D analogues.
  • Examples of androgen receptor modulators include finasteride and other 5 ⁇ -reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
  • bisphosphonates include alendronate, cimadronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, piridronate, risedronate, tiludronate, and zolendronate, and pharmaceutically acceptable salts and esters thereof.
  • cathepsin K inhibitors examples include VEL-0230, AAE581 (balicatib), MV061194, SB-462795 (relacatib), MK-0822 (odanacatib), and MK-1256.
  • estrogen and estrogen receptor modulators include naturally°Ccurring estrogens (e.g., 7-estradiol, estrone, and estriol), conjugated estrogens (e.g., conjugated equine estrogens), oral contraceptives, sulfated estrogens, progestogen, estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424, tamoxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzo
  • integrin binders examples include vitaxin (MEDI-522), cilengitide and L-000845704.
  • This invention encompasses pharmaceutical compositions comprising one or more compounds of the invention, and optionally one or more other drugs, such as those described above.
  • compositions are single unit dosage forms suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), or transdermal administration to a patient.
  • mucosal e.g., nasal, sublingual, vaginal, buccal, or rectal
  • parenteral e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial
  • transdermal administration e.g., transdermal administration to a patient.
  • dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
  • suspensions e.g., aqueous
  • the formulation should suit the mode of administration.
  • oral administration requires enteric coatings to protect the compounds of this invention from degradation within the gastrointestinal tract.
  • a formulation may contain ingredients that facilitate delivery of the active ingredient(s) to the site of action.
  • compounds may be administered in liposomal formulations, in order to protect them from degradative enzymes, facilitate transport in circulatory system, and effect delivery across cell membranes to intracellular sites.
  • composition, shape, and type of a dosage form will vary depending on its use.
  • a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease.
  • a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease.
  • compositions of this invention are preferably administered orally.
  • Discrete dosage forms suitable for oral administration include tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
  • Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18 th ed. (Mack Publishing, Easton Pa.: 1990).
  • Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • tablets and capsules represent the most advantageous oral dosage unit forms.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • Such dosage forms can be prepared by conventional methods of pharmacy.
  • pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • Disintegrants may be incorporated in solid dosage forms to facility rapid dissolution.
  • Lubricants may also be incorporated to facilitate the manufacture of dosage forms (e.g., tablets).
  • compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
  • dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
  • Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • tablets and capsules represent the most advantageous oral dosage unit forms.
  • tablets can be coated by standard aqueous or non-aqueous techniques.
  • Such dosage forms can be prepared by conventional methods of pharmacy.
  • pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • Disintegrants may be incorporated in solid dosage forms to facility rapid dissolution. Lubricants may also be incorporated to facilitate the manufacture of dosage forms (e.g., tablets).
  • Parenteral dosage forms can be administered to patients by various routes including subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection
  • water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol
  • mice homozygous for a genetically engineered mutation in the murine ortholog of the human NOTUM gene were generated using corresponding mutated embryonic stem (ES) cell clones from the OMNIBANK collection of mutated murine ES cell clones (see generally, U.S. Pat. No. 6,080,576).
  • ES cell clones containing a mutagenic viral insertion into the murine NOTUM locus were microinjected into blastocysts which were in turn implanted into pseudopregnant female hosts and carried to term.
  • the resulting chimeric offspring were subsequently bred to C57 black 6 female mice and the offspring checked for the germline transmission of the knocked-out NOTUM allele.
  • mice homozygous ( ⁇ / ⁇ ) for the disruption of the NOTUM gene were studied in conjunction with mice heterozygous (+/ ⁇ ) for the disruption of the NOTUM gene and wild-type (+/+) litter mates.
  • mice were subject to a medical work-up using an integrated suite of medical diagnostic procedures designed to assess the function of the major organ systems in a mammalian subject.
  • homozygous ( ⁇ / ⁇ ) “knockout” mice in the described numbers and in conjunction with heterozygous (+/ ⁇ ) and wild-type (+/+) litter mates, more reliable and repeatable data were obtained.
  • These differences which were measured by microCT (Scanco ⁇ CT40), were: 28% (p ⁇ 0.001) at midshaft femur; 19% (p ⁇ 0.001) at midshaft humerous; 17% (p ⁇ 0.001) at midshaft tibia; and 11% (p ⁇ 0.001) at tibia-fibula junction.
  • FIGS. 3 and 4 show results of femur breaking strength tests (performed by SkeleTech, now Ricerca Biosciences) using a standard 4-point bending test.
  • spine compression tests of both NOTUM homs and hets did not show a significant reduction in maximum spine compression loads as compared to wildtype controls.
  • EC 50 values were determined using this assay, which utilized conditioned media that was prepared as follows. Plasmid containing human notum pectinacetyltransferase in pcDNA3.1(+) vector was transfected into HEK (293 cells and clones were selecting by growing in presence of 400 ug/mL of G418. The clone containing highest expression of human notum pectinacetyltransferase in the conditioned media was maintained for all future activity assays. L cells overexpressing and secreting Wnt3a into the conditioned media were purchased from ATCC.
  • the assay protocol was as follows. Approximately 5 million CellSensor®LEF/TCF-blaFreeStyleTM 293Fcells were grown to near confluency in 15-cm plates.
  • the cell growth medium consisted of DMEM with 10% Dialyzed FBS, 5 ⁇ g/ml Blasticidin (Invitrogen, R210-01), 0.1 mM NEAA, 25 mM HEPES and 1 ⁇ GPS. Cells were then trypsinized by first rinsing with PBS, followed by addition of 5 mL of trypsin and incubation of plates at room temperature for two minutes.
  • a total of 10 mL of assay media (Opti-MEM, plus 0.5% dialyzed FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 10 mM HEPES, 1 ⁇ GPS) was then added per 15 cm plate.
  • Cells were counted and suspended at 0.75 million cells per mL.
  • Cells were seeded into Biocoat 384-well plates (Fisher, Catalogue #356663) at a density of 15000 cells per 20 ⁇ L per well. After incubation of cells at 37° C. for 3 hours, 10 ⁇ L of 30 mMLiCl in assay medium was added per well, followed by incubation at 37° C. for another 3 hours.
  • mice C57BI/6-Albino/129SvEv mice were injected intravenously (i/v) via the lateral tail vein with a single sublethal dose of Concanavalin A from Canavalia ensiformis (Jack bean, Type IV-S, lyophilized powder, aseptically processed; Sigma) administered at 10-16 mg/kg mouse body weight in a total volume of 0.1-0.3 ml of pyrogen-free PBS.
  • the tail vein injections of mice restrained in a Plexiglas mouse restrainer were performed without anesthesia, using a 1 ml syringe with a 27 gauge needle.
  • blood samples were collected by retro-orbital bleeding.
  • mice were sacrificed and sera from the blood samples were analyzed for the presence of IL-12, TNF- ⁇ , MCP-1, IFN- ⁇ , IL-10, and IL-6 using a mouse inflammation cytometric bead array (CBA) kit (BD Biosciences, Mountain View, Calif.), according to the manufacturer's instructions. Data were acquired with a FACSCalibur flow cytometer and analyzed with BD CBA Software (BD Biosciences). Biochemical markers of liver failure were assessed by measuring serum liver damage enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), using a standard clinical biochemical analyzer. Livers of ConA-treated mice were sectioned and stained with H&E to evaluate the degree of T cell-mediated immune inflammation.
  • CBA mouse inflammation cytometric bead array
  • IC 50 values were determined using this assay, which utilized trisodium 8-octanoyloxypyrene-1,3,6-trisulfonate (OPTS), a water soluble enzyme substrate for fluorimetric assays of esterases and lipases.
  • Plasmid containing human notum pectinacetyltransferase in pcDNA3.1(+) vector was transfected into HEK (293 cells and clones were selecting by growing in presence of 400 ug/mL of G418. Condition media from these cells was used for the assay.
  • ECHO was used to acoustically dispense 75 mL of compounds into dry Greiner 384-well plates (catalog #781076), followed by addition of 10 uL of 50 mMTris/HCl (pH 6.8) to every well of these 384-well assay plates.
  • Conditioned media containing human notum pectinacetyltransferase was diluted 75 ⁇ with Assay Buffer (50 mMTris, pH 6.8, 5 mM CaCl (2, 0.5 mM MgCl (2), and 25 ⁇ L of this “Enzyme Mix” was added to each well followed by a 10 minute pre-incubation.
  • Enzyme reactions were initiated by addition of 15 ⁇ L OPTS substrate (Sigma, catalog #74875) to a final concentration of 5 ⁇ M and reaction times were for 10 minutes at room temperature. All plates were read on an Envision Plate Reader with an excitation wavelength of 485 nm and emission wavelength of 535 nm.
  • 6-chloro-8-fluorochroman-4-one 1 (5 g, 24.9 mmol) was added dropwise to a solution of phosphorous oxychloride (2.3 mL, 24.9 mmol) in 15 mL of DMFat 0° C. The reaction mixture was stirred at 0° C. for 30 minutes then heated to 80° C. for 1.5 hours. Reaction was then cooled to room temperature and quenched with 1N NaOAc solution and extracted with dichloromethane (2 ⁇ 25 mL). The organic layer was concentrated in vacuo and carried to the next step without further purification.
  • ester 3 was taken up in THF and excess 1N NaOH solution and stirred at 50° C. for 24 hr.
  • the reaction mixture was cooled to room temperature, washed with CH 2 Cl 2 (2 ⁇ 50 mL) and acidified with 1 N HCl.
  • the resulting solid product was filtered, washed with water and dried to obtain 8-chloro-6-fluoro-4H-thieno[3,2-c]chromene-2-carboxylic acid 4 (3.89 g, 55% yield) as an off white solid.
  • the crude mixture was purified by silica gel chromatography (ethyl acetate-hexanes) to give ethyl 7-methoxy-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate 7 (0.292 g, 1.01 mmol, 71% yield) as a pale yellow waxy solid.
  • the ester could be carried to next step without chromatographic purification.
  • ester 11 was taken up in THF and excess 1N NaOH solution and stirred at 50° C. for 24 hr.
  • the reaction mixture was cooled to room temperature and washed with dichloromethane then acidified with 1 N HCl.
  • the resulting solid product was filtered, washed with water and dried to obtain 6,7-difluoro-4,5-dihydronaptho[1,2-b]thiophene-2-carboxylic acid 12 (7.69 g, 65% yield) as a white solid.
  • Phosphorous oxychloride (58.99 mmol, 5.4 mL) was added drop wise to dry DMF (26 mL) at 0° C.
  • 10.4 g 5,8-difluorothiochroman-4-one 13 (51.74 mmol) was added slowly while keeping the temperature less than 5° C.
  • the reaction mixture was stirred for 0.5 hours and then heated at 80° C. for 1.5 hours.
  • the reaction mixture was cooled to room temperature, and then poured into 9 mL ice cold aqueous sodium acetate (25%, W/V) solution. The mixture was extracted by diethyl ether twice.
  • reaction mixture was filtered, rinsed with 1:3 MeOH/H2O solution, and dried under vacuum to afford 8.91 g ethyl 6,9-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylate 15 as an off-white solid.
  • ketone 5 (5 g, 28.4 mmol) and N-chlorosuccinimide (3.78 g, 28.4 mmol) were suspended in water and stirred at 60° C. Then 40% aqueous sulfuric acid (7.6 mL, 56.7 mmol) was added slowly and the reaction mixture stirred at 60° C. for 6 hours. Then the reaction mixture was cooled to room temperature, filtered and washed with water.
  • ketone 5 (1 g, 5.68 mmol) was taken up in acetonitrile (8 mL) and selectfluor (3.02 g, 8.5 mmol) was added and the reaction stirred at room temperature for 2 hours before heated to 40° C. for 48 hours. Then the reaction mixture was cooled to room temperature and concentrated in vacuo then re-dissolved in methanol and filtered. The methanol layer was concentrated in vacuo and purification by silica gel column chromatography with hexane/ethyl acetate (0 to 30%) to produce 5-fluoro-6-methoxy-3,4-dihydronapthalen-1(2H)-one 18 (395 mg, 36% yield).
  • ketone 5 (5 g, 28.4 mmol) and N-bromosuccinimide (5.05 g, 28.4 mmol) were taken up in water and stirred at 60° C. Then 40% aqueous sulfuric acid (7.6 mL, 56.7 mmol) was added slowly and the reaction mixture was stirred at 60° C. for 5 hours. The reaction mixture was cooled to room temperature and product filtered off and washed twice with water.
  • the solid mixture was purified by silica gel chromatography (hexane:ethyl acetate) to obtain pure 2-methoxy-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carbonitrile 21 (0.155 g, 30%) and 3-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalene-2-carbonitrile 22 (0.06 g, 12%).
  • crude acid 27 (7.02 g, 30 mmol) was suspended in polyphosphoric acid (15 mL) and heated at 100° C. for 20 minutes. Then the reaction mixture was cooled to room temp and ice was added while stirring. The resulting solid precipitate was filtered and washed twice with cold water. This crude mixture was purified by silica gel chromatography (ethyl acetate-hexanes) to afford 7-(trifluoromethyl)chroman-4-one 28 (0.899 g, 14.4% yield) and 5-(trifluoromethyl)chroman-4-one 29 (0.214 g, 3.4% yield).
  • Carboxylic acid compounds such as 16 were prepared by Synthetic Methods A-H.6,9-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid 16 (90 mg, 0.32 mmol), NH 4 Cl (125 mg, 2.36 mmol), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (168 mg, 0.38 mmol), and Et 3 N (0.27 mL, 1.94 mmol) were dissolved in DMF (2 mL) and stirred at room temperature for 2 hours. Addition of water to the reaction mixture precipitated the product. The precipitate was filtered, rinsed by 1:3 MeOH/H2O, H2O, heptanes, and dried in vacuo to afford carboxamide 30 (69.4 mg, 77%) as a white solid.
  • Carboxylic acid compounds such as 31 were prepared by Synthetic Methods A-H.
  • acid 31 (0.154 g, 0.57 mmol) was suspended in DMF (3 mL).
  • ammonium chloride 0.307 g, 5.7 mmol
  • HATU 0.327 g, 0.86 mmol
  • triethyl amine 960 ⁇ L, 6.9 mmol
  • Carboxylic acid compounds such as 33 were prepared by Synthetic Methods A-H.
  • a solution of acid 33 (0.04 g, 0.155 mmol) in dichloromethane (0.8 mL) was treated with oxalyl chloride (0.14 mL, 1.55 mmol) and 1 drop DMF. After stirring for 1 hour the reaction was concentrated thrice from dichlormethane.
  • the crude yellow orange solid (0.052 g, quant.) was dissolved in THF (1 mL) and added to an ice cold solution of ammonium hydroxide (30 ⁇ L, 0.755 mmol) in THF. The reaction slowly warmed to room temperature and stirred overnight. The reaction was then poured into 1 N HCl.
  • Acid 33 (0.06 g, 0.232 mmol, 1 eq.) was dissolved in dichloromethane (1.2 mL). This solution was treated with oxalyl chloride (0.20 mL, 2.32 mmol, 10 eq.) and 1 drop DMF. After stirring for 1 hour the reaction was concentrated thrice from dichloromethane. The crude yellow orange solid (0.0.0782 g, quant.) was dissolved in THF (1.5 mL) and was added to a solution of methyl 2-aminoacetate (0.0437 g, 0.348 mmol, 1.5 eq) in THF (0.5 mL).
  • Carboxylic acid compounds such as 36 were prepared by Synthetic Methods A-H. Acid 36 (25 mg, 1 eq, 0.11 mmol), 2-amino-ethanol (7 mg. 1 eq), EDCI (26 mg, 1.2 eq) and HOBT (18 mg, 1.2 eq) were combined in dichloromethane (1.5 mL) and the mixture stirred at room temperature over night. The mixture was diluted with Ethyl acetate, washed with H2O, dried over MgSO 4 and concentrated. Purification by preparative HPLC afforded the desired product 37 (10 mg, 33%). Compounds were purified by silica gel chromatography or preparative HPLC.
  • Synthesis of 38 Compounds such as 35 were prepared using Synthetic Method I. Ester 35 (0.043 g, 0.130 mmol) was dissolved in THF (0.2 mL) and methanol (0.3 mL) and aqueous sodium hydroxide (1.0 N, 0.14 mL, 0.143 mmol) was added. After stirring at ambient temperature for 30 minutes the reaction was concentrated. The solid residue was suspended in 1 N aqueous NaOH and water, cooled to 0° C. and acidified with 1 N aqueous HCl to pH ⁇ 2. The solid was collected via vacuum filtration, washed with water and dried, yielding acid 38 as an off-white solid (0.035 g, 86%).
  • Method 2 A solution of acid 41 (100 mg, 0.35 mmol) in peracetic acid (35% in acetic acid, 0.4 mL) was stirred at room temperature for 1 hr. The reaction mixture was quenched with dimethyl sulfide and the resulting mixture was stirred overnight. No peroxide was observed by a low level peracetic acid test strip and a peroxide test strip. The reaction was diluted with water, extracted with ethyl acetate twice and concentrated in vacuo. Preparative HPLC purification afforded 43 (65 mg, 59%) as a white solid.
  • bromide 59 (1.00 g, 4.44 mmol), potassium cyanide (0.318 g, 4.88 mmol), NiCl 2 (PPh 3 ) 2 (0.145 g, 0.222 mmol), triphenyl phosphine (0.116 g, 0.444 mmol) and zinc (0.087 g, 1.33 mmol) were suspended in acetonitrile (10 mL) then heated at 70° C. overnight. The reaction was cooled to room temperature then diluted with DCM (50 mL) and washed with H2O, brine solution, dried over MgSO 4 then concentrated in vacuo. The crude product was crystallized from hot ethyl acetate/hexanes to give nitrile 60 (0.380 g, 2.21 mmol, 50% yield) as a crystalline white solid.
  • ether 64 (0.700 g, 3.97 mmol) was added to a suspension of aluminum chloride (1.32 g, 9.92 mmol) in toluene (10 mL) at room temperature, then refluxed for 30 minutes. The reaction was cooled to room temperature and quenched with H2O (50 mL). The mixture was extracted with ethyl acetate thrice and the combined organic layers were washed with brine solution, dried over MgSO 4 and concentrated in vacuo.
  • triflate 66 (0.924 g, 3.14 mmol), potassium cyanide (0.224 g, 3.45 mmol), NiCl 2 (PPh 3 ) 2 (0.102 g, 0.157 mmol), PPh 3 (0.082 g, 0.314 mmol) and Zn (0.065 g, 1.00 mmol) were suspended in acetonitrile (10 mL) and heated at 70° C. for 4 hours. The reaction was cooled to room temperature then diluted in DCM (50 mL) and washed with H 2 O, brine solution, dried over MgSO 4 and concentrated in vacuo. The crude oil was purified by silica gel chromatography (ethyl acetate-hexanes gradient) to give nitrile 67 (0.408 g, 75%) as a crystalline white solid.
  • 3-Bromo-2-fluorotoluene 72 (0.465 g, 2.46 mmol) was taken up in THF (25 mL) and cooled to ⁇ 78° C. n-Butyllithium (2.5 M in hexanes, 1.2 mL, 2.95 mmol) was added and the reaction stirred for 30 minutes.
  • Another round bottom flask was charged with succinic anhydride (0.320 g, 3.20 mmol) and THF (4 mL), cooled to ⁇ 78° C. and equipped with a jacketed addition funnel, which was cooled to ⁇ 78° C. After the stirring period, the anion solution was transferred quickly to the cold addition funnel. The anion solution was added to the anhydride solution over 10 minutes.
  • Keto-acid 74 (1.62 g, 7.71 mmol) was dissolved in TFA (8 mL) and triethylsilane (3.7 mL, 23.1 mmol) was added. After stirring at ambient temperature overnight, the reaction was concentrated. The residue was taken up in ethyl acetate and washed twice with water. The organic phase was thrice extracted with 3 N NaOH and the combined aqueous layers were cooled to 0° C. and acidified with concentrated HCl to pH ⁇ 1. This acidic aqueous portion was thrice extracted with ethyl acetate and the combined organic layers were washed with water and brine, dried over MgSO 4 , filtered and concentrated. Acid 75 was isolated as a yellow solid (1.06 g, 70%) and used without further purification.
  • Acid 75 (0.204 g, 1.04 mmol) was dissolved in DCM (5 mL) and treated with oxalyl chloride (0.9 mL, 10.4 mmol) and 1 drop DMF. After stirring for 1 hour at ambient temperature this reaction was thrice concentrated from DCM. The isolated red-brown oil was taken up in dichloroethane (26 mL) and cooled to 0° C. Aluminum chloride (0.277 g, 2.08 mmol) was added in portions. After stirring at 0° C. for 1 hour the reaction was quenched by the dropwise addition of water and 1 N aqueous HCl.
  • Keto-acid 73 (0.433 g, 2.06 mmol) was dissolved in THF (12 mL) and 2 drops of concentrated sulfuric acid were added. 10% Palladium on carbon (50% wetted, 0.150 g, 35 mol %) was added and the flask was sealed. The flask was evacuated and backfilled three times with nitrogen, and then evacuated and backfilled three times with hydrogen; hydrogen was added to the flask until the pressure reached 50 psi. After 68 hours, the catalyst was filtered off using Celite and ethyl acetate. The filtrate was washed with water and brine, dried over MgSO 4 , filtered and concentrated, yielding acid 77 as a yellow oil (0.333 g, 83%), which was used without further purification.
  • Acid 77 (0.320 g, 0.163 mmol) was dissolved in DCM (8.5 mL) and treated with oxalyl chloride (1.5 mL, 16.3 mmol) and 1 drop DMF. After stirring for 1 hour at ambient temperature this reaction was thrice concentrated from DCM. The isolated clear yellow-brown oil (0.419 g, quant.) was taken up in dichloromethane (48 mL) and cooled to 0° C. Aluminum chloride (0.435 g, 3.26 mmol) was then added in portions. The reaction slowly warmed to room temperature with stirring overnight and was quenched by dropwise addition of water and 1 N aqueous HCl.
  • This aqueous phase was thrice extracted with ethyl acetate and the combined organic phase was washed with saturated aq. NaHCO 3 and brine, dried over MgSO 4 , filtered and concentrated.
  • the crude yellow-brown oil was purified by silica gel column chromatography (0-2% ethyl acetate-hexanes) yielding ketone 78 as a yellow oil (0.171 g, 59%).
  • Nitro-ester 79 (0.2060 g, 0.6491 mmol) was dissolved in ethyl acetate (6.5 mL). 10% palladium on carbon (50% wet, 0.0412 g, 10 mol %) was added and the flask was sealed. The flask was evacuated and backfilled three times with nitrogen, and then evacuated and backfilled three times with hydrogen (via balloon). Hydrogen was bubbled through the reaction for 5 minutes and the reaction was heated to 55° C. After 6 hours at 55° C., more 10% palladium on carbon (50% wet, 0.0412 g, 10 mol %) was added and the reaction was equipped with a fresh hydrogen balloon, following the same evacuation/backfilling/purging procedure used previously. The reaction was heated at 55° C. overnight. The catalyst was filtered off using Celite with ethyl acetate. The filtrate was concentrated, yielding 80 as a yellow-brown solid (0.190 g, quant.).
  • Method 1 Aniline 80 (0.129 g, 0.448 mmol) was taken up in water (0.6 mL) and concentrated HCl (0.3 mL) and cooled to 0° C. A solution of sodium nitrite (0.040 g, 0.584 mmol) in water (0.3 mL) was added and the reaction stirred at 0° C. for 10 minutes. After the stirring period, a solution of copper (I) cyanide (0.048 g, 0.538 mmol) and sodium cyanide (0.075 g, 1.52 mmol) in water (0.6 mL) was added dropwise to the reaction flask. After stirring at 0° C. for 30 minutes, the reaction was heated at 60° C. for 30 minutes.
  • the reaction was diluted with water and thrice extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over MgSO 4 , filtered and concentrated. The crude dark orange oil was purified by silica gel column chromatography (0-5% ethyl acetate-hexanes), yielding nitrile 81 as a pale yellow solid (0.010 g, 8%).
  • Method 2 Copper (I) cyanide (0.014 g, 0.156 mmol) was taken up in dry DMSO (0.2 mL) and warmed to 60° C. t-Butylnitrite (50.6 ⁇ L, 0.426 mmol) was added in one portion. A solution of ethyl 6-amino-7-methyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate 80 (0.041 g, 0.142 mmol) in DMSO (0.2 mL) was then added dropwise and the reaction stirred at 60° C. for 1 hour. The reaction was cooled to 45° C. and 5 M aqueous HCl was slowly added.
  • Compound 89 was prepared by General Method B. A mixture of mg 4,5-dihydrobenzo[1,2-b:6,5-b]dithiophene-2-carboxylic acid (100 mg, 0.42 mmol), NCS (83 mg, 0.63 mmol), and 1.5 ml THF was stirred at 55° C. for 1.5 hour. The reaction was followed by LC-MS. Upon completion of the reaction, the mixture was purified by Prep HPLC, to recover 90 as a white solid (15 mg).
  • HPLC Method & Time (min) refers to the following HPLC conditions:
  • the column “IC 50 ” provides the compounds' IC 50 as measured using the binding assay described herein, wherein: **** means a value of less than or equal to 0.05 ⁇ M; *** means a value of less than or equal to 0.1 ⁇ M; ** means a value of less than or equal to 1.0 ⁇ M; * means a value of less than or equal to 2.0 ⁇ M; and - means that the IC 50 was not determined.
  • the column “EC 50 ” provides the compounds' EC 50 as measured using the reporter assay described herein, wherein: **** means a value of less than or equal to 0.05 ⁇ M; *** means a value of less than or equal to 0.1 ⁇ M; ** means a value of less than or equal to 0.5 ⁇ M; * means a value of less than or equal to 1.0 ⁇ M; and - means that the EC 50 was not determined.
  • FIG. 5 shows the in vivo effect of 6,9-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid (“Compound B”) as compared to control and the NOTUM inhibitor 2-((5-chloro-6-methylthieno[2,3-d]pyrimidin-4-yl)thio)acetic acid (“Compound A”).
  • Compound B 6,9-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid
  • Compound A 2-((5-chloro-6-methylthieno[2,3-d]pyrimidin-4-yl)thio)acetic acid
  • mice treated with 102 mg/kg exhibited a 9% (p ⁇ 0.001) increase in midshaft femur cortical thickness.
  • FIG. 7 shows the in vivo effect of 6,8-difluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid (“Compound C”).

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US13/478,529 2011-05-27 2012-05-23 4h-thieno[3,2-c]chromene-based inhibitors of notum pectinacetylesterase and methods of their use Abandoned US20120302562A1 (en)

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US20140256784A1 (en) * 2011-05-27 2014-09-11 Joseph Barbosa 4h-thieno[3,2-c]chromene-based inhibitors of notum pectinacetylesterase and methods of their use
US9624238B2 (en) * 2011-05-27 2017-04-18 Lexicon Pharmaceuticals, Inc. 4H-thieno[3,2-C]chromene-based inhibitors of Notum Pectinacetylesterase and methods of their use
CN111065621A (zh) * 2017-08-31 2020-04-24 第一三共株式会社 制备抗体-药物缀合物的新方法
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CN103649072A (zh) 2014-03-19
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US20140256784A1 (en) 2014-09-11
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AU2012262748B2 (en) 2017-03-02
JP6166718B2 (ja) 2017-07-19
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US9624238B2 (en) 2017-04-18
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