WO2016004028A1 - Dérivés 2-oxopyrimidine-5-carboxylate pour le traitement de maladies de la thyroïde - Google Patents

Dérivés 2-oxopyrimidine-5-carboxylate pour le traitement de maladies de la thyroïde Download PDF

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Publication number
WO2016004028A1
WO2016004028A1 PCT/US2015/038538 US2015038538W WO2016004028A1 WO 2016004028 A1 WO2016004028 A1 WO 2016004028A1 US 2015038538 W US2015038538 W US 2015038538W WO 2016004028 A1 WO2016004028 A1 WO 2016004028A1
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alkyl
hydrogen
thyroid
alkoxy
mammal
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PCT/US2015/038538
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English (en)
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Rauf LATIF
Terry Davies
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Latif Rauf
Terry Davies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0459—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two nitrogen atoms as the only ring hetero atoms, e.g. piperazine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/046—Thyroid disorders

Definitions

  • the invention relates to 2-oxopyrimidine-5-carboxylic esters and amides that are agonists at the TSH receptor (TSHR).
  • TSHR TSH receptor
  • rhTSH is expensive and must be administered parenterally.
  • Small molecule agonists to the TSHR have potential for clinical use as a cost-effective method of diagnosing and treating thyroid cancer metastases when used as a substitute for expensive recombinant TSH. They also have possible uses in the treatment of thyroid dysfunction.
  • Thyroid-stimulating hormone is a heterodimeric glycoprotein hormone secreted from the anterior pituitary. Its action is mediated through the TSHR, which is a member of the class A GPCR family. The ho lo receptor has been fully characterized. TSHR, in addition to being the major regulator of thyroid gland function, is also expressed in bone. In vitro and in vivo studies of TSHR regulation in osteoclasts and osteoblasts have defined the importance of TSH and the TSHR in bone remodeling (see Abe et al., Cell.
  • TSHR also happens to be a primary autoantigen in autoimmune thyroid disease, especially Graves' disease. Modulating the function of the receptor either orthosterically or allosterically, using small molecule ligands (SMLs), therefore has therapeutic potential.
  • SMLs small molecule ligands
  • the invention relates to method for the treatment of a thyroid disease or condition comprising administering to a mammal a therapeutically effective amount of a compound of formula I:
  • R 1 is selected from hydrogen, (Ci-C6)alkyl, fluoro(Ci-C6)alkyl, (Ci-C6)alkoxy, fluoro(Ci-C6)alkoxy, (Ci-C6)alkylthio, fluoro(Ci-C6)alkylthio, aryl, aryloxy, arylthio, cyano, and alkoxycarbonyl;
  • R 2 is selected from O and S;
  • R 3 is selected from hydrogen and (Ci-Ce)alkyl
  • R 4 is (Ci-Cio)hydrocarbyl optionally substituted with one, two, or three substituents independently selected from -OH, halogen, (Ci-Ce)alkyl, (Ci-C6)alkoxy, cyano, and nitro;
  • R 5 is selected from -OR 5a and -NR 5b R 5c ;
  • R 5a is hydrogen or (Ci-Cio)hydrocarbyl, optionally substituted with one, two, or three substituents independently selected from -OH, halogen, (Ci-C6)alkyl, (Ci-C6)alkoxy, methylenedioxy, ethylenedioxy, cyano, and nitro;
  • R 5b is (Ci-Cio)hydrocarbyl, optionally substituted with one, two, or three substituents independently selected from -OH, halogen, (Ci-Ce)alkyl, (Ci-C6)alkoxy, methylenedioxy, ethylenedioxy, cyano, and nitro; and
  • R 5c is selected from hydrogen and (Ci-C6)alkyl.
  • the invention relates to a method for treating hyperthyroid diseases such as Graves' disease, thyroid cancer, or hyperthyroid as a consequence of pituitary cancer.
  • hyperthyroid diseases such as Graves' disease, thyroid cancer, or hyperthyroid as a consequence of pituitary cancer.
  • the compound of formula I is administered in combination with radioactive iodine.
  • the invention relates to a method for treating a hypothyroid condition, such as Hashimoto's thyroiditis.
  • a hypothyroid condition such as Hashimoto's thyroiditis.
  • the compound of formula I functions directly to stimulate thyroid output.
  • the invention in another aspect, relates to a method for determining the success of thyroid ablation in a mammal.
  • the method comprises the sequential steps of:
  • the observation of an increase between the first and second measurement indicates metastasis or incomplete ablation.
  • the measure of thyroid output is thyroglobulin concentration.
  • the invention in another aspect, relates to a method for activating a thyroid stimulating hormone receptor in a mammal comprising administering to the mammal an amount of a compound of formula I.
  • the invention in another aspect, relates to a method for activating a thyroid stimulating hormone receptor in at least one mammalian cell comprising obtaining a sample of thyroid tissue from a mammal and bringing a compound of formula I into contact with the sample.
  • the invention in another aspect, relates to a method for treating a bone degenerative disorder, such as osteoporosis, comprising administering to a mammal a therapeutically effective amount of a compound of formula I.
  • the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of formula I or one of the subgenera described below.
  • R 2 is O, and in some it is S.
  • R 3 is hydrogen or methyl.
  • R 4 is selected from methyl and optionally substituted phenyl; usually R 4 is methyl.
  • R 1 is selected from hydrogen, methyl, ethyl, propyl, butyl, methoxy, ethoxy, -CF3, -CF2CF3, -SCH3, -SCF3, -OCF3, phenyl, phenyloxy, benzyloxy, phenylthio, benzylthio, -CN, and -CO2CH3.
  • R 1 is selected from -CF3, ethyl, isopropyl, and -SCH3.
  • R 5 is -OR 5a .
  • R 5a is selected from (Ci-C6)alkyl and benzyl, and benzyl may be optionally substituted with (Ci-C4)alkyl or -OCH2O-.
  • R 5a is selected from (Ci-C4)alkyl, benzyl, and methylenedioxybenzyl.
  • R 2 is O
  • R 3 is hydrogen
  • R 4 is methyl.
  • R 5 is -NR 5b R 5c .
  • R 5b is selected from hydrogen, methyl, phenyl, and benzyl.
  • R 2 is O
  • R 3 is hydrogen
  • R 4 is methyl
  • R 5b is benzyl or substituted benzyl
  • R 5c is hydrogen or methyl.
  • Ci to C20 hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthylethyl. Hydrocarbyl refers to any substituent comprised of hydrogen and carbon as the only elemental constituents.
  • Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched. Examples of aliphatic hydrocarbons include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbornyl, etc.
  • Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
  • alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof.
  • Alkyl refers to alkyl groups from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s- butyl, t-butyl and the like.
  • the use of the generic term includes all isomers; thus, for example, "butyl” encompasses all four-carbon alkyls: w-butyl, sec-butyl, isobutyl and i-butyl.
  • Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cy-propyl, cy-butyl, cy-pentyl, norbornyl and the like.
  • carbocycle is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state.
  • C3-C10 carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene;
  • Cs-Ci 2 ) carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene.
  • Carbocycle if not otherwise limited, refers to monocycles, bicycles and polycycles.
  • Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • a heterocycle may be non- aromatic (heteroaliphatic) or aromatic (heteroaryl).
  • heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzo furan, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like.
  • heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and
  • Hydrocarbyloxy refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms attached to the parent structure through an oxygen.
  • Alkoxy is a subset of hydrocarbyloxy and includes groups of a straight or branched configuration. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like.
  • alkoxy (and hydrocarbyloxy) includes methyl enedioxy and ethylenedioxy.
  • Lower-alkoxy refers to groups containing one to four carbons.
  • halogen means fluorine, chlorine, bromine or iodine atoms.
  • acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. Examples include acetyl, benzoyl, propionyl, isobutyryl and the like. Lower- acyl refers to groups containing one to four carbons.
  • the double bonded oxygen, when referred to as a substituent itself is called "oxo".
  • substituted refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical.
  • cycloalkylaminoalkyl dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyloxyphenyl, and benzyloxy.
  • Oxo is also included among the substituents referred to in "optionally substituted”; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl).
  • 1, 2, or 3 hydrogen atoms are replaced with a specified radical.
  • more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine.
  • substituents are halogen, halo(Ci-C4)hydrocarbyl, halo(Ci-C4)hydrocarbyloxy, cyano, thiocyanato, (Ci-C4)hydrocarbylsulfinyl, (Ci-C4)hydrocarbyl-sulfonyl, aminosulfonyl, nitro, acetyl, and acetamido.
  • R 2 be sulfur
  • the pyrimidinone above can be treated with P2S5 or Lawesson's reagent.
  • the nitrogen may be alkylated to provide compounds in which R 3 is other than hydrogen.
  • the ester in which R 5a is methyl or ethyl may be saponified and the resulting acid condensed with the appropriate amine by methods well-known in the peptide art.
  • the compounds described herein may contain an asymmetric center (depending on substitution) and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-.
  • the present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms.
  • Optically active (R)- and (S)- isomers may be prepared using homo- chiral synthons or homo-chiral reagents, or optically resolved using conventional techniques. All tautomeric forms are intended to be included.
  • salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric,
  • ethanedisulfonic ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like.
  • suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine,
  • chloroprocaine choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.
  • composition comprising a compound disclosed above, or a pharmaceutically acceptable salt form thereof, and a pharmaceutically acceptable carrier or diluent.
  • the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients.
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of formula I with the carrier, which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the compound of formula I may be administered in combination with radioactive iodine.
  • the compound of formula I acts as an agonist (analogously to natural TSH or Thyrogen®) to stimulate the thyroid to take up the radioiodine, resulting in chemical ablation.
  • the initial therapy for most patients with well differentiated thyroid cancer is total or near-total thyroidectomy.
  • Thyroidectomy is followed by radioactive iodine ( 131 I) thyroid remnant ablation to destroy residual thyroid tissue.
  • the compound of formula I is given either concurrently with 13 l I, or the two are administered separately, usually with the 131 I administered 12- 48 hours after one or more doses of the compound of formula I.
  • the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number most abundant in nature.
  • Radioisotopes of hydrogen, carbon, phosphorous, fluorine, and chlorine include 2 H, 3 H, 13 C, 14 C, 15 N, 35 S, 18 F, and 36 C1, respectively.
  • Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention.
  • Tritiated, i.e. 3 H, and carbon-14, i.e., 14 C, radioisotopes are particularly preferred for their ease in preparation and detectability.
  • Compounds that contain isotopes n C, 13 N, 15 0 and 18 F are well suited for positron emission tomography.
  • Radiolabeled compounds of formula I of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.
  • CHO-HA - TSHR luciferase cells for primary screening were generated by trans fecting pGL4.29 [luc2P/CRE/Hygro] construct into a highly selected stable line of HATSHR CHO cells as described by Nagayama et al., [J Clin Invest. 1991 ;88(l):336-340] and selecting them with hygromycin.
  • TSHR/LHR chimeric luciferase cells were prepared using a construct pSV2-neo-ECE-TSH-LHR-l 1 (Kindly provided us by Dr. Basil Rapoport, Cedars-Sinai Research institute and University of California, Los Angeles, CA).
  • a 367 amino acid insert containing the homologous regions of the rat LH/CG receptor sequence replaced the TSHR ectodomain, which was then co-transfected with the pGL4.29 [luc2P/CRE/Hygro] construct in CHO cells and further selected for double transfectants with optimal concentrations of neomycin sulphate and hygromycin.
  • Parent CHO luciferase cells were generated by transfecting pGL4.29 [luc2P/CRE/Hygro] construct into CHO PSVL cells (JP02) and selecting with hygromycin for stable trans formants. The best stable clone was selected based on different concentrations of forskolin and unresponsiveness to TSH. All of these above mentioned stable cell lines were cultured in Ham's F-12 medium with 10% fetal bovine serum (FBS) and lOOIU/ml of penicillin and streptomycin and 50ug/ml of hygromycin
  • TM4 primary Sertoli cells obtained from ATCC (CRL-1715) and cultured in DMEM: F12 medium (cat # 30-2006) with 2.5% FBS and 5% horse serum (ATCC; cat #30-2040).
  • the specificity against the LH/hCG receptor was tested using stable line of rat hCGR in HEK 293 cells that we obtained from Dr K.M.J Menon, University of Michigan, Ann Arbor, Michigan.
  • a high expressing stable line of CHO-HA TSHR cells carrying an amino terminus HA tagged TSHR was selected. These stable CHO cells were transfected with the construct pGL4.29 [CRE/minP/luc2P] carrying a minimal promoter driving a CREB response element (CRE) tagged to a modified form of luciferase reporter gene luc2P.
  • Luc2P is a synthetically derived luciferase sequence with humanized codon optimization that is designed for high expression and reduced anomalous transcription.
  • the luc2P gene contains hPEST, a protein destabilization sequence, which further reduces background, transcribed protein.
  • TSH receptor Activation of the TSH receptor by TSH or an agonist results in Gsa-adenylate cyclase coupling and increase in intracellular cAMP, which binds to the CRE element and results in the transcription of luciferase gene and accumulation of luciferase enzyme within the activated cells. Luciferase in these cells was detected after lysing the cells using the commercial substrate Bright Glow (Promega Corporation, Madison, WI).
  • RNA isolation Total RNA was isolated from FRTL5 untreated with 1 ⁇ and 10 ⁇ of the compound of example 1 for 4hrs using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) and chromosomal DNA from this was removed in accordance with the manufacturer's instructions. The RNA concentration was determined on the basis of absorbance at 260 nm, and its purity was evaluated by the ratio of absorbance at 260/280 nm (>1.9). RNAs were kept frozen at -70°C until analyzed.
  • RNA (1 ⁇ g) was reverse-transcribed into cDNA with random hexamers using Advantage RT-for- PCR kit (Clontech).
  • Quantitative Reverse Transcription-PCRs The qRT-PCRs were performed using the Applied Biosystem StepOnePlus Real-time PCR system. The reactions were established with 10 of SYBR Green master mix (Applied Biosystems, Foster City, CA), 0.4 ⁇ (2 ⁇ ) of sense/anti-sense gene-specific primers, 2 ⁇ of cDNA and DEPC- treated water to a final volume of 20 ⁇ . The PCR reaction mix was denatured at 95 °C for 60 s before the first PCR cycle. The thermal cycle profile was: denaturizing for 30 s at 95 °C; annealing for 30 s at 57- 60 °C(dependent on primers); and extension for 60 s at 72°C.
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • mice Female C57BL/6 mice (Jackson Laboratory) of 6-8 weeks old with mean body weight of 20 g maintained on standard diet were injected intraperitoneal (IP) with 100 ⁇ g/mouse of the compound of example 1 for three consecutive days in a fluid volume of 60-90 ⁇ ⁇ containing a final concentration of - 25% DMSO.
  • IP intraperitoneal
  • the control animals received diluted vehicle (DMSO) or bovine TSH 30ug/mouse by the same route.
  • Thyroid hormone (T4) levels were estimated in serum from blood collected by
  • T4 was measured with Neonatal free T4 RIA kit (Coat-A-Count, Siemens Medical Solutions Diagnostics, CA) according to the manufacturer's protocol. All experiments involving animals were carried out according to the institutional animal care committee guidelines.
  • TSHR cell lines expressing various reporter vectors (CRE-, NFAT-RE, SRE-, SRF-RE-). These stable lines were characterized and optimized for responses using positive (TSH, inomycin, PMA) and negative controls.
  • 20,000 cells Prior to measurement of signaling, 20,000 cells were seeded in square bottom white plates (Nunc cat # 164610) in 20 ⁇ 1 of Ham's F12 complete medium and incubated overnight at 37°C. Following the complete medium was replaced with serum free medium for 2hrs and then treated with 10 ⁇ of compound and the appropriate controls for 4hrs. At the end of incubation period the cells were lysed using ⁇ , of Bright Glow reagent and incubated for 2 minutes at RT and the plates were read using BMG Pherastar microplate reader.
  • Example 1 To analyze the specificity of compounds of formula I, Example 1 was tested against cells that expressed the LH receptor and FSH receptor.
  • LH receptor cells we used HEK 293 transfected with rat hCG receptor and for FSH receptor cells we used primary murine Sertoli cells (line TM4) that express the FSHR and that respond to FSH in a dose dependent manner. Intracellular cAMP was measured in these cells after stimulation with 0.1, 1 and 10 ⁇ of the test compound and corresponding positive and negative controls.
  • the compound of example 1 did not show any activity against the LH-receptor- nor the FSH- receptor-expressing cells, even at the highest concentration used (10 ⁇ ) and in contrast to the response of the cells against their respective ligands hCG and FSH incorporated as positive controls.
  • Example 1 showed no significant activation of RhoA kinase via SRF luciferase nor ERK1/2 by SRE luciferase, indicating that it did not engage ⁇ nor Gal2.
  • the activation of Gs and Gq by example 1 similar to TSH, would strongly suggest that compounds of formula I are able to initiate iodine organification and thyroid hormone secretion and promote thyroid growth by their ability to engage in Gq activation in the same manner as TSH itself.
  • Example 1 was tested for its effect on expression of mRNA for thyroglobulin (TG), sodium-iodide symporter (NIS) thyroid peroxidase (TPO) and TSH receptor expression using FRTL5. Prior to exposure the cells were deprived of TSH for 48hrs and starved in serum free medium for another 2hrs. Single-dose treatment of ⁇ ⁇ of Example 1 for 4hrs showed a 2-8 fold increase in thyroid specific gene expression (Tg, NIS and TSHR) when measured by qPCR.
  • Tg, NIS and TSHR thyroid specific gene expression
  • Example 1 In vivo potency of Example 1 : T4 levels were measured at different time points in Balb/c mice that received a single ip or iv injection of 20mg/kg body weight of example 1. T4 release was observed at 2hrs after the compounds reached peak levels in the blood but with no sustained action during the course of the study. T4 levels measured after 3 ip injections of lOC ⁇ g/mouse of example 1 dissolved in DMSO showed a sustained two-fold increase serum T4 levels. These in vivo studies clearly indicate the effectiveness of compounds of formula I as agonists to the TSHR.
  • Example 1 The compound of example 1 was also subjected to standard pharmacokinetic studies. A single injection of example 1 at 20mg/kg was given to Balb/c mice intravenously and intraperitoneally and their plasma was analyzed by mass spectrometry at different points after reaching Tmax. The half-life (Tl/2) was 1 hour by both routes of administration. Example 1 showed moderate plasma clearance of 29.63 mL/min/kg and a high volume of distribution (4.6 fold higher than total body water) indicating extravascular distribution.
  • ALP Alkaline phosphatase
  • collagen were used as osteoblast differentiation markers.
  • ALP measured after treating the cells with 10 ⁇ of Example 1 in the presence of osteogenic differentiation factor (ODF) for 10 days, increased in a statistically significant manner.
  • ODF osteogenic differentiation factor
  • H89 N-[2-((p-bromocinnamyl)amino)ethyl]- 5-isoquinolinesulfonamide
  • PKA/cAMP pathway a known inhibitor of PKA/cAMP pathway
  • Example 1 indeed induced proliferation of osteoblast cells in a manner similar to TSH.

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Abstract

L'invention concerne l'utilisation d'esters et d'amides 2-oxopyrimidine-5-carboxyliques de formule (I). Les composés sont des agonistes au niveau du récepteur TSH (TSHR) et sont par conséquent utiles dans le traitement de patients souffrant d'un dysfonctionnement de la thyroïde et dans la gestion du cancer différencié de la thyroïde.
PCT/US2015/038538 2014-07-01 2015-06-30 Dérivés 2-oxopyrimidine-5-carboxylate pour le traitement de maladies de la thyroïde WO2016004028A1 (fr)

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CN114478396A (zh) * 2022-03-18 2022-05-13 河南省人民医院 二氢嘧啶(硫)酮类化合物及其制备方法与应用
CN114957379A (zh) * 2021-06-28 2022-08-30 河南省人民医院 一种二氢嘧啶硫酮类化合物及其制备方法与应用
US11912683B1 (en) 2023-09-13 2024-02-27 King Faisal University (6-methyl-4-substitutedphenyl-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(piperidin-1-yl)methanones as antitubercular agents
US11986476B1 (en) 2023-09-13 2024-05-21 King Faisal University (6-methyl-4-substitutedphenyl-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(piperidin-1-yl)methanones as antitubercular agents

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114957379A (zh) * 2021-06-28 2022-08-30 河南省人民医院 一种二氢嘧啶硫酮类化合物及其制备方法与应用
CN114957379B (zh) * 2021-06-28 2024-03-26 河南省人民医院 一种二氢嘧啶硫酮类化合物及其制备方法与应用
CN114478396A (zh) * 2022-03-18 2022-05-13 河南省人民医院 二氢嘧啶(硫)酮类化合物及其制备方法与应用
CN114478396B (zh) * 2022-03-18 2023-12-01 河南省人民医院 二氢嘧啶(硫)酮类化合物及其制备方法与应用
US11912683B1 (en) 2023-09-13 2024-02-27 King Faisal University (6-methyl-4-substitutedphenyl-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(piperidin-1-yl)methanones as antitubercular agents
US11986476B1 (en) 2023-09-13 2024-05-21 King Faisal University (6-methyl-4-substitutedphenyl-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(piperidin-1-yl)methanones as antitubercular agents

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