Thyroid Stimulating Hormone (TSH) is a heterodimeric glycoprotein hormone that regulates thyroid homeostasis upon interaction with the TSH receptor (TSHR). TSH binds to the TSH receptor, which couples preferentially to the G-alpha (s) (Gs) protein, resulting in activation of adenylate cyclase and an increase in cyclic adenosine 3′, 5′ monophosphate (cAMP). CID 2887926 is the first example of a small molecule TSHR inverse agonist. Preliminary structure-activity relationship studies led to the discovery of a selective TSHR inverse agonist, ML224, which could be a useful tool for studying TSHR functions; it could also be a potential lead for development of drugs to treat TSHR-mediated hyperthyroidism caused by constitutively activating mutations or stimulating auto-antibodies associated with Graves’ disease.
Assigned Assay Grant #: R03MH090855
Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin
Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin
Assay Submitter & Institution: National Institute of Diabetes and Digestive and Kidney Diseases, Marvin Gershengorn
PubChem Summary Bioassay Identifier (AID): 488990
Recommendations for Scientific Use of the Probe
Thyroid Stimulating Hormone (TSH) is a heterodimeric glycoprotein hormone that regulates thyroid homeostasis upon interaction with the TSH receptor (TSHR). TSH binds to the TSH receptor, which couples preferentially to the G-alpha (s) (Gs) protein, resulting in activation of adenylate cyclase and an increase in cyclic adenosine 3′, 5′ monophosphate (cAMP)1. TSHR exhibits basal (agonist-independent or constitutive) signaling, and the current state of the art is lacking any small-molecule TSHR inverse agonists. The probe molecule (ML224) is a member of a series of TSHR modulators and selectively inhibits TSH-stimulated cAMP production with an IC50 = 2.3 μM, and TSHR basal activity with an IC50 = 6 μM. This probe can be used to study TSHR functions in vitro; it could also be used as a lead for development of drugs to treat hyperthyroidism caused by TSHR constitutively activating mutations or stimulating auto-antibodies associated with Graves’ disease.
Thyroid Stimulating Hormone (TSH) is an α/β heterodimeric glycoprotein hormone secreted from the anterior pituitary gland. It belongs to the glycoprotein hormone family, which includes Chorionic Gonadotropin (CG), Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH)2. TSH binds to the TSH receptor (TSHR), which couples preferentially to the G-alpha (s) (Gs) protein, resulting in activation of adenylate cyclase and an increase in cyclic adenosine 3′, 5′ monophosphate (cAMP). TSHR was first identified in the thyroid cell plasma membrane in 19663, and since that time, it has been found in bone4, brain5, kidney6, testis7, endometrium8, and the immune system9. The primary function of TSHR in thyroid follicular cells is the regulation of thyroid hormone synthesis and secretion, and thyrocyte size and number1,2, but its role in other organs and tissues is not fully understood. The identification of small-molecule TSHR modulators could provide valuable pharmacological tools for researchers interested in elucidating the roles and importance of this receptor in a variety of these tissues. TSHR antagonists could be used to treat patients with recurrent or metastatic thyroid cancer10, nonautoimmune hyperthyroidism11,12, or Graves’ disease13–15.
Graves’ disease affects close to 0.5% of the population, with a majority of the cases seen in females, and it is the major cause of hyperthyroidism. This disease shares many features with autoimmune hypothyroidism, and its cause is the presence of IgG antibodies that target and activate TSHR16. Some side effects include Graves’ ophthalmopathy, weight loss, heat intolerance, difficulty sleeping, and tremors. Current treatments for Graves’ disease include antithyroid drugs17, radioactive iodine, or surgery. Each of these therapies has been shown to be effective in clinical trials and are generally well tolerated by patients, but the amount of relapse is significant, and there are patients who require alternative treatments18,19. A bioavailable low molecular weight antagonist would present a cost effective, non-invasive, and readily manufactured and administered option for these patients.
Unlike receptors for CG/LH and FSH, TSH receptor exhibits basal (agonist-independent or constitutive) signaling. 7TMR antagonists that inhibit basal signaling are referred to as inverse agonists20. Antagonists that inhibit agonist-stimulated signaling, but do not inhibit agonist-independent signaling are termed neutral antagonists. An initial screen was first performed to identify antagonists. After these antagonists were identified, a follow up screen was performed to determine which members from this group also inhibited basal signaling. The molecules that were originally identified as antagonists, and later found to also inhibit basal signaling, were then described as inverse agonists. Although antibodies are used as TSHR inverse agonists and antagonists21, to our knowledge, there is currently no small molecule TSHR inverse agonist present in literature. The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), which has been described as a thyroid disruptor, inhibited cAMP accumulation in Chinese hamster ovary (CHO) cells expressing TSHRs22. However, because they also found that DDT-inhibited cAMP accumulation stimulated by forskolin, it is not correct to conclude that DDT is a TSHR inverse agonist. CBE-52 (Figure 1) is the only known small-molecule TSHR antagonist. This compound was active not only in the model cell system (IC50 of 4.2 μM), but it also inhibited both TSH-and thyroid-stimulating antibody (TsAb)-induced up-regulation of mRNA transcripts for thyroperoxidase under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs23. NCGC00168126 was originally identified as the first selective TSHR agonist from an NCGC qHTS screen of 73,180 compounds (AID 1401)24,25. Over 100 analogs were synthesized for structure activity relationship (SAR) studies, and as would be expected, some of the analogs were inactive. Among the 66 inactive analogs, NCGC00161856 (CID 2887926) was the only inverse agonist26. However, it showed no selectivity for TSHR over LHR and FSHR. While another library screen to identify selective TSHR antagonists is ongoing, here we wish to report the biological evaluation and preliminary structure-activity relationship study of this series of THSR antagonists.
2. Materials and Methods
All commercially available reagents and solvents were purchased and used without further purification. All compounds for biological testing were purified by silica gel column chromatography. 1H spectra were recorded using an Inova 400 MHz spectrometer (Varian). LCMS was used to analyze the samples’ purity.
Method 1: A 7 minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 ml/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50°C. Method 2: A 3 minute gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 ml/min. Determination of purity was performed using an Agilent Diode Array Detector with both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode.
See Appendix 1 for a listing of assay names and corresponding AIDs.
HTRF Assay for antagonists of the TSH receptor
TSHR is a seven-transmembrane receptor that couples to the Gs protein, resulting in activation of adenylate cyclase and an increase in intracellular cAMP level. Recently, a time resolved fluorescence resonance energy transfer (TR-FRET) based cAMP assay has been made available for high-throughput screens from Cisbio. It uses an anti-cAMP antibody labeled with Eu3+, which binds to a d2-dye labeled cAMP tracer, resulting in TR-FRET. The cAMP from the cell lysate can displace the d2 labeled cAMP tracer that interrupts the TR-FRET (Figure 2).
Elisa Assays for antagonists of the TSH, LH and FSH receptors
Generation of stable cell-lines expressing TSHR, LHCGR or FSHR: cDNA from human TSHR was amplified by PCR from hTSHR-pSVL27 and inserted into the pcDNA3.1(−)/hygromycin vector using restriction sites XhoI and BamHI. cDNA from human LHCGR was amplified by PCR from hLHR-pGS528 and inserted into the pcDNA3.1(+)/hygromycin vector using restriction sites BamHI and XhoI. The FSHR cDNA in pcDNA3.1 was obtained from the Missouri S&T cDNA Resource Center (www.cDNA.org) and was subcloned into the pcDNA3.1(−)/hygromycin vector. Constructs were confirmed by sequencing (MWG Biotech). HEK-EM 293 cells were transfected with the cDNA of TSHR, LHCGR or FSHR using FuGENE 6 Transfection reagent (Roche Diagnostics) according to the manufacturer’s protocol. Two days after transfection, the cells were passaged and grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 10 μg/ml streptomycin (Life Technologies Inc.), with hygromycin (250 μg/ml) as a selection marker. After 7 to 10 days, hygromycin-resistant clones were selected, and after a few days of further growth, they were submitted for a cAMP assay to identify clones that stably express the appropriate receptor.
Determination of intracellular cyclic AMP accumulation: Transiently transfected cells were cultured for 48 h before the cAMP assay. HEK-EM293 cells stably expressing TSHR, LHCGR or FSHR were seeded into 24-well plates with a density of 2.2× 105 cells/well 24 h before the cAMP assay. After removal of growth medium, cells were incubated for 1 h in HBSS (Cellgro) with 10 mM HEPES (Cellgro) containing 1mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma) and the ligand of interest in a humidified 5% CO2 incubator at 37°C. Following aspiration of HBSS, cells were lysed using lysis buffer of the cAMP-Screen Direct System (Applied Biosystems). The cAMP content of the cell lysate was determined using the manufacturer’s protocol. Data were analyzed using GraphPad Prism 4 for Windows. Highly purified human LH and FSH were purchased from Dr. A. Parlow (Harbor-UCLA Medical Center, CA). Bovine TSH was purchased from Sigma.
RT-PCR Analysis of TSH-dependent transcripts in primary cultures of human thyrocytes
Culture of primary human thyrocytes: Thyroid tissue samples were obtained through the NIH Clinical Center during surgery for unrelated reasons. Patients provided informed consent for an IRB approved protocol, and materials were received anonymously via approval of research activity through the Office of Human Subjects Research. The specimens were maintained in HBSS on ice, and isolation of cells was initiated within 4 h after surgery. All preparations were performed under sterile conditions. Tissue samples were minced into small pieces by fine surgical forceps and scissors in a 10 cm dish with a small volume of HBSS. Tissue pieces were transferred to a 15 ml tube (Falcon) and washed a minimum of 3 times with HBSS. Afterwards, tissue pieces were incubated with HBSS containing 3 mg/ml Collagenase Type IV (Gibco). Enzymatic digestion proceeded for 30 min or longer with constant shaking in a water bath at 37°C until a suspension of isolated cells was obtained. After centrifugation for 5 min at 1000 rpm, the supernatant was removed and cells were resuspended in 10 ml DMEM with 10% FBS. Cells were plated in 10 cm tissue culture dishes and incubated at 37°C in a humidified 5% CO2 incubator. After 24 h, the supernatant containing non-adherent cells was removed. The primary cultures of thyroid cells formed a confluent monolayer within 5–7 days. For determination of TPO mRNA expression, thyrocytes were seeded into 24-well plates at a density of 6× 104 cells/well 24 h before the experiment.
Total RNA was purified using RNeasy Micro kits (Qiagen). First strand cDNA was prepared using a High Capacity cDNA Archive Kit (Applied Biosystems). RT-PCR was performed in 25 μl reactions using cDNA prepared from 100 ng of total RNA and Universal PCR Master Mix (Applied Biosystems). Primers and probes were Assays-on-Demand (Applied Biosystems). Quantitative RT-PCR results were normalized to GAPDH to correct for differences in RNA input.
2.2. Probe Chemical Characterization
N-(4-(5-(3-(furan-2-ylmethyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)-3,5-dimethylphenyl)acetamide (7a): 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.15 (s, 3 H), 2.21 (s, 6 H), 3.77 (d, J=15.7 Hz, 1 H), 3.84 (s, 3 H), 4.41 (s, 1 H), 4.70 – 4.87 (m, 2 H), 5.35 (d, J=15.7 Hz, 1 H), 5.78 (s, 1 H), 6.20 (d, J=2.9 Hz, 1 H), 6.24 – 6.32 (m, 1 H), 6.53 (d, J=8.0 Hz, 1 H), 6.76 – 6.91 (m, 2 H), 7.03 (br. s., 1 H), 7.13 (s, 2 H), 7.30 – 7.37 (m, 2 H), 7.49 (d, J=2.0 Hz, 1 H), 7.92 – 8.03 (m, 1 H); LC/MS (electrospray + ve), m/z 526.2 (MH)+, Retention time t = 5.71 min; Purity: UV220 > 98%, UV254 > 98%; Solubility: (PBS, pH 7.4, 23°C) = 21 μM.
2.3. Probe Preparation
General procedure for the synthesis of 2-aminobenzamides from isatoic anhydride
A solution of isatoic anhydride 2 (0.16 g, 1.0 mmol, 1.0 equiv) in 10 ml of anhydrous acetonitrile at room temperature was treated with amine R2NH2 (1.05 mmol, 1.05 equiv). The reaction mixture was stirred at room temperature for 2 h, heated at 50 °C for 4 h and then concentrated under reduced pressure to afford amide 3 as a solid in 90–99% yield.
Synthesis of 2,3-dihydroquinazolin-4(1H)-ones (7)
A solution of benzyl chloride 5 in CH3CN was treated with phenol 4 and K2CO3 and heated to 150°C for 10 min in the microwave. The reaction mixture was filtered, concentrated under reduced pressure and used crude in the next reaction with 3. 2-Aminobenzamide 3 was prepared either by coupling of 2-aminobenzoic acid 1 with amine R2NH2 or reaction of isatoic anhydride 2 with amine R2NH2 (see Scheme 2). A solution of aldehyde 6 and 2-aminobenzamide 3 in EtOH was treated with Yb(OTf)3 and heated to 80°C for 2–4 h. The reaction mixture was concentrated under reduced pressure and purified via column chromatography on SiO2 (EtOAc and Hexanes) to afford 2,3-dihydroquinazolin-4-one 7.
1H NMR (400 MHz, CHLOROFORM-d) δ 4.61 (s, 1 H), 4.63 (s, 1 H), 5.58 (br. s., 2 H), 6.33 (br. s., 1 H), 6.62–6.66 (m, 1 H), 6.69–6.71 (m, 1 H), 7.19–7.25 (m, 1 H), 7.28 – 7.43 (m, 6 H); LCMS: (electrospray +ve), m/z 227.1 (MH)+; HPLC: tR = 4.38 min,UV254 = 96%.
1H NMR (400 MHz, CHLOROFORM-d) δ 4.60 (s, 1 H), 4.61 (s, 1 H), 5.57 (br. s., 2 H), 6.24 – 6.42 (m, 3 H), 6.59 – 6.74 (m, 2 H), 7.16 – 7.25 (m, 1 H), 7.33–7.39 (m, 2 H); LCMS: (electrospray +ve), m/z 217.1 (MH)+; HPLC: tR = 3.77 min, UV254 = 98%.
A solution of 3-(chloromethyl)-4-methoxybenzaldehyde (85 mg, 0.46 mmol, 1.0 equiv) and 2,6-dimethylphenol (62 mg, 0.51 mmol, 1.1 equiv) in 3.0 ml of anhydrous acetonitrile was treated with K2CO3 (320 mg, 2.3 mmol, 5.0 equiv) and heated in a microwave reactor for 30 min at 150°C. The solid was filtered, and then the filtrate was concentrated under reduced pressure and treated with 2-amino-N-(furan-2-ylmethyl)benzamide (110 mg, 0.51 μmol, 1.1 equiv) in 5 ml of EtOH, followed by ytterbium trifluoromethanesulfonate (57 mg, 0.02 μmol, 0.2 equiv). The reaction mixture was heated at 80°C for 2 h. The product was isolated via preparative HPLC purification, and the solvent was removed under reduced pressure to afford 2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one (64.5 mg, 30%) as a white solid after triturating with diethyl ether. 1HNMR (400 MHz, DMSO-d6) δ ppm 2.16 (s, 6 H), 3.77 (s, 3 H), 3.86 (d, J=15.6 Hz, 1 H), 4.69 (d, J=1.9 Hz, 2 H), 5.19 (d, J=15.4 Hz, 1 H), 5.75 (d, J=2.3, Hz, 1 H), 6.30 (d, J=3.2 Hz, 1 H), 6.39 (dd, J=3.2, 1.9 Hz, 1 H), 6.62–6.70 (m, 2 H), 6.86 – 6.97 (m, 1 H), 6.99–7.04 (m, 3 H), 7.19 – 7.29 (m, 2 H), 7.32 (d, J=2.3 Hz, 1 H), 7.49 (d, J=2.4, Hz, 1 H), 7.57 (dd, J=1.8, 0.7 Hz, 1 H), 7.68 (dd, J=7.9, 1.5 Hz, 1 H); HPLC: t = 6.88 min, UV254 = 97%; HRMS (ESI): m/z calcd for C29H28N2O4 [M+1]+ 469.2132, found 469.2138.
Potassium carbonate (1.10 g, 8.12 mmol, 5.0 equiv) was added to a solution of 3-(chloromethyl)-4-methoxybenzaldehyde (300 mg, 1.625 mmol, 1.0 equiv) and 2,6-dimethylphenol (218 mg, 1.79 mmol, 1.1 equiv) in 10 ml of acetonitrile. The reaction mixture was heated to 150°C in the microwave for 30 min. Upon completion, the mixture was filtered and concentrated under reduced pressure to afford 3-((2,6-dimethylphenoxy)methyl)-4-methoxybenzaldehyde (400 mg, 91% yield) as a yellow solid. A portion of this (100 mg, 0.370 mmol, 1.0 equiv) was dissolved in ethanol (4 ml), and treated with 2-amino-N-(pyridin-3-ylmethyl)benzamide (92 mg, 0.41 mmol, 1.1 equiv) followed by ytterbium(III) trifluoromethanesulfonate (45.9 mg, 0.074 mmol, 0.2 equiv). The reaction mixture was heated to 80°C for 2 h. Upon completion, the mixture was concentrated under reduced pressure and purified via column chromatography on silica gel with 0–30% EtOAc/Hexanes gradient elution to afford the desired 2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(pyridin-3-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one (110 mg, 62.0 % yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.39 – 8.45 (m, 2 H), 7.60 – 7.67 (m, 1 H), 7.48 (d, J=2.35 Hz, 1 H), 7.15 – 7.35 (m, 4 H), 6.94 – 7.02 (m, 3 H), 6.84 – 6.93 (m, 1 H), 6.60 – 6.69 (m, 2 H), 5.82 (d, J=2.35 Hz, 1 H), 5.07 (d, J=15.65 Hz, 1 H), 4.64 (d, J=2.74 Hz, 2 H), 3.99 (d, J=15.45 Hz, 1 H), 3.73 (s, 3 H), 2.12 (s, 6 H); LCMS: (electrospray +ve), m/z 480.2 (MH)+; HPLC: tR = 5.05 min,UV254 = 100%; HRMS (ESI): m/z calcd for C30H30N3O3 [M+H]+ 480.2282, found 480.2291.
Potassium carbonate (0.55 g, 4.0 mmol, 8.0 equiv) was added to a solution of 3-(chloromethyl)-4-methoxybenzaldehyde (91.0 mg, 0.5 mmol, 1.0 equiv) and 2,6-dimethylbenzenethiol (68.4 mg, 0.5 mmol, 1.0 equiv) in 4 ml of acetonitrile. The mixture was heated to 150°C in the microwave for 10 min, filtered, and the filtrate was concentrated under reduced pressure. The crude product was treated with 2-amino-N-(furan-2-ylmethyl)benzamide (107 mg, 0.5 mmol, 1.0 equiv) in 5 ml of EtOH, followed by ytterbium trifluoromethanesulfonate (62 mg, 0.1 mmol, 0.2 equiv). The reaction mixture was heated at 80°C for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure and purified via column chromatography on silica gel with 10–60% EtOAc/Hexanes gradient elution to afford 2-(3-((2,6-dimethylphenylthio)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one (101 mg, 42%) as a white solid after triturating with diethyl ether. 1HNMR (400 MHz, DMSO-d6) δ ppm 2.26 (s, 6 H), 3.56 (d, J=15.7 Hz, 1 H), 3.66 (s, 3 H), 3.72 (d, 1 H), 3.79 (d, 1 H), 5.12 (d, J=15.7 Hz, 1 H), 5.55 (d, J=2.2 Hz, 1 H), 6.25 (d, J=3.1 Hz, 1 H), 6.41 (dd, J=3.0, 1.9 Hz, 1 H), 6.59 (d, J=8.0 Hz, 1 H), 6.66 (t, J=7.5 Hz, 1 H), 6.78 – 6.94 (m, 2 H), 7.00 – 7.28 (m, 6 H), 7.59–7.66 (m, 2 H); LCMS: (electrospray + ve), m/z 485.2 (MH)+, tR = 7.16 min, UV254 = 98%.
A suspension of 2,6-dimethyl-4-nitrophenol (1.58 g, 9.45 mmol) in AcOH (20 ml), MeOH (15 ml), and THF (10 ml) in a hydrogenator vessel was treated with acetic anhydride (6 ml, 63.6 mmol) and PtO2 (200 mg, 0.881 mmol), pressurized to 50 p.s.i. with H2 and shaken for 24 h. The reaction mixture was returned to atmospheric pressure, diluted with EtOAc, washed with H2O, dried (MgSO4), filtered and concentrated under reduced pressure. N-(4-hydroxy-3,5-dimethylphenyl)acetamide was isolated as a white solid that was pure based upon LCMS analysis and used without further purification.
Potassium carbonate (810 mg, 5.34 mmol) was added to a solution of tetrabutylammonium iodide (78.9 mg, 0.534 mmol), 3-(chloromethyl)-4-methoxybenzaldehyde (197 mg, 1.07 mmol), and N-(4-hydroxy-3,5-dimethylphenyl)acetamide (211 mg, 1.18 mmol) in 20 ml of acetonitrile. The reaction mixture was heated to 150°C in the microwave for 1 h, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with 5–50% EtOAc/DCM gradient to afford the desired product (196 mg, 56%) as a white solid. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.15 (s, 3 H), 2.28 (s, 6 H), 3.93 (s, 3 H), 4.83 (s, 2 H), 7.02 (d, J=8.4 Hz, 1 H), 7.13 (s, 1 H), 7.17 (s, 2 H), 7.89 (dd, J=8.4, 2.0 Hz, 1 H), 8.12 (d, J=1.8 Hz, 1 H), 9.94 (s, 1 H); LCMS: (electrospray + ve), m/z 328.2 (MH)+, tR = 3.16 min, UV254 = >95%.
N-(4-(5-formyl-2-methoxybenzyloxy)-3,5-dimethylphenyl)acetamide (145 mg, 0.441 mmol), 2-amino-N-(furan-2-ylmethyl)benzamide (105 mg, 0.486 mmol), and 0.2 equiv of Yb(OTf)3 in 5 ml of EtOH were heated at 80°C for 4 h. The reaction mixture was concentrated under reduced pressure and the residue was purified via column chromatography on silica-gel with 7–60% ethyl acetate in hexanes to afford the desired product as a white solid (40.9 mg, 0.078 mmol, 17.6% yield). 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.15 (s, 3 H), 2.21 (s, 6 H), 3.77 (d, J=15.7 Hz, 1 H), 3.84 (s, 3 H), 4.41 (s, 1 H), 4.70 – 4.87 (m, 2 H), 5.35 (d, J=15.7 Hz, 1 H), 5.78 (s, 1 H), 6.20 (d, J=2.9 Hz, 1 H), 6.24 – 6.32 (m, 1 H), 6.53 (d, J=8.0 Hz, 1 H), 6.76 – 6.91 (m, 2 H), 7.03 (br. s., 1 H), 7.13 (s, 2 H), 7.30 – 7.37 (m, 2 H), 7.49 (d, J=2.0 Hz, 1 H), 7.92 – 8.03 (m, 1 H); LC/MS (electrospray + ve), m/z 526.2 (MH)+, Retention time t = 5.71 min; Purity: UV220 > 98%, UV254 > 98%; HRMS (ESI): m/z calcd for C31H31N3O5 [M+H]+ 526.2351, found 526.2350; Solubility: (PBS, pH 7.4, 23°C) = 21 μM. Stability profile over 48 h (PBS, pH 7.4, 23°C) is shown in Figure 3.
3.1. Summary of Screening Results
The hit compound (CID 2887926 and AID 504389) came from a small library of 120 compounds prepared for SAR study of a TSHR agonist series (AID 1401). A screen of the entire MLSMR library to identify selective TSHR antagonists was recently concluded. The hits from that screen are currently being confirmed for selectivity against other receptors in that class, but the primary screening results are available (AID 504810).
3.2. Dose Response Curves for Probe
Figure 4Activity of ML224 at the three glycoprotein hormone receptors in the ELISA cAMP assays
3.3. Scaffold/Moiety Chemical Liabilities
2,3-Dihydroquinazolin-4-ones are quite stable in the solid state. Stability profile of ML224 in PBS at pH 7.4 over 48 h at room temperature showed >90% of the compound remained at 48 h. However, in solution, 2,3-dihydroquinazolin-4-ones could be slowly oxidized by air. In addition, they are not stable under acidic conditions and are hydrolyzed to the amine and aldehyde. Despite the stability issues, 2,3-dihydroquinazolin-4-ones are found in at least two known drugs, i.e. Fenquizone and Metolazone. Precautions have been and should be taken to maintain the integrity of the physical sample.
3.4. SAR Tables
3.5. Cellular Activity
TSHR antagonists from this series were found to be active against the THSH expressed stably in HEK-EM 293 cells and primary cultures of human thyrocytes. No toxicity has been seen in in vitro studies in HEK cells stably expressing the TSHR and in primary cultures of human thyrocytes.
3.6. Profiling Assays
This probe was selective against two closely related receptors, LHR and FSHR. We are planning to profile this probe against a panel of GPCRs.
CID 2887926 (A, Figure 5) is the first example of a small-molecule TSHR inverse agonist, which is an antagonist that also inhibits basal signaling. In HEK-EM 293 cells stably expressing TSHRs, it inhibits TSH-stimulated cAMP production by 86% with an IC50 value of 0.78 μM and inhibits basal cAMP production by the TSHRs by 58% with an IC50 value of 3.0 μM. It is an allosteric modulator that most likely binds to the TSHR serpentine domain. Basal signaling of constitutively activating mutations (CAM) increased thyroid function in a small percentage of hyperthyroid patients29. We have tested the inverse agonist at four CAMs that are located in different TSHR domains: S281N in the amino-terminal ectodomain, I568T in extracellular loop 2, and M453T and F631I in transmembrane helices 2 and 6, respectively. Constitutive signaling is up to 27-fold higher in these CAMs than in wild-type TSHR. The basal activities and cAMP production in HEK-EM 293 cells transiently expressing TSHRs of wild-type TSHR and the four CAMs were measured over 60 min. We found that CID 2887926 inhibited basal signaling of S281N, I568T, F631I and M453T with the following IC50 values and maximum inhibition levels, respectively: 1.4 μM and 78%, 3.7 μM and 77%, 0.5 μM and 36%, and 0.6 μM and 42%. S281N and I568T have residue substitutions in the amino-terminal ectodomains or extracellular loops, and were inhibited to a greater extent30. M453T and F631I have transmembrane domaine substitutions and were inhibited to a lesser degree, which may be secondary to the altered conformation of the transmembrane helices where CID 2887926 likely binds31. These findings support the TSHR model where the extracellular loops and ectodomain region cooperate to generate a structural module functioning as an agonist of the serpentine domain32.
The inverse agonist activities of CID 2887926 were also measured in primary cultures of human thyrocytes to determine its effect on the expression of genes important in thyroid function and thyroid hormone synthesis33. CID 2887926 decreased cAMP accumulation in human thyrocytes by 72% with a potency of 6.6 μM, but did not decrease cAMP accumulation in these cells stimulated by isoproterenol or forskolin. Thyrocytes treated with CID 2887926 in the presence of IBMX for 48 h decreased thyroperoxidase (TPO), TSHR, thyroglobulin (TG), and sodium/iodide-symporter (NIS) mRNA levels between 33 and 71%, but did not decrease deiodinase type 2 (DIO2) mRNA levels. Combined, these results establish CID 2887926 as an inverse agonist that can decrease the levels of mRNAs for several genes expressed in differentiated thyrocytes. Three groups that could especially benefit from small molecule inverse agonists are patients with Graves’ disease, non-autoimmune hyperthyroidism or metastatic thyroid cancer34–38. The use of SML inverse agonists in children with hyperthyroidism where radioiodine or surgical ablation is less attractive could be most beneficial. Patients with recurrent or metastatic thyroid cancer who are receiving thyroid hormones for TSH suppression could especially benefit from TSHR inverse agonists. (Text adapted from Neumann et al, Endocrinology 2010, 3454).
Previously, we found that 2,3-dihydroquinazolin-4-one TSHR agonists were highly selective for the TSH over LH receptor and FSH receptor24. ML109 (Figure 1; another highly related, recently declared probe from NCGC) is a potent TSHR agonist (EC50 = 90 nM) with no activity against LHR and FSHR. However, we found that CID 2887926 (A, Figure 5) was active against all three receptors (Table 3, entry 1). Our initial optimization led to CID 50897816 (B, Figure 5), which had a better selectivity for TSHR over LHR and FSHR. Thus, CID 50897816 was used to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. Graves’ hyperthyroidism is generally treated with antithyroid drugs, radioactive iodine, or surgery39,40. An orally bioavailable TSHR antagonist with minimal side effects could be used as initial chronic therapy for GD. Effective GD therapy should inhibit TSHR activation by the majority of TSAbs. The inhibition of TSHR with CID 50897816 mediated up-regulation of TPO mRNA in human thyrocytes in primary culture, and TSH-induced increases in TPO mRNA were determined. For all 30 GD sera tested, CID 50897816 inhibited GD sera up-regulation of TPO mRNA by 65 ± 2.0% (mean ± SEM). CID 50897816 decreased the basal levels of TPO mRNA and the mRNAs of thyroglobulin and TSHR, but it had no effect on the level of deiodinase type 2 mRNA, indicating that the reduction was not simply due to toxicity. Thus, CID 50897816 is a nontoxic antagonist of TSHR activation by TSAbs in all 30 GD patient sera in human thyrocytes. TSAbs, like TSH, bind primarily to the amino-terminal ectodomain of TSHR at multiple epitopes and a small molecule, like CID 50897816, would unlikely inhibit binding of TSAbs (or TSH) due to the known difficulty of inhibiting protein-protein interactions with small molecules41. CID 50897816 was previously shown to not inhibit TSAb (or TSH) binding to TSHR, but its activity may be derived from restraints on conformational changes that occur once it binds at the allosteric site. This could explain the inhibition of TSHR activation and account for its ability to inhibit TSHR signaling by all GD sera. In conclusion, CID 50897816 is an antagonist of activation of the TSHR by TSAbs, and it is the lead molecule for the development of a therapeutic agent to treat the hyperthyroidism of GD (text adapted from Neumann et al, Journal of clinical endocrinology & metabolism, 2011, 548).
As shown in Figure 6, several areas were explored in order to identify a selective TSHR antagonist. All analogs were first evaluated for antagonizing TSH-stimulated c-AMP activity in an Elisa assay (AID 504387). The active analogs were further evaluated for inhibition of TSHR basal activity (AID 504379) and for selectivity over LHR and FSHR (AID 504385 and AID 504384, respectively). The SAR and the selectivity are summarized as follows: (1) In area A, furan-2-ylmethane and 3-pyridiyl-methane groups at the R2 position are important for the inverse agonist activity; (2) The OH group at the R1 position, which is important for the TSHR agonistic activity, diminishes the antagonistic activity; (3) In area B, a methoxy group at the R3 position is not important for TSHR inhibition; however, it is important for TSHR selectivity over LHR; (4) Replacement of the oxygen at the Y position with a sulfur resulted in loss of activity against TSHR basal activity. CID 50897794 (Entry 5, Table 3) is a TSHR neutral antagonist; (5) In area C, the 2,6-dimethyl group is important for antagonistic activity and the NHAc group at the 4 position is important for the selectivity of TSHR over FSHR. Among the tested analogs, CID 50897809 (C, Figure 5) was the most selective TSHR inverse agonist. It has no activity for LHR and weak activity for FSHR (Entry 3, Table 3), and therefore, is nominated as a probe molecule for the TSH receptor.
4.1. Comparison to Existing Art and How the New Probe is an Improvement
To our knowledge, the probe molecule is the first selective TSHR inverse agonist. Several compounds from this series are the most potent TSHR antagonists reported to date. For the first time, we have demonstrated that a small molecule (CID 2887926) not only can inhibit basal signaling by wild-type TSHRs and four constitutively active mutants of TSHR expressed transiently in HEK-EM 293 cells; however, is also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs, where it inhibits basal levels of mRNA transcripts for thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR.
4.2. Mechanism of Action Studies
A Schild analysis of TSH-stimulated cAMP production shows that CID 2887926 acts as a competitive antagonist of TSH signaling. The Schild plot of these data was linear with a slope of 1.0, which is typical for a competitive ligand. CID 2887926 had no effect on [125I]TSH binding to TSHRs on the surface of HEK-EM 293 cells. A lack of effect of CID 2887926 on TSH binding was expected because we have previously shown that CID 25246343, a TSHR agonist that contains the same scaffold as CID 2887926, does not affect TSH binding and provided evidence that it binds in the transmembrane domain of TSHR24. We assume that CID 2887926 and other TSHR antagonists from these series bind in the serpentine region of TSHR.
4.3. Planned Future Studies
The probe molecule is the first selective TSHR inverse agonist and the most potent TSHR antagonist reported to date. This probe was selective for TSHR over two closely related receptors, LHR and FSHR. We are planning to profile this probe against a panel of GPCRs to determine its selectivity for TSHR over non-relevant receptor targets. In addition, we plan to develop the probe into a lead compound that can decrease thyroid hormone secretion in mice. The lead compound could be used for development of drugs for the treatment of hyperthyroidism caused by TSHR constitutively activating mutations or stimulating auto-antibodies associated with Graves’ disease.
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Appendix 1. Assay Listing and Corresponding AIDs
Wenwei Huang,a,* Erika Englund,a Steve Titus,a Noel Southall,a Wei Zheng,a Marc Ferrer,a Juan Marugan,a Susanne Neumann,b and Marvin Gershengornb.
Received: March 31, 2011; Last Update: February 28, 2013.
National Center for Biotechnology Information (US), Bethesda (MD)
Huang W, Englund E, Titus S, et al. Identification of Thyroid Stimulating Hormone Receptor Inverse Agonists. 2011 Mar 31 [Updated 2013 Feb 28]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.