HK1184785A - Inhibitors of notum pectinacetylesterase and methods of their use - Google Patents

Description

Inhibitors of notum pectinacetylesterase and methods of use thereof

This application claims priority to U.S. provisional patent application No.61/382,526, filed on 9/14/2010, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to small molecule inhibitors of notum pectinacetylesterase, compositions comprising said inhibitors and methods of use thereof.

Background

Bone health depends on the coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts. "bone turnover reflects the balance between these anabolic and catabolic cellular functions and ensures that mature bone is able to repair itself when damaged and maintain its endocrine function by releasing minerals such as calcium and phosphorus into the blood circulation. "Allen, J.G., etc.,J.Med. Chem.53(June10,2010), pp.4332-4353,4332. Many disease states alter this balance, resulting in an increase or decrease in bone mass or a change in bone quality. The gradual loss of bone mineral density is known as osteopenia; severe loss of bone is called osteoporosis. As above.

The current standard of care for the treatment and prevention of osteoporosis utilizes oral small molecule antiresorptive agents of the diphosphonates type. As above, at page 4333. Zoledronic acid, raloxifene, calcium and vitamin D supplements are also typically used in the treatment of osteoporosis. As above. Although anti-resorptive agents can help prevent bone loss, anabolic agents "can increase bone mass to a greater extent, … …, and also have the ability to improve bone quality and increase bone strength. "Guo, H, etc.,J.Med. Chem.53(February25,2010), pp.1819-1829,1819. In the united states, human PTH is the only FDA-approved assimilating agent. The same as above; allen, at page 4333. Due to the lack of useful assimilators for the treatment of osteoporosis, there is an urgent need to develop non-toxic, cost-effective and easy-to-administer small molecule compounds for the treatment of this disease. "Guo, at page 1819.

"although development of agents stimulating bone formation has progressed less than that of antiresorptive therapy, several approaches are known to promote osteoblast function. "Allen, at page 4338. These pathways include bone morphogenic proteins, transforming growth factor beta, parathyroid hormone, insulin-like growth factor, fibroblast growth factor, and wingless MMTV integration site (WNT) signaling. As above. Guo and co-workers have recently reported results on the first of these pathways. Guo, supra. In particular, they reported that certain substituted benzothiophene and benzofuran compounds enhance bone morphogenetic protein 2 expression in mice and rats. Two of the reported compounds stimulate bone formation and the restoration of trabecular connectivity in vivo. Page 1819, supra.

Another of these pathways is the WNT pathway, which is associated with a variety of developmental and regenerative processes. Allen, at page 4340. However, this approach is complex, and it is unclear as to more knowledge of it and as to how its components affect bone. For example, LRP-5, whose mutations are associated with increased bone mass in humans, and the beta-catenin through which typical WNT signaling occurs, "may not be directly linked via WNT signaling to control bone mass, has been proposed. The same as above.

Recent analysis of gene expression data has led to the identification of new targets for WNT signaling. See, e.g., Torisu, Y, etc.,Cancer Sci.,99(6):1139-1146,1143(2008). One such target is NOTUM pectinacetylesterase, also known as NOTUM and LOC 174111.

Summary of The Invention

The present invention encompasses compounds of the formula:

wherein: one of E and G is nitrogen and the other of E and G is nitrogen or CR₄(ii) a One of Y and Z is CR₅And the other of Y and Z is O or S (O)_mWherein m is 0, 1 or 2; r₁Is halogen, -R_1A、-OR_1A、-S(O)_nR_1A、–S(O)_nOR_1Aor-S (O)_nN(R_1A)₂Wherein n is 0, 1 or 2; each R_1AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2A、-N(R_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocycle, or with R₅Together with the atoms to which they are attached form an optionally substituted cyclic moiety; each R_3AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₄Is hydrogen, halogen or optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; r₅Is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl or heterocyclic, or with R₃Together with the atoms to which they are attached form an optionally substituted cyclic moiety; and each R_5AIs hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle.

The present invention encompasses pharmaceutical compositions comprising the compounds disclosed herein.

The invention also encompasses methods of inhibiting NOTUM pectinacetylesterase ("NOTUM"), methods of stimulating endocortical formation, and methods of treating, managing and preventing diseases and disorders associated with bone loss, such as osteoporosis, using the compounds disclosed herein.

Drawings

Certain aspects of the invention may be understood with reference to the drawings.

FIG. 1 provides a graphical representation of the differences between cortical thickness at various bone sites in NOTUM homozygous knockout mice ("HOMs") and their wild-type littermates ("WT").

Figure 2 provides a graphical representation of the increase in cortical bone thickness observed in both NOTUM homozygous and heterozygous ("HET") knockout mice compared to their wild type littermates.

Figure 3 provides a graphical representation of results obtained from femoral fracture strength and spinal compression tests performed on bones of male NOTUM homozygous and heterozygous knockout mice and their wild type littermates.

Figure 4 provides a graphical representation of results obtained from femoral fracture strength and spinal compression tests performed on bones of female NOTUM homozygous and heterozygous knockout mice and their wild type littermates.

FIG. 5 provides a graphical representation of cortical thickness measurements of the mid-femoral shaft obtained 25 days after administration of the F1 male-crossed (129 x C57) mice with 1mg/kg, 8mg/kg, and 24mg/kg of the NOTUM inhibitor 2- ((5, 6-dimethylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid.

FIG. 6 provides a graphical representation of cortical thickness measurements of the mid-femoral shaft obtained 5 weeks after Fischer344 ovariectomized rats dosed with 2- ((5-chloro-6-methylthioeno [2,3-d ] pyrimidin-4-yl) thio) acetic acid, where "sham-operated controls" refer to rats that have not had their ovaries removed and have not been administered compound; "sham-operated plus compound" refers to rats that have not had their ovaries removed but were administered compound; "OVX control" refers to ovariectomized rats to which no compound has been administered; "OVX plus compound" refers to ovariectomized rats to which compound is administered.

Fig. 7 provides a graphical representation of cortical thickness measurements of the medial tibial shaft obtained from the same experiment associated with fig. 6.

FIG. 8 provides a graphical representation of cortical thickness measurements of the mid-femoral shaft obtained 25 days after administration of the F1 male-crossed (129 x C57) mice with the NOTUM inhibitor 2- ((6-chloro-7-cyclopropylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid at 3mg/kg, 10mg/kg and 30 mg/kg.

FIG. 9 provides a graphical representation of cortical thickness measurements of the mid-femoral shaft obtained 7 days and 18 days after administration of F1 male-crossed (129 x C57) mice with the 34mg/kg NOTUM inhibitor 2- ((6-chloro-7-methylthio [3,2-d ] pyrimidin-4-yl) thio) acetic acid.

Detailed Description

The present 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 the study of mice lacking a functional NOTUM gene ("knockout mice"), the discovery of compounds that inhibit NOTUM, and the discovery that such compounds can be used to stimulate cortical bone formation in mice and rats.

Definition of

Unless otherwise indicated, the term "alkenyl" refers to straight, branched, and/or cyclic hydrocarbons having from 2 to 20 (e.g., 2 to 10 or 2 to 6) carbon atoms and including at least one carbon-carbon double bond. Representative alkenyl moieties include vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 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.

Unless otherwise indicated, the term "alkoxy" refers to an-O-alkyl group. Examples of alkoxy groups include, but are not limited to, -OCH₃、-OCH₂CH₃、-O(CH₂)₂CH₃、-O(CH₂)₃CH₃、-O(CH₂)₄CH₃and-O (CH)₂)₅CH₃。

Unless otherwise indicated, the term "alkyl" refers to straight-chain, branched-chain, and/or cyclic ("cycloalkyl") hydrocarbons. The acyclic alkyl moiety can have 1 to 20 (e.g., 1 to 10 or 1 to 4) carbon atoms; the cyclic alkyl moiety may have 3 to 20 (e.g., 3 to 10 or 3 to 6) carbon atoms. Alkyl moieties having 1 to 4 carbon atoms are referred to as "lower alkyl". Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4-dimethylpentyl, octyl, 2, 4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl. Cycloalkyl moieties may be monocyclic or polycyclic, examples of which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Other examples of alkyl moieties have linear, branched, and/or cyclic moieties (e.g., 1-ethyl-4-methyl-cyclohexyl). The term "alkyl" includes saturated hydrocarbons as well as alkenyl and alkynyl moieties.

Unless otherwise indicated, the term "alkylaryl" or "alkyl-aryl" refers to an alkyl moiety bound to an aryl moiety.

Unless otherwise indicated, the term "alkylheteroaryl" or "alkyl-heteroaryl" refers to an alkyl moiety bound to a heteroaryl moiety.

Unless otherwise indicated, the term "alkyl heterocycle" or "alkyl-heterocycle" refers to an alkyl moiety bound to a heterocyclic moiety.

Unless otherwise indicated, the term "alkynyl" refers to a straight, branched, or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2 to 6) carbon atoms and including at least one carbon-carbon triple bond. Representative alkynyl moieties include ethynyl, 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-decynyl.

Unless otherwise indicated, the term "aryl" refers to an aromatic ring or an aromatic or partially aromatic ring system composed of carbon and hydrogen atoms. The aryl moiety may comprise multiple rings joined or fused together. Examples of aryl moieties include, but are not limited to, anthracenyl, azulenyl, biphenyl, fluorenyl, indane, indenyl, naphthyl, phenanthryl, phenyl, and 1,2,3, 4-tetrahydro-naphthalene.

Unless otherwise indicated, the term "arylalkyl" or "aryl-alkyl" refers to an aryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the terms "halogen" and "halo" encompass fluorine, chlorine, bromine, and iodine.

Unless otherwise indicated, the term "heteroalkyl" refers to an alkyl moiety (straight, branched, or cyclic) in which at least one carbon atom has been replaced with a heteroatom (e.g., N, O or S).

Unless otherwise indicated, the term "heteroalkylaryl" or "heteroalkyl-aryl" refers to a heteroalkyl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term "heteroalkylheterocycle" or "heteroalkyl-heterocycle" refers to a heteroalkyl moiety bound to a heterocycle moiety.

Unless otherwise indicated, the term "heteroaryl" refers to an aryl moiety in which at least one carbon atom has been replaced with a heteroatom (e.g., N, O or S). Examples include, but are not limited to, acridinyl, benzimidazolyl, benzofuranyl, benzisothiazolyl, benzisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furanyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and triazinyl.

Unless otherwise indicated, the term "heteroarylalkyl" or "heteroaryl-alkyl" refers to a heteroaryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term "heterocycle" refers to an aromatic, partially aromatic or non-aromatic, monocyclic or polycyclic ring or ring system composed of carbon, hydrogen, and at least one heteroatom (e.g., N, O or S). The heterocyclic ring may include a plurality of (i.e., two or more) rings that are fused or joined together. Heterocycles include heteroaryls. Examples include, but are not limited to, 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, tetrahydrothienyl, tetrahydrothiopyranyl, and valerolactanyl.

Unless otherwise indicated, the term "heterocyclyl-alkyl" or "heterocycle-alkyl" refers to a heterocyclic moiety bound to an alkyl moiety.

Unless otherwise indicated, the term "heterocycloalkyl" refers to a non-aromatic heterocycle.

Unless otherwise indicated, the term "heterocycloalkylalkyl" or "heterocycloalkyl-alkyl" refers to a heterocycloalkyl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term "managing" encompasses preventing the recurrence of a particular disease or disorder in a patient already suffering from the disease or disorder, and/or prolonging the time in which a patient suffering from the disease or disorder is in remission. The term encompasses modulating the threshold, progression and/or duration of the disease or disorder, or altering the way in which a patient responds to the disease or disorder.

Unless otherwise indicated, the term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and inorganic bases, and organic acids and organic bases. Suitable pharmaceutically acceptable base addition salts include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N' -benzhydrylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Suitable non-toxic acids include, but are not limited to, 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. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Thus, examples of specific salts include hydrochloride and mesylate salts. Other pharmaceutically acceptable salts are well known in the art. See, e.g., Remington's Pharmaceutical Sciences 18 th edition (Mack Publishing, Easton PA: 1990) and Remington: pharmaceutical sciences and practices (Remington: The science and Practice of Pharmacy) 19 th edition (Mack Publishing, Easton PA: 1995).

Unless otherwise indicated, the term "prevent" means an action that inhibits or reduces the severity of a particular disease or disorder that occurs before a patient begins to develop the disease or disorder. In other words, the term encompasses prophylaxis.

Unless otherwise indicated, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder or one or more symptoms associated with the disease or disorder, or to prevent relapse thereof. A "prophylactically effective amount" of a compound refers to the amount of a therapeutic agent that, alone or in combination with other agents, is capable of providing a prophylactic benefit in preventing disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

Unless otherwise indicated, the term "substituted" when used to describe a chemical structure or moiety refers to a derivative in which one or more hydrogen atoms in the structure or moiety are replaced by a chemical moiety or functional group such as, but not limited to, an alcohol, aldehyde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, cyclopropyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC (O) alkyl), amide (e.g., -C (O) NH-alkyl-, -alkylNHC (O) alkyl), amidino (e.g., -C (NH) NH-alkyl-, -C (NR) NH)₂) Amines (primary, secondary and tertiary amines such as alkylamino, arylamino, arylalkylamino), aroyl, aryl, aryloxy, azo, carboxamide groups (e.g. -NHC (O) O-alkyl-, -OC (O) NH-alkyl), carbamoyl groups (e.g. -CONH₂CONH-alkyl, CONH-aryl, CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid, carboxylic anhydride, carboxylic acid chloride, cyano, ester, epoxide, ether (e.g. methoxy, ethoxy), guanidino, halogen, haloalkyl (e.g. -CCl)₃、-CF₃、-C(CF₃)₃) Heteroalkyl, hemiacetal, imine (primary and secondary), isocyanate, isothiocyanate, ketone, nitrile, nitro, oxo, phosphodiester, sulfide, sulfonamide (e.g., SO)₂NH₂) Sulfones, sulfonyl groups (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl groups), sulfoxides, thiols (e.g. mercapto, thioether) and ureas(e.g., -NHCONH-alkyl-). Specific substituents are alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, haloalkyl, heterocycle, and hydroxy.

Unless otherwise indicated, 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 disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A "therapeutically effective amount" of a compound refers to an amount of a therapeutic agent that, alone or in combination with other therapies, is capable of providing a therapeutic benefit in the treatment or management of a disease or disorder. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

Unless otherwise indicated, the term "treatment" means the action that occurs when a patient suffers from a particular disease or disorder to lessen the severity of the disease or disorder or to delay or slow the progression of the disease or disorder.

The term "comprising" has the same meaning as "including, but not limited to," unless otherwise specified. Similarly, the term "such as" has the same meaning as the term "such as, but not limited to".

Unless otherwise indicated, one or more adjectives immediately preceding a series of nouns should be construed as applying to each noun. For example, the phrase "optionally substituted alkyl, aryl or heteroaryl" has the same meaning as "optionally substituted alkyl, optionally substituted aryl or optionally substituted heteroaryl".

It should be noted that a chemical moiety that forms part of a larger compound may be described herein using the name usually assigned to it or the name of a group usually assigned to it when it exists as a single molecule. For example, when the terms "pyridine" and "pyridyl" are used to describe a moiety linked to other chemical moieties, they are given the same meaning. Thus, the two phrases "XOH, wherein X is pyridyl" and "XOH, wherein X is pyridine" are given the same meaning and encompass the compounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.

It should also be noted that a structure or a portion of a structure should be construed as encompassing all stereoisomers of it if the stereochemistry of the structure or portion of the structure is not indicated by, for example, bold or dashed lines. Further, any atom shown in the figures having unsatisfied valences is assumed to have enough hydrogen atoms attached to satisfy the valences. Furthermore, chemical bonds drawn with a solid line parallel to a dashed line encompass both single and double bonds (e.g., aromatic bonds), if valency permits.

Compound (I)

The present invention encompasses compounds of the formula:

wherein: one of E and G is nitrogen and the other of E and G is nitrogen or CR₄(ii) a One of Y and Z is CR₅And the other of Y and Z is O or S (O)_mWherein m is 0, 1 or 2; r₁Is halogen, -R_1A、-OR_1A、-S(O)_nR_1A、–S(O)_nOR_1Aor-S (O)_nN(R_1A)₂Wherein n is 0, 1 or 2; each R_1AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2A、-N(R_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocycle, or with R₅Together with the atoms to which they are attached form an optionally substituted cyclic moiety; each R_3AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₄Is hydrogen, halogen or optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; r₅Is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl or heterocyclic, or with R₃Together with the atoms to which they are attached form an optionally substituted cyclic moiety; and each R_5AIs hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle. It should be noted that at Y, R₃The internal bond drawn between the attached carbon and Z is dashed.

In certain compounds, both E and G are nitrogen.

In certain compounds, Z is S.

In certain compounds, Y is S.

In certain compounds, R₁is-R_1Aor-S (O)_nR_1A. In certain compounds, R_1AIs a substituted alkyl group. In a specific compound, R_1AOptionally substituted by one or more cyano, halogen, hydroxy, -N (R)_1B)₂、-S(O)_pR_1B、–S(O)_pOR_1B、-S(O)_pN(R_1B)₂、–C(O)OR_1B、-C(O)N(R_1B)₂or-C (O) N (R)_1B)S(O)_pR_1BSubstitution; wherein each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and p is 0, 1 or 2. In certain compounds, R_1BOptionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, haloalkyl, heteroCyclic or hydroxy substituted.

In certain compounds, R_1AIs a substituted alkyl group. In certain compounds, R_1AIs represented by-C (O) OR_1BAnd (4) substitution. In other compounds, R_1AIs represented by-C (O) N (R)_1B)₂And (4) substitution. In a specific compound, R_1BIs hydrogen or lower alkyl.

In certain compounds, R₂Is hydrogen, halogen or lower alkyl. In a specific compound, R₂Is hydrogen.

In certain compounds, R₃Is cyano, halogen, hydroxy or optionally substituted alkyl.

In certain compounds, R₄Is hydrogen.

In certain compounds, R₅Is cyano, halogen, hydroxy or optionally substituted alkyl.

Certain compounds have the formula:

other compounds have the formula:

certain compounds have the formula:

wherein X is OR_1AOr N (R)_1A)₂(ii) a Each R_1BIndependently hydrogen or optionally substituted alkyl, aryl, heteroalkylA group or a heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen, -C (O) N (R)_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 0, 1 or 2.

In particular compounds of this formula, n is 1 or 2. In certain compounds, n is 0.

In certain compounds, X is N (R)_1A)₂。

One embodiment of the present invention encompasses compounds of the formula:

wherein: r_1BIs hydrogen or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen, -C (O) N (R)_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle.

Specific compounds are such compounds: when R is₂Is hydrogen, R₃Is methyl and R₅When it is chlorine, R_1BIs not ethyl; and when R is₃When it is methyl, R₅Is not methyl or optionally substituted phenyl. In certain compounds, R₃Is not lower alkyl (e.g., methyl). In certain compounds, R₅Is not lower alkyl (e.g., methyl).

In certain compounds, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In a specific compound, R_1BIs hydrogen or alkyl.

In certain compounds, R₃Is alkyl or halogen (e.g. chlorine).

In certain compounds, R₅Is alkyl (e.g., lower alkyl) or halogen.

One embodiment of the present invention encompasses compounds of the formula:

wherein: r_1BIs hydrogen or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2Aor-N (R)_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_2AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 1 or 2.

In certain compounds of this formula, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In a specific compound, R_1BIs hydrogen or alkyl.

In certain compounds, R₂Is hydrogen or optionally substituted alkyl.

In certain compounds, R₃Is alkyl or halogen (e.g. chlorine). In certain compounds, R₃Is not lower alkyl (e.g., methyl).

In certain compounds, R₅Is alkyl (e.g., lower alkyl) or halogen. In certain compounds, R₅Is not lower alkyl (e.g., methyl).

Certain compounds of the present invention have the formula:

wherein X is OR_1AOr N (R)_1A)₂(ii) a Each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen,-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 0, 1 or 2.

In particular compounds of this formula, n is 1 or 2. In certain compounds, n is 0.

In certain compounds, X is N (R)_1A)₂。

One embodiment of the present invention encompasses compounds of the formula:

wherein: r_1BIs hydrogen or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3Aor-N (R)_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and each R_5AIndependently of each otherIs hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle.

In certain compounds of this formula, R₂Is hydrogen or optionally substituted alkyl.

In certain compounds, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In a specific compound, R_1BIs hydrogen or alkyl.

In certain compounds, R₃Is alkyl or halogen (e.g. chlorine). In certain compounds, R₃Is not lower alkyl (e.g., methyl).

In certain compounds, R₅Is alkyl (e.g., lower alkyl) or halogen. In certain compounds, R₅Is not lower alkyl (e.g., methyl).

One embodiment of the present invention encompasses compounds of the formula:

wherein: r_1BIs hydrogen or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2Aor-N (R)_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_2AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₄Is hydrogen, halogen or optionally substituted alkyl, arylA group, heteroalkyl or heterocyclic ring; r₅Is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 1 or 2.

In certain compounds of this formula, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In a specific compound, R_1BIs hydrogen or alkyl.

In certain compounds, R₂Is hydrogen or optionally substituted alkyl.

In certain compounds, R₃Is alkyl or halogen (e.g. chlorine). In certain compounds, R₃Is not lower alkyl (e.g., methyl).

In certain compounds, R₅Is alkyl (e.g., lower alkyl) or halogen. In certain compounds, R₅Is not lower alkyl (e.g., methyl).

One embodiment of the present invention encompasses compounds of the formula:

wherein: each R_1AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3Aor-N (R)_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently isHydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 0, 1 or 2.

Specific compounds are such compounds: when R is₂When is hydrogen, R₃And R₅Both are not phenyl; when R is_1AIs a substituted pyrimidine, R₂Is hydrogen and R₃When it is a substituted phenyl group, R₅Is not methyl; when R is₂When is hydrogen, R₃And R₅Both are not methyl; and when R is_1AIs substituted phenyl, R₂Is hydrogen and R₃When it is a substituted phenyl group, R₅Is not hydrogen.

In certain compounds, R_1AIs a substituted alkyl group. In a specific compound, R_1ABy one or more cyano, halogen, hydroxy, -N (R)_1B)₂、-S(O)_pR_1B、–S(O)_pOR_1B、-S(O)_pN(R_1B)₂、-C(O)OR_1B、-C(O)N(R_1B)₂or-C (O) N (R)_1B)S(O)_pR_1BSubstitution; wherein each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and p is 0, 1 or 2.

In certain compounds, R_1BOptionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, heterocycle, or hydroxyl groups. In a specific compound, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In certain compounds, R_1BIs hydrogen or alkyl.

In certain compounds, R₂Is hydrogen or optionally substituted alkyl.

In certain compounds, R₃Is alkyl or halogen.

In certain compounds, R₅Is alkyl or halogen.

One embodiment of the present invention encompasses compounds of the formula:

wherein: each R_1AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₂Is hydrogen or optionally substituted alkyl; each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₃Is halogen, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3Aor-N (R)_3A)₂Optionally substituted alkyl, aryl, heteroalkyl or heterocyclic; each R_3AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; r₅Is halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; each R_5AIndependently hydrogen, optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and n is 0, 1 or 2.

In a particular embodiment, the compound is not 2- ((6, 7-diphenylfuro [3, 2-d)]Pyrimidin-4-yl) thio) -1-morpholinoethanone. In one embodiment, R₃And R₅Both are not phenyl.

In certain compounds, R_1AIs a substituted alkyl group. In a specific compound, R_1ABy one or more cyano, halogen, hydroxy, -N (R)_1B)₂、-S(O)_pR_1B、–S(O)_pOR_1B、-S(O)_pN(R_1B)₂、-C(O)OR_1B、-C(O)N(R_1B)₂or-C (O) N (R)_1B)S(O)_pR_1BSubstitution; wherein each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and p is 0, 1 or 2.

In certain compounds, R_1BOptionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, heterocycle, or hydroxyl groups. In a specific compound, R_1BIs hydrogen or optionally substituted alkyl (e.g. lower alkyl) or aryl. In certain compounds, R_1BIs hydrogen or alkyl.

In certain compounds, R₂Is hydrogen or optionally substituted alkyl.

In certain compounds, R₃Is alkyl or halogen.

In certain compounds, R₅Is alkyl or halogen.

Specific compounds have an IC of less than about 0.1, 0.05, or 0.025 μ M when determined by a binding assay described herein₅₀. A particular compound has an EC of less than about 5, 2.5, or 1 μ Μ, when determined by the reporter assay described herein₅₀。

The compounds of the present invention (i.e., the compounds disclosed herein) can be prepared by the methods disclosed herein as well as by methods known in the art. See, for example, U.S. patent No.6,579,882 to Stewart et al, european patent No.0447891 to Wiesenfeldt et al.

Method of treatment

The present invention encompasses methods of stimulating endocortical formation in a patient comprising administering to a patient in need thereof an effective amount of a compound of the present invention. The invention also encompasses methods of increasing cortical bone thickness comprising administering to a patient in need thereof an effective amount of a compound of the invention.

The present invention encompasses methods of treating, managing or preventing a disease or disorder associated with bone loss comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the present invention. Examples of diseases and disorders include osteoporosis (e.g., postmenopausal osteoporosis, steroid or glucocorticoid-induced osteoporosis), osteopenia, and paget's disease.

The invention also encompasses methods of treating, managing or preventing bone fractures comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention. Specific fractures are associated with metastatic bone disease, i.e., cancer that has metastasized to bone. Examples of cancers capable of metastasizing to bone include prostate, breast, lung, thyroid, and renal cancers.

The invention also encompasses methods of treating, managing or preventing bone loss associated with or caused by a disease or disorder comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of a compound of the invention. Examples of diseases and disorders include celiac disease, crohn's disease, cushing's syndrome, hyperparathyroidism, inflammatory bowel disease, and ulcerative colitis.

Examples of patients who may benefit from the methods of the invention include men and women over the age of 55 years, postmenopausal women, and patients with renal insufficiency.

The compounds of the invention may be administered in combination (e.g., at the same time or at different times) with other drugs known to be useful in the treatment, management, or prevention of diseases or conditions affecting bone. Examples of such other drugs include: androgen receptor modulators, bisphosphonates, calcitonin, calcium sensitive receptor antagonists, cathepsin K inhibitors, estrogen and estrogen receptor modulators, conjugates of integrins, antibodies and receptor antagonists, parathyroid hormone (PTH) and its analogs and mimetics, and vitamin D and synthetic vitamin D analogs.

Examples of androgen receptor modulators include finasteride and other 5 α -reductase inhibitors, nilutamide, flutamide, bicalutamide, liazole, and abiraterone acetate.

Examples of diphosphonates include alendronate, incadronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, pyridronate, risedronate, tiludronate and zoledronate, and pharmaceutically acceptable salts and esters thereof.

Examples of cathepsin K inhibitors include VEL-0230, AAE581 (Barlica), MV061194, SB-462795 (Raracatin), MK-0822 (odanacatib) and MK-1256.

Examples of estrogens and estrogen receptor modulators include naturally occurring estrogens (e.g., 7-estradiol, estrone, and estriol), conjugated estrogens (e.g., conjugated equine estrogens), oral contraceptives, sulfated estrogens, progestins, 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-benzopyran-3-yl ] -phenyl-2, 2-dimethylpropionate, 4' -dihydroxybenzophenone-2, 4-dinitrophenylhydrazone and SH 646.

Examples of conjugates of integrins, antibodies and receptor antagonists include vitaxin (MEDI-522), cilengitide and L-000845704.

Pharmaceutical preparation

The present invention encompasses pharmaceutical compositions comprising one or more compounds of the invention and optionally one or more other drugs, such as the drugs described above.

Certain pharmaceutical compositions are in single dose 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. Examples of dosage forms include, but are not limited to: a tablet; a caplet; capsules, such as soft elastic gelatin capsules; a cachet; a lozenge; rhombus ingot; a dispersant; suppositories; ointment; cataplasm (cataplasm); a paste; powder preparation; a dressing; a cream; a plaster; a solution; pasting a piece; aerosols (e.g., nasal sprays or inhalants); gelling; 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 water-in-oil liquid emulsions), solutions, and elixirs; a liquid dosage form 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.

The formulation should be suitable for the mode of administration. For example, oral administration requires an enteric coating to protect the compounds of the invention from degradation in the gastrointestinal tract. Similarly, the formulations may contain ingredients that facilitate the delivery of the active ingredient to the site of action. For example, the compounds may be administered in a liposome formulation in order to protect them from degrading enzymes, facilitate transport in the circulatory system, and achieve delivery across the cell membrane to intracellular sites.

The composition, shape and type of dosage form vary with its use. For example, a dosage form used in the acute treatment of a disease may contain a greater amount of one or more active ingredients than a dosage form used in the chronic treatment of the same disease. Similarly, parenteral dosage forms may contain smaller amounts of one or more active ingredients than those contained in oral dosage forms used to treat the same disease. These and other aspects of the particular dosage forms encompassed by the present invention that differ from one another will be apparent to those skilled in the art. See, for example, Remington's pharmaceutical Sciences 18 th edition (Mack Publishing, Easton PA: 1990).

Preferably, the pharmaceutical composition of the invention is 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 a predetermined amount of active ingredient and may be prepared by pharmaceutical methods well known to those skilled in the art. See, e.g., Remington's pharmaceutical sciences 18 th edition (Mack Publishing, Easton PA: 1990).

Typical oral dosage forms are prepared by combining the active ingredient with at least one excipient in an intimate mixture according to conventional pharmaceutical compounding techniques. Excipients may take a wide variety of forms depending on the form of preparation desired for administration.

Because of their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form. Tablets may be coated, if desired, by standard aqueous or non-aqueous techniques. Such dosage forms may be prepared by conventional pharmaceutical methods. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired presentation. Disintegrants may be incorporated into the solid dosage form to facilitate rapid dissolution. Lubricants may also be incorporated to facilitate manufacture of the dosage form (e.g., tablet).

Examples

Knockout mice

Corresponding mutant Embryonic Stem (ES) cell clones from the mutant murine ES cell clone OMNIBANK (OMNIBANK collection of mutated muscle ES cell lines) were used to generate mice homozygous for the genetically engineered mutation in the murine ortholog of the human NOTUM gene (see generally U.S. patent No.6,080,576). Briefly, ES cell clones containing the mutagenized viral insert in the murine NOTUM locus were microinjected into embryo sacs, which were in turn implanted into pseudopregnant female hosts and committed to term. The resulting chimeric offspring were then crossed with C57black6 female mice and offspring were examined for germline transmission of the knockout NOTUM allele. Animals heterozygous for the mutant NOTUM allele will then be bred to breed about 1: 2: a ratio of 1 results in progeny that are homozygous for the mutant NOTUM allele, progeny that are heterozygous for the mutant NOTUM allele, or wild-type progeny.

Mice homozygous for the disruption of the NOTUM gene (-/-) were studied in combination with mice heterozygous for the disruption of the NOTUM gene (+/-) and wild-type littermates (+/+). During this analysis, mice were subjected to medical diagnostic examinations using a full suite of medical diagnostic procedures designed to assess the function of major organ systems in mammalian subjects. By studying homozygous (-/-) "knockout" mice in the numbers described in combination with heterozygous (+/-) and wild type (+/- +) littermates, more reliable and reproducible data were obtained.

As shown in fig. 1, male mice with homozygous disruption of the NOTUM gene ("homozygous mice") exhibited greater cortical thickness at various bone sites at 16 weeks of age compared to their wild-type littermates (number of mice N =10 for both groups). These differences measured by micct (Scanco μ CT 40) were: the mid-femoral shaft is 28% (p < 0.001); the mid-humeral shaft is 19% (p < 0.001); the mid-tibial shaft is 17% (p < 0.001); the tibia-fibula junction is 11% (p < 0.001). As shown in figure 2, at 16 weeks of age, mice heterozygous for the NOTUM mutation ("heterozygous mice") also had cortical bone thickness in the mid-femoral shaft greater than their wild-type littermates: male heterozygous mice (N = 50) showed a 6% (p = 0.007) increase compared to their wild type littermates (N = 23); female heterozygous mice (N = 57) showed a 9% (p < 0.001) increase compared to their wild type littermates (N = 22).

The actual manifestation of the redistribution of bone formation observed in NOTUM animals is reflected in figures 3 and 4, which show the results of a femoral fracture strength test (performed by skelmetech, now Ricerca Biosciences) using a standard 4-point bending test. As shown in figure 3, the graph provides results obtained for 16-week-old male mice, heterozygous mice (N = 20) showed a 5% (p = 0.54) increase in femoral fracture strength compared to their wild-type littermate (N = 23), while homozygous mice (N = 17) showed a 28% (p < 0.001). On the other hand, spinal compression tests of both NOTUM homozygous and heterozygous mice did not show a significant reduction in maximum spinal compression load compared to wild-type controls. Similar results were obtained for 16 week old female mice. As shown in figure 4, heterozygous mice (N = 20) showed a 12% increase in femoral fracture strength (p = 0.04) compared to their wild type littermates (N = 21), while homozygous mice (N = 18) showed a 28% increase (p < 0.001). Analysis of these and other data revealed a strong correlation between cortical thickness and femoral fracture strength.

Reporter assay

Determination of compound EC using this assay₅₀The assay utilizes conditioned media prepared as follows. The plasmid containing human backplate-glial acetyltransferase in pcDNA3.1(+) vector was transfected into HEK293 cells and clones were selected by growth in the presence of 400ug/mL G418. The highest expressing clones containing human notum glial acetyltransferase in conditioned medium were maintained for all future activity assays. L cells that overexpress Wnt3a and secrete it into conditioned media were purchased from ATCC.

The measurement procedure is as follows. About 500 ten thousandLEF/TCF-bla FreeStyle^TM293F cells were grown to near confluence in 15-cm plates. The cell growth medium was composed of DMEM containing 10% dialyzed FBS, 5. mu.g/ml blasticidin (Invitrogen, R210-01), 0.1mM AEA, 25mM HEPES and 1 XPPS. Cells were then trypsinized by first washing with PBS, followed by the addition of 5mL trypsin, and incubating the plates for 2 minutes at room temperature. Then a total of 10mL was added to each 15cm plateAssay medium (Opti-MEM, addition of 0.5% dialyzed FBS, 0.1mM NEAA, 1mM sodium pyruvate, 10mM HEPES, 1 XPPS). Cells were counted and suspended at a density of 75 ten thousand cells per mL. Cells were seeded at a density of 15000 cells per 20 μ L per well in Biocoat384 well plates (Fisher, catalog No. 356663). After incubating the cells at 37 ℃ for 3 hours, 10. mu.L of assay medium containing 30mM LiCl was added to each well, followed by incubation at 37 ℃ for an additional 3 hours. Meanwhile, compounds were acoustically pulsed into Greiner384 well plates (catalog No. 781076) using ECHO followed by the addition of 10 μ L per well of Wnt3a modulated medium and 10 μ L per well of notum pectinacetylesterase modulated medium. Then 10 μ L of Wnt3 a/notum glial acetyltransferase mixture was transferred from Greiner plates to each well containing CellSensor cells in 384-well plates. After incubating the cells at 37 ℃ overnight, the reaction was developed by adding 5 μ L of 1xCCF4 (Invitrogen, catalog No. K1085) to each well, covering the entire 384-well plate, and shaking gently at room temperature for 3 hours in the dark. The plates were then read on an Envision plate reader using an excitation wavelength of 400nm and emission wavelengths of 460nm and 535 nm.

Binding assays

Determination of IC of Compounds Using this assay₅₀The assay utilizes trisodium 8-octanoyloxypyrene-1, 3, 6-trisulfonate (OPTS), a water soluble enzyme substrate for fluorometric assays of esterases and lipases. The plasmid containing human backplate-glial acetyltransferase in pcDNA3.1(+) vector was transfected into HEK293 cells and clones were selected by growth in the presence of 400ug/mL G418. Conditioned media from these cells was used in the assay.

75nL of compound was acoustically dispensed using ECHO into dry Greiner 384-well plates (cat. No. 781076), and 10uL of 50mM Tris/HCl (pH 6.8) was then added to each well of these 384-well assay plates. The conditioned medium containing human notum glial acetyltransferase was assayed in assay buffer (50 mM Tris, pH6.8, 5mM CaCl)₂，0.5mM MgCl₂) Diluted 75-fold and 25. mu.L of this "enzyme mix" was added to each wellCompound ", then a 10 min preincubation was performed. The enzymatic reaction was initiated by adding 15. mu.L of OPTS substrate (Sigma, cat. No. 74875) to a final concentration of 5. mu.M for 10 minutes at room temperature. All plates were read on an Envision reader using a 485nm excitation wavelength and a 535nm emission wavelength.

General synthetic method a: preparation of 3-chloro-2-methylthioeno [2,3-b ] pyridin-4-ol (2) and 3-bromo-2-methylthioeno [2,3-b ] pyridin-4-ol (3)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

in a 100mL round-bottom flask, 6-methylthieno [2,3-d ] pyrimidin-4-ol 1 (1 g, 6.2 mmol) was taken in acetic acid (12 mL) and stirred at room temperature. Bromine (0.3 mL, 6.2 mmol) was added thereto, and the reaction mixture was stirred at room temperature overnight. The reaction was then quenched with ice and stirred until the ice melted. The product was then filtered off, washed with water and dried to give 3-bromo-2-methylthioeno [2,3-b ] pyridin-4-ol 2 as a dry product (1 g, 66% yield).

Taking 6-methylthieno [2,3-d ] in a 3L round-bottom flask]Pyrimidin-4-ol 1 (50 g, 310 mmol) was placed in acetic acid (525 mL) and NCS (37.5 g, 372 mmol) was added. The reaction was heated at 55 ℃ for 8 hours. The reaction was then cooled to room temperature, quenched with water, and the compound was extracted with dichloromethane (2 x250 mL). The combined organic layers were washed with brine, over MgSO₄Dried, filtered and concentrated to give 3-chloro-2-methylthioeno [2,3-b ] as solid 3]Pyridin-4-ol (42.15g, 70% yield).

General synthetic method B: preparation of 2- (5-chloro-6-methylthio [2,3-d ] pyrimidin-4-ylthio) acetic acid (6)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

4, synthesis: reacting 3-chloro-2-methylthiophene [2,3-b ]]Pyridin-4-ol 3 (40 g, 2000mmol, 1 eq.) was suspended in POCl₃(368 mL, 4000mmol, 20 equiv.) and heated to 90 ℃ for 4 hours. After cooling, the reaction was concentrated and the residue was cooled to 0 ℃. Ice cold water was then added slowly to ensure POCl₃And completely consumed. The black solution was extracted 3 times with ethyl acetate. The combined organic extracts were washed with saturated NaHCO₃The aqueous solution was washed twice, once with brine over MgSO₄Drying, filtering and concentrating to obtain light brown solid 4, 5-dichloro-6-methylthiophene [2,3-d]Pyrimidine 4 (41.6 g, 93% yield), which was used immediately after complete drying.

5, synthesis: 4, 5-dichloro-6-methylthioeno [2,3-d ] pyrimidine 4 (40 g, 182.2mmol, 1 eq.) was suspended in methanol (500 mL) and cooled to 0 ℃. Methyl 2-mercaptoacetate (27 mL, 191.5mmol, 1.05 equiv.) was added slowly followed by TEA (53 mL, 382.6mmol, 2.1 equiv.) added slowly. After stirring for 90 minutes, the reaction was concentrated. The residue was taken up in ethyl acetate and filtered through a plug of silica gel to remove salt and some colour. The filtrate was concentrated and purified by silica gel chromatography using 0-15% ethyl acetate in hexane to give methyl 2- (5-chloro-6-methylthioeno [2,3-d ] pyrimidin-4-ylthio) acetate 5 (47.30 g, 91% yield) as an off-white solid.

6, synthesis: taking 2- (5-chloro-6-methylthiophene [2, 3-d)]Pyrimidin-4-ylthio) Methyl acetate 5 (45 g, 150mmol, 1 equiv.) was taken up in THF (350 mL), 1N aqueous NaOH (300 mL, 300mmol, 2 equiv.) was added and stirring was effective. After 40 min, the reaction was concentrated and the residue taken up in water, cooled to 0 ℃ and acidified to low pH with 1N HCl. The precipitate was collected by vacuum filtration and washed with water. It was dried under vacuum overnight to give pure 2- (5-chloro-6-methylthioeno [2,3-d ] as a white solid]Pyrimidin-4-ylthio) acetic acid 6 (40.5 g, 94% yield). MS m/z C₉H₇ClN₂O₂S₂[M+1]⁺=275 and [ M +1]⁺³=277。¹H NMR (400MHz, methanol-d)₄)δppm8.66(s,1H),4.05(s,2H),2.54(s,3H)。¹³C NMR(101MHz,DMSO-d₆)δppm170.04(s),162.55(s,1C),162.48(s,1C),152.55(s,1C),135.03(s,1C),125.03(s,1C),114.36(s,1C),32.17(s,1C),14.05(s,1C)。

General synthetic method C: preparation of 2- (6-bromo-5-methylthio [2,3-d ] pyrimidin-4-ylthio) acetic acid (10)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

8, synthesis: 4-chloro-5-methylthioeno [2,3-d ] pyrimidine 7 (1 g, 5.4 mmol), prepared according to general synthesis B, was taken in acetic acid and stirred at room temperature. To the reaction mixture was added bromine (1 eq =275 μ L, 5.4 mmol) and the reaction mixture was stirred overnight. The reaction was then quenched with ice and stirred until the ice melted. The product was then filtered off, washed with water and dried to yield 1g 6-bromo-4-chloro-5-methylthioeno [2,3-d ] pyrimidine 8 (786 mg, 55% yield) as a pale yellow solid.

Compounds 9 and 10 were prepared according to general synthetic procedure B.

General synthetic method D: preparation of methyl 2- (5-cyclopropyl-6-methylthio [2,3-d ] pyrimidin-4-ylthio) acetate (12)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

a100 mL round-bottom flask was charged with 2- (5-bromo-6-methylthioeno [2,3-d ]]Pyrimidin-4-ylthio) acetic acid methyl ester 11 (0.100 g, 0.3mmol, 1 equiv.), cyclopropylboronic acid (51 mg, 0.6 mmol) and tricyclohexylphosphine (8.4 mg, 0.03 mmol), and K is taken₃PO₄(212 mg, 1.05 mmol) was placed in a mixture of toluene and water (4 mL, 1: 1). The mixture was cooled to room temperature and N₂Stirring under an atmosphere. To the stirred solution was added palladium diacetate (4 mg, 0.015 mmol). The reaction mixture was then heated at 100 ℃ for 8 hours. The reaction was quenched by addition of 1N HCl and the aqueous portion was extracted three times with EtOAc. The combined organic fractions were washed with MgSO 2₄Dried, filtered and concentrated to give 2- (5-cyclopropyl-6-methylthioeno [2.3-d ] as a pale yellow solid]Pyrimidin-4-ylthio) acetic acid methyl ester (40 mg, 45% yield) 12.

General synthetic method E: preparation of methyl 2- (6-ethyl-5-methylthio [2,3-d ] pyrimidin-4-ylthio) acetate (13)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

in a cell containing 2- (6-bromo-5-methylthioeno [2,3-d ] prepared according to general synthetic method C]Pyrimidin-4-ylthio) acetic acid methyl ester 9 (50 mg, 0)15 mmol) and diethyl zinc (1.1M in toluene, 204. mu.L, 0.23 mmol) in a 100mL round-bottomed flask, Pd (dba)₃(4 mg, 0.0075 mmol) and 2-dicyclohexylphosphine-2 ', 6' -isopropoxydiphenyl (13 mg, 0.03 mmol) were placed in anhydrous DMF (2.5 mL) in a microwave vessel and heated under microwave irradiation at 160 ℃ for 10 min. After cooling to room temperature, it was filtered through celite and concentrated. It was placed in EtOAc and washed with water. The organic layer was washed with brine, dried over magnesium sulfate and concentrated to give 2- (6-ethyl-5-methylthioeno [2, 3-d)]Pyrimidin-4-ylthio) acetic acid methyl ester 13 (17 mg, 40% yield).

General synthetic method F: preparation of 2- (5- (dimethylamino) -6-methylthio [2,3-d ] pyrimidin-4-ylthio) acetic acid (14)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

methyl 2- (5-bromo-6-methylthio [2.3-d ] pyrimidin-4-ylthio) acetate 11 (100 mg, 0.3 mmol), prepared using general synthetic method C, was placed in 1mL (excess, as solvent) of water containing 40% dimethylamine. A catalytic amount of copper oxide was then added and the reaction was heated under microwave irradiation at 140 ℃ for 15 min. The reaction was stopped and then purified by preparative HPLC (aqueous ammonium acetate/acetonitrile). 14 was isolated as a white solid (60 mg, 71% yield).

General synthetic method G: preparation of 2- (5, 6-dimethylthieno [2,3-d ] pyrimidin-4-ylthio) -N- (2-methoxyethyl) acetamide (17)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

2- (5, 6-Dimethylthiophene [2,3-d ], commercially available and also preparable using general synthetic method B]Pyrimidin-4-ylthio) acetic acid 15 (0.050 g, 0.2mmol, 1 equiv.) was placed in DMF (2.2 mL). To the mixture was added 2-methoxyethylamine 16 (0.019 g, 0.24mmol, 1.2 equivalents), HATU (0.076 g, 0.2mmol, 1 equivalent), and N, N-diisopropylethylamine (0.08 mL, 0.44mmol, 2.2 equivalents), and the reaction mixture was stirred at room temperature for 8 hours. Water was added for quenching and the aqueous portion was extracted three times with EtOAc. The combined organic layers were washed with MgSO 2₄Dried, filtered and concentrated. The residue was purified by preparative HPLC using neutral conditions to give 2- (5, 6-dimethylthieno [2,3-d ] as an off-white solid]Pyrimidin-4-ylthio) -N- (2-methoxyethyl) acetamide 17 (56 mg, 90% yield).

General synthesis method H: preparation of isopropyl 2- (5, 6-dimethylthieno [2,3-d ] pyrimidin-4-ylthio) acetate (19)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

compound 15 (100 mg, 0.4 mmol), which is commercially available and can also be prepared using general synthetic method B, is dissolved in anhydrous DCM, thionyl chloride (56 uL, 0.8 mmol) is added and stirred at room temperature for 1 hour. Excess 2-propanol 18 was then added and stirring continued at room temperature for 1 hour. The reaction mixture was then concentrated and purified using neutral phase preparative HPLC (aqueous ammonium acetate/acetonitrile) to give 19 (100 mg, 85% yield) as a solid.

General synthetic method I: preparation of 2- (5-chloro-6-isopropylthieno [2,3-d ] pyrimidin-4-ylthio) acetic acid (24)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

21, synthesis: commercially available pyrimidinone 20 (10 g, 51.4mmol, 1 eq) which can also be prepared by general synthetic method D is suspended in POCl₃(95 mL, 1028mmol, 20 equivalents) and heated to 90 ℃ for 4 hours. After cooling, the reaction was concentrated and the residue was cooled to 0 ℃. Ice cold water was then added slowly to ensure POCl₃And completely consumed. The black solution was extracted 3 times with ethyl acetate. The combined organic extracts were washed with saturated NaHCO₃The aqueous solution was washed twice, once with brine over MgSO₄Drying, filtering and concentrating to obtain light brown solid 4-chloro-6-isopropyl thieno [2,3-d]Pyrimidine 21 (10.16 g, 93% yield), which was used immediately after complete drying.

22 synthesis: taking solid 4-chloro-6-isopropyl thieno [2,3-d]Pyrimidine 21 (9.2 g, 43.2mmol, 1 eq) was placed in acetic acid (75 mL) and NCS (8.65 g, 86.5mmol, 2 eq) was added. The reaction was heated at 55 ℃ for 6 hours. The reaction was cooled, quenched with water, and the compound was extracted with dichloromethane (2 × 50 mL). The combined organic layers were washed with brine, over MgSO₄Dried, filtered and concentrated to give 4, 5-dichloro-6-isopropylthieno [2,3-d ] as a solid]Pyrimidine 22 (6.2 g, 58% yield).

Synthesis of 23: 4, 5-dichloro-6-isopropylthieno [2,3-d ] pyrimidine 22 (11.6 g, 47mmol, 1 eq.) was suspended in methanol (125 mL) and cooled to 0 ℃. Methyl 2-mercaptoacetate (6.86 mL, 49.35mmol, 1.05 equiv.) was added slowly followed by TEA (13.7 mL, 98.7mmol, 2.1 equiv.) slowly. After stirring for 90 minutes, the reaction was concentrated. The residue was taken up in ethyl acetate and filtered through a plug of silica gel to remove salt and some colour. The filtrate was concentrated and purified by silica gel chromatography using 0-15% ethyl acetate in hexane to give methyl 2- (5-chloro-6-isopropylthieno [2,3-d ] pyrimidin-4-ylthio) acetate 23 as an off-white solid (13.6 g, 92% yield).

Synthesis of 24: compound 23 (5.0 g, 15.8mmol, 1 equiv.) is taken up in THF (50 mL), 1N aqueous NaOH (32 mL, 32mmol, 2 equiv.) is added and stirring is effected. After 40 min, the reaction was concentrated and the residue taken up in water, cooled to 0 ℃ and acidified to low pH with 1N HCl. The precipitate was collected by vacuum filtration and washed with water. It was dried under vacuum overnight to give pure 2- (5-chloro-6-isopropylthieno [2,3-d ] as a white solid]Pyrimidin-4-ylthio) acetic acid 24 (40.5 g, 94% yield). MS m/zC₁₁H₁₁ClN₂O₂S₂[M+1]⁺=303 and [ M + 1%]⁺³=305。¹H NMR(400MHz,DMSO-d₆)δppm12.83(s,1H),8.81(s,1H),4.12(s,2H),3.52-3.76(m,1H),1.32(d,J=7.03Hz,6H)。¹³C NMR(101MHz,DMSO-d₆)δppm170.0(s,1C),162.8(s,1C),162.2(s,1C),152.6(s,1C),146.9(s,1C),125.1(s,1C),112.3(s,1C),32.2(s,1C),28.6(s,1C),23.4(s,1C),23.1(s,1C)。

General synthetic method J: preparation of 2- (6-chloro-7-cyclopropylthieno [2,3-d ] pyrimidin-4-ylthio) acetic acid (31)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

26, synthesis: taking 7-bromo-4-methoxythieno [3,2-d ] in a 500mL round-bottom flask]Pyrimidine 25 (4 g, 16.32mmol, 1 eq.) and cyclopropylboronic acid (2.81 g, 32.6 mmo)l, 2 equiv.) and potassium carbonate (6.76 g, 49mmol, 3 equiv.) and the catalyst PdCl₂(Ph₃P)₂(575 mg, 0.816mmol, 0.05 eq.) in toluene (60 mL) and H₂O (16 mL). With N₂The reaction mixture was purged vigorously for 5 minutes and heated at 100 ℃ for 6 hours. After cooling to room temperature, the reaction mixture was filtered and the two layers were separated. The aqueous layer was washed with toluene and the combined organic solvents were concentrated. The crude material was purified by silica gel column chromatography using EtOAc and heptane (10-30%) solvents to afford 7-cyclopropyl-4-methoxythieno [3,2-d as a pale yellow solid]Pyrimidine 26 (3 g, 90% yield).

Compound 27 was synthesized using general synthesis method a.

28 Synthesis: 6-chloro-7-cyclopropyl-4-methoxythieno [3,2-d ] pyrimidine (2 g, 8.25 mmol) was taken up in 12N HCl (6 mL, 72.5 mmol) and the reaction mixture was heated at 70 ℃ for 40 min. After cooling to room temperature, water (15 mL) was added to the light brown slurry and mixed appropriately. The precipitate was filtered off and washed with water and heptane. After drying it at 40 ℃ overnight, it gave a pale yellow solid (1.6 g, 90% yield).

Compound 29 was synthesized using general synthesis method B. Compound 30 was synthesized using general synthesis method B. MS m/z C₁₁H₉ClN₂O₂S₂[M+1]⁺=301 and [ M + 1%]⁺³=303。¹HNMR(400MHz,DMSO-d₆)δppm8.92(s,1H),4.22(s,2H),2.10-2.20(m,1H),1.45-1.52(m,2H),0.96-1.06(m,2H)。¹³C NMR(101MHz,DMSO-d₆)δppm169.6(s,2C),161.4(s,1C),156.2(s,1C),154.1(s,2C),134.4(s,1C),125.9(s,1C),31.9(s,1C),10.2(s,1C),6.3(s,2C)。

General synthesis method K: preparation of 2- (7-cyclopropyl-6-methylthio [2,3-d ] pyrimidin-4-ylthio) acetic acid (35)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

compound 26 was synthesized using general synthesis method J.

31, synthesis: to N, N-diisopropylethylamine (14 mL, 80 mmol) in THF (60 mL) at-60 deg.C was added N-BuLi (37.5 mL, 1.6M, 60 mmol) dropwise. After stirring at the same temperature for 15 minutes, compound 31 (4.14 g, 20 mmol) was added as one portion and allowed to warm to-45 ℃ over 45 minutes. It was then cooled back to-60 ℃ and quenched by slow dropwise addition of MeI (5 mL, 80 mmol), and the reaction mixture was warmed to room temperature and stirred for 2 hours. After 2 hours, it was quenched with a mixture of brine and water (100 mL) and the THF layer was separated. The aqueous layer was extracted with dichloromethane and the combined organic solvents were washed again with brine and passed over MgSO₄. The crude product was purified by silica gel chromatography using ethyl acetate/hexane mixture (10% -30%) to give compound 32 (2.64 g, 60% yield).

Compounds 32-35 were synthesized using general synthesis method B. MS m/z C₁₂H₁₂N₂O₂S₂[M+1]⁺=281。¹H NMR(400MHz,DMSO-d₆)δppm12.86(br.s.,1H),8.85(s,1H),4.20(s,2H),2.65(s,3H),1.99(tt,J=8.63,5.43Hz,1H),1.23-1.30(m,2H),0.88-0.97(m,2H)。

General synthetic method L: preparation of 4- (carboxymethylthio) -7-methylthio [3,2-d ] pyrimidine-6-carboxylic acid (40)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

compound 36 is commercially available. Compound 37 was synthesized using general synthesis method B.

38 synthesis: to a stirred solution of thiophene 37 (368 mg, 2mmol, 1.0 equiv) in anhydrous THF (5 mL) at-30 ℃ was added a solution of LDA in THF (2.2 mmol, 1.1 equiv) (freshly prepared by mixing 0.34mL of diisopropylamine with 0.96mL of 2.5mnbuli in 5mL of THF at-30 ℃). The reaction changed from clear to yellow. After 20 min, ethyl chloroformate (0.28 mL, 3mmol, 1.5 equiv.) was added dropwise. After the reaction was warmed to room temperature in a cold water bath, it was quenched with water. The mixture was diluted with 10mL of ethyl acetate and extracted with ethyl acetate (10 mL × 2 times). The combined organic layers were washed with brine, over Na₂SO₄Drying and concentrating. The crude mixture was then adsorbed on silica gel and purified by flash chromatography (eluting with 10% DCM/EA) to give ester 38 (127 mg, 25% yield) as a white solid.

Compounds 39 and 40 were synthesized using general synthesis method B.

General synthetic method M: preparation of 2- (6-cyano-7-methylthio [3,2-d ] pyrimidin-4-ylthio) acetic acid (44) and 2- (6-carbamoyl-7-methylthio [3,2-d ] pyrimidin-4-ylthio) acetic acid (46)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

41, synthesis: to a solution of 37 (368 mg, 2mmol, 1.0 equiv.) in anhydrous THF (5 mL) prepared using general synthetic method B at-30 deg.C was added a solution of LDA (2.2 mmol, 1.1 equiv.) in THF (by dissolving at-30 deg.C)Freshly prepared by mixing 0.34mL diisopropylamine with 0.96mL2.5m nBuLi in 5mL THF). The reaction changed from clear to yellow. After 20 min, iodine (609 mg, 2.4mmol, 1.2 equiv) was added dropwise. After the reaction was warmed to room temperature in a cold water bath, it was quenched with water. The reaction was diluted with 10mL ethyl acetate and extracted with ethyl acetate (10 mL x2 times). The combined organic layers were washed with brine, over Na₂SO₄Drying and concentrating. The crude mixture was then adsorbed on silica gel and purified by flash chromatography (eluting with 10% hexane/ethyl acetate) to give compound 41 as a white solid (407 mg, 65% yield).

Compounds 42 and 43 were synthesized using general synthesis method B.

44, synthesis: to DMF (1 mL) containing Compound 43 (70 mg, 0.19mmol, 1 eq) was added Pd (PPh)₃)₄(44 mg, 0.038mmol, 0.2 equiv.) and Zn (CN)₂(30 mg, 0.25mmol, 1.1 equiv.). Using the small bottle with N₂The gas was purged for 5 minutes and then capped. The reaction was then heated at 100 ℃ for 1.5 hours under microwave irradiation. All solvents were removed and the residue was purified by preparative thin layer chromatography to give a white solid (28 mg, 42% yield). MS m/z C₁₀H₇N₃O₂S₂[M+1]⁺=266.0；¹H NMR(400MHz,DMSO-d₆)δppm9.09(s,1H),4.12(br.s.,2H)2.60(s,3H)。

46 synthesis: compound 45 prepared using general Synthesis procedure M (40 mg, 0.142mmol, 1 eq.) was taken in 1mL MeOH. To this solution was added 1N NaOH (2.1 mL). The resulting mixture was then stirred at room temperature and then neutralized by the addition of 1N HCl. The reaction was purified by preparative HPLC to give compound 46 (12 mg, 29% yield) as a white solid. MS m/z C₁₀H₉N₃O₃S₂[M+1]⁺=284.0, observed value 224.¹H NMR (400MHz, chloroform-d) delta ppm8.83(s,1H)6.06-6.36(m,2H)4.20(s,2H)2.77(s, 3H).

General synthesis method N: preparation of 2- (3-chloro-2-methylthio [3,2-c ] pyridin-4-ylthio) acetic acid (50)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

compound 47 is commercially available. Compound 48 was synthesized using general procedure a. Compounds 49 and 50 were synthesized using general procedure B.

General synthetic method O: preparation of 2- (5, 6-dimethylfuro [2,3-d ] pyridin-4-ylthio) acetic acid (55)

This general method of synthesizing the compounds encompassed by the present invention is illustrated by the specific examples described below:

52, synthesis: in a 50mL round-bottom flask, 2-amino-4, 5-dimethylfuran-3-carbonitrile 51 (1 g,7.4 mmol) was taken and placed in formic acid (15 mL) as a solvent. The mixture was heated to reflux temperature for 6 hours. The reaction was cooled to room temperature and concentrated under vacuum pump. It is then placed in dichloromethane with H₂And O washing. The crude product was then passed over Na₂SO₄And concentrated to give crude dimethylfuropyrimidinol 52 as a crude product, which was used directly in the next step.

Synthesis of 53: reacting 5, 6-dimethylfuro [2,3-d ]]Pyrimidin-4-ol 52 (700 mg, 4.3mmol, 1 eq.) was suspended in POCl₃(10 mL, 100mmol, 23 equiv.) and heated to 90 ℃ for 3 hours. After cooling, the reaction was concentrated and the residue was takenThe residue was cooled to 0 ℃. Ice cold water was then added slowly to ensure POCl₃And completely consumed. The black solution was extracted 3 times with ethyl acetate. The combined organic extracts were washed with saturated NaHCO₃The aqueous solution was washed twice, once with brine over MgSO₄Drying, filtering and concentrating to obtain light brown solid 4-chloro-5, 6-dimethylfuro [2,3-d]Pyrimidine 53 (350 mg, 55% yield), which was used immediately after complete drying.

54, synthesis: 4-chloro-5, 6-dimethylfuro [2,3-d ] pyrimidine 53 (350 mg, 1.91mmol, 1 eq.) was suspended in methanol (10 mL) and cooled to 0 ℃. Methyl 2-mercaptoacetate (0.13 mL, 2.0mmol, 1.05 equiv.) was added slowly followed by TEA (0.39 mL, 3.84mmol, 2.1 equiv.) added slowly. After stirring for 90 minutes, the reaction was concentrated. The residue was taken up in ethyl acetate and filtered through a plug of silica gel to remove salt and some colour. The filtrate was concentrated and purified by silica gel chromatography using 0-15% ethyl acetate in hexane to give methyl 2- (5, 6-dimethylfuro [2,3-d ] pyrimidin-4-ylthio) acetate 54 as a solid (420 mg, 90% yield).

55, synthesis: methyl 2- (5, 6-dimethylfuro [2,3-d ] pyrimidin-4-ylthio) acetate 54 (400 mg, 1.58mmol, 1 eq) was taken in THF (10 mL), and 1N aqueous NaOH (3.2 mL, 3.2mmol, 2 eq) was added with efficient stirring. After 40 min, the reaction was concentrated and the residue taken up in water, cooled to 0 ℃ and acidified to low pH with 1N HCl. The precipitate was collected by vacuum filtration and washed with water. This was dried under vacuum overnight to give pure 2- (5, 6-dimethylfuro [2,3-d ] pyrimidin-4-ylthio) acetic acid 55 (353 mg, 94% yield) as a solid.

General synthetic method P: preparation of 2- (7-cyclopropyl-6- (trifluoromethyl) thieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid (60):

compounds 26, 56, 57, 58 and 60 were synthesized using general synthesis method J.

59 Synthesis: a100 mL round-bottom flask was charged with Ru (phen)₃Cl₂(14 mg, 0.01 eq.) and K₂HPO₄(930 mg, 3 equiv.) and the reaction mixture degassed by alternating evacuation and nitrogen backfilling (. times.3) and then adding MeCN (20 mL, 0.125M) and 2- (7-cyclopropylthieno [3,2-d ] via syringe]Pyrimidin-4-yl) thio) acetic acid ethyl ester 58 (500 mg, 1 eq). The resulting solution was degassed by alternating evacuation and nitrogen backfilling (× 3) at-78 ℃, allowing the solution to warm to room temperature under nitrogen between each repetition. Trifluoromethanesulfonyl chloride (0.4 mL, 2 equiv.) was added via syringe, the vial was sealed with parafilm and placed approximately 2cm from a 25W small fluorescent bulb. After 48 h, the reaction was quenched with water (2 mL) and CH₂Cl₂Extract (. times.2), combine the organic layers over MgSO₄Dried and concentrated in vacuo. The crude material was then purified by preparative HPLC using neutral conditions to give 2- (7-cyclopropyl-6- (trifluoromethyl) thieno [3,2-d ] in 10% yield]Pyrimidin-4-yl) thio) acetic acid methyl ester 59 (62 mg).

Representative Compounds

A large number of compounds were prepared and tested for their activity in one or more of the assays described herein. Some of these compounds are listed in table 1 below, where the "preparation" column indicates the general synthetic method used to prepare the compounds. The column "HPLC method and time (min)" refers to the following HPLC conditions:

a: sunfire c185u4.6x50mm, 0% to 90% B, gradient time =2min, flow rate =3.5mL/min, wavelength =220 and 254nm, solvent a =10mM aqueous ammonium acetate, solvent B = acetonitrile.

B: sunfire c185u4.6x50mm, 10% to 90% B, gradient time =2min, flow rate =3.5mL/min, wavelength =254 and 280nm, solvent a = pure water, solvent B =95% methanol/5% water and 0.1% trifluoroacetic acid (v/v).

“IC₅₀The column provides the IC of the compound determined using the binding assay described herein₅₀Wherein: represents a value less than or equal to 0.025 μ Μ; represents a value less than or equal to 0.05 μ Μ; denotes a value less than or equal to 0.1 μ Μ; denotes a value less than or equal to 0.25 μ Μ; - - -represents the undetermined IC₅₀。“EC₅₀The column provides the EC for the compound determined using the reporter assay described herein₅₀Wherein: represents a value less than or equal to 1 μ Μ; represents a value less than or equal to 5 μ Μ; represents a value less than or equal to 10 μ Μ; denotes a value less than or equal to 15 μ Μ; - - -represents undetermined EC₅₀。

TABLE 1

Pharmacology of 2- ((5, 6-dimethylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid

In the first study, the treatment was initiated at 10.7 weeks of age with 2- ((5, 6-dimethylthieno [2, 3-d)]Pyrimidin-4-yl) thio) acetic acid the in vivo effect of the compound was determined by 25 days of treatment of F1 male hybrid (129 x C57) mice. The compound is administered in the diet of the animal. Four groups of mice were used: control (N = 12); 25mg/kg of compound (N = 9); 83mg/kg of compound (N = 9); and administration of Li in the diet₂CO₃(0.1%) of the other group (N = 9).

Cortical thickness of the mid-femoral shaft was measured using Scanco μ CT 40. An increase in cortical bone thickness was observed at both compound doses compared to the control group: 9% (p = 0.04) in the 25mg/kg group; 83mg/kg group 8% (p = 0.07). For Li₂CO₃In group, a 2% reduction in cortical bone thickness was observed (p = 0.90).

In a second study, the in vivo effect of the compounds was determined by 25 days of treatment of F1 male hybrid (129 x C57) mice, again starting at 10.7 weeks of age, with 2- ((5, 6-dimethylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid. The compound is administered in the diet of the animal. Four groups of mice were used: control (N = 12); 1mg/kg of compound (N = 9); 8mg/kg of compound (N = 9); and 24mg/kg of compound (N = 9). As shown in fig. 5, mice treated with 8mg/kg showed a 4% (p = 0.19) increase in cortical thickness of the mid-femoral shaft and mice treated with 24mg/kg showed a 6% (p = 0.05) increase.

Pharmacology of 2- ((5-chloro-6-methylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid

The pharmacology of 2- ((5-chloro-6-methylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid was studied in Fischer344 ovariectomized rats. Rats underwent ovariectomy or sham surgery at 43 or 46 weeks of age, and after 37 or 40 weeks, treatment with compound was initiated at 83 weeks of age.

4 treatment groups were used: sham group given control diet (N = 14); sham group to which compound was administered (N = 12); OVX surgery group given control diet (N = 14) and OVX surgery group given compound (N = 12). The compounds were administered by incorporation into the diet of rats: 0.46 g compound per kg diet. The target dose was 30 mg/kg. Dosing was performed for 5 weeks.

Bone mass and structure were determined by micct using Scanco μ CT 40. As shown in fig. 6, treatment increased cortical bone thickness of the mid-femoral shaft in both the sham and OVX groups: increased by 3% in intact rats and 5% in ovariectomized rats. The difference between cortical bone thickness of the sham-operated control group and OVX control group was-13%. The two-factor ANOVA value was: for OVX surgery, p < 0.001; for treatment, p = 0.05; for the interaction, p = 0.65.

As shown in fig. 7, treatment also increased cortical thickness of the medial tibial shaft in both the sham and OVX groups: increased by 2% in intact rats and 5% in ovariectomized rats. The two-factor ANOVA value was: for OVX surgery, p = 0.79; for treatment, p = 0.05; for the interaction, p = 0.52.

Pharmacology of 2- ((5-chloro-6-isopropylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid

The in vivo effect of the compounds was determined by starting 25 days of treatment of F1 male hybrid (129 x C57) mice with 2- ((5-chloro-6-isopropylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid at 8.7 weeks of age. The compound was administered twice daily by oral gavage (vehicle = water containing 0.1% Tween 80). 4 groups of mice were used: control (N = 13); 5mg/kg of compound (N = 13); 10mg/kg of compound (N = 13); 15mg/kg of compound (N = 13).

Cortical thickness of the mid-femoral shaft was measured by micct (Scanco μ CT 40). An increase in cortical bone thickness was observed at all doses compared to the control group: 6% increase in the 5mg/kg group (p = 0.002); an increase of 5% (p = 0.007) in the 10mg/kg group; an increase of 6% (p = 0.001) in the 15mg/kg group.

Pharmacology of 2- ((6-chloro-7-cyclopropylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid

The in vivo effect of the compounds was determined by starting 25 days of treatment of F1 male hybrid (129 x C57) mice with 2- ((6-chloro-7-cyclopropylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid at 8.7 weeks of age. The compound was administered daily by oral gavage (vehicle = water containing 0.1% Tween 80). 4 groups of mice were used: control (N = 13); 3mg/kg of compound (N = 13); 10mg/kg of compound (N = 13); 30mg/kg of compound (N = 13).

Cortical thickness of the mid-femoral shaft was measured by micct (Scanco μ CT 40). As shown in fig. 8, an increase in cortical bone thickness was observed at all doses compared to the control: 7% increase in the 3mg/kg group (p = 0.003); 10% increase in the 10mg/kg group (p < 0.001); an increase of 13% (p < 0.001) in the 30mg/kg group.

Pharmacology of 2- ((6-chloro-7-methylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid

In the first experiment, the in vivo effect of the compounds was determined by 25 days of treatment of F1 male hybrid (129 x C57) mice with 2- ((6-chloro-7-methylthio [3,2-d ] pyrimidin-4-yl) thio) acetic acid starting at 54 weeks of age. The compound was administered daily by oral gavage (vehicle = water containing 0.1% Tween 80). 3 groups of mice were used: control (N = 10); 10mg/kg of compound (N = 10); 100mg/kg of compound (N = 10). An increase in cortical bone thickness of the mid-femoral shaft measured by micct (Scanco μ CT 40) was observed at both doses compared to the control: 6% increase in the 10mg/kg group (p = 0.06); an increase of 7% (p = 0.03) in the 100mg/kg group. In this experiment, cortical thickness data of the mid-femoral shaft was obtained using the right femur of each mouse, as is usually the case. In this experiment, the results of the 10mg/kg dose group included left femoral measurements from one mouse with abnormal right femoral measurements.

In a second experiment, the in vivo effect of the compounds was determined by starting treatment of F1 male hybrid (129 x C57) mice with 2- ((6-chloro-7-methylthio [3,2-d ] pyrimidin-4-yl) thio) acetic acid at 8.1 weeks of age for 7 and 18 days. The compound is administered in the diet of the animal. 3 groups of mice were used: control (N = 9); 34mg/kg of compound for 7 days (N = 9); 34mg/kg of compound for 18 days (N = 9). As shown in fig. 9, an increase in cortical bone thickness of the mid-femoral shaft was observed in both treatment groups compared to the control: an increase of 6% (p = 0.13) after 7 days of treatment; increased by 10% (p < 0.01) after 18 days of treatment.

All references (e.g., patents and patent applications) cited above are hereby incorporated by reference in their entirety.

Claims (15)

1. A compound of the formula:

wherein:

x is OR_1BOr N (R)_1B)₂；

Each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₂is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2A、-N(R_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₃is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_3AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₅is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_5AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and is

n is 1 or 2;

wherein "optionally substituted" means optionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, haloalkyl, heterocycle, or hydroxy.

2. A compound of the formula:

wherein:

x is OR_1BOr N (R)_1B)₂；

Each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₂is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2A、-N(R_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₃is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_3AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₄is hydrogen, halogen or optionally substituted alkyl, aryl, heteroalkyl or heterocyclic;

R₅is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_5AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and is

n is 0, 1 or 2;

wherein "optionally substituted" means optionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, haloalkyl, heterocycle, or hydroxy.

3. The compound of claim 2, wherein n is 0;

4. the compound of claim 1 OR 2, wherein X is OR_1B。

5. The compound of any one of claims 1-4, wherein R_1BIs hydrogen orOptionally substituted alkyl or aryl.

6. The compound of claim 5, wherein R_1BIs hydrogen or alkyl.

7. The compound of any one of claims 1-4, wherein R₂Is hydrogen or optionally substituted alkyl.

8. The compound of any one of claims 1-4, wherein R₃Is alkyl or halogen.

9. The compound of claim 8, wherein R₃Is chlorine.

10. The compound of any one of claims 1-4, wherein R₅Is alkyl or halogen.

11. The compound of claim 10, wherein R₅Is C_1-4An alkyl group.

12. A compound which is 2- ((5, 6-dimethylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid, 2- ((5-chloro-6-isopropylthieno [2,3-d ] pyrimidin-4-yl) thio) acetic acid, 2- ((6-chloro-7-cyclopropylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid or 2- ((6-chloro-7-methylthieno [3,2-d ] pyrimidin-4-yl) thio) acetic acid, or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient or diluent.

14. Use of a compound according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment or management of a disease or disorder characterized by bone loss.

15. Use of a compound of the formula:

wherein:

x is OR_1BOr N (R)_1B)₂；

Each R_1BIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₂is hydrogen, halogen, -CO₂R_2A、-C(O)N(R_2A)₂、-SR_2A、-OR_2A、-N(R_2A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_2AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₃is hydrogen, halogen, cyano, -CO₂R_3A、-C(O)N(R_3A)₂、-SR_3A、-OR_3A、-N(R_3A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

each R_3AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

R₅is hydrogen, halogen, -CO₂R_5A、-C(O)N(R_5A)₂、-SR_5A、-OR_5A、-N(R_5A)₂Or optionally substituted alkyl, aryl, heteroalkyl, or heterocycle; and is

Each R_5AIndependently hydrogen or an optionally substituted alkyl, aryl, heteroalkyl, or heterocycle;

wherein "optionally substituted" means optionally substituted with one or more alkoxy, alkyl, amino (including alkylamino, dialkylamino), aryl, carboxylic acid, cyano, halogen, haloalkyl, heterocycle, or hydroxy.