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| Targets |
AH-3960 acts on the type-1 parathyroid hormone receptor (PTHR1). It binds to the receptor's transmembrane domain (TMD) region and does not require interactions with the amino-terminal extracellular domain (ECD). [1]
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| ln Vitro |
AH-3960 functioned as a weak agonist at the PTHR1. In HEK-293-derived GS-22A cells transiently transfected to express the PTHR1, the compound induced a significant cAMP response, albeit considerably weaker than that of PTH(1-34). The maximal increase vs basal was ~22-fold at 3×10⁻⁵ M ligand (P = .0007). [1]
In GP-2.3 cells (stably expressing PTHR1 and Glosensor cAMP reporter), AH-3960 induced clear and dose-dependent increases in cAMP over the dose range of 1 μM to 300 μM (maximum increase vs basal = 73-fold, P = .002). [1] AH-3960 exhibited much less activity in G2R-5 cells (stably expressing the PTHR2), indicating selectivity for PTHR1 over PTHR2. [1] AH-3960 exhibited agonist activity not only on wild-type PTHR1 and single mutant receptors (PTHR1-H223R, PTHR1-T410P), but also on the double-mutant PTHR1-HR/TP (H223R/T410P). It was the only ligand found to function as a clear cAMP agonist on PTHR1-HR/TP. Dose-response analysis confirmed that AH-3960 acted as an agonist at the double-mutant receptor and resulted in maximum cAMP levels that were, if anything, greater than those attained with PTH(1-34) acting on the wild-type PTHR1. [1] In dissociation time-course assays, AH-3960 (1×10⁻⁵ M) did not cause a significant change in the dissociation rate of either ¹²⁵I-PTH(1-34) or ¹²⁵I-M-PTH(1-15) from the PTHR1-WT, although it tended to slightly slow the rate of dissociation of ¹²⁵I-PTH(1-34). [1] |
| Enzyme Assay |
Radioligand competition assays were performed in membranes prepared from COS-7 cells transiently transfected to express either the intact wild-type PTHR1 or PTHR1-delNT (which lacks the ECD). The tracer radioligands used were ¹²⁵I-PTH(1-34) and ¹²⁵I-M-PTH(1-15). On the intact PTHR1, AH-3960, at the highest concentration tested (100 μM), only partially (~40%) inhibited the binding of ¹²⁵I-PTH(1-34), whereas it more completely (~90%) inhibited binding of ¹²⁵I-M-PTH(1-15), with an apparent affinity (pIC50) of 4.78 ± 0.22 (corresponding to ~16 μM). On PTHR1-delNT, AH-3960 inhibited binding of ¹²⁵I-M-PTH(1-15) with a maximum efficacy and apparent affinity (pIC50 = 5.47 ± 0.04, corresponding to ~3.4 μM) that was similar to that observed on the intact PTHR1. [1]
A dissociation time-course assay was performed. Membranes prepared from GP-2.3 cells (stably expressing PTHR1-WT) were prebound with ¹²⁵I-PTH(1-34) or ¹²⁵I-M-PTH(1-15) radioligands for 90 minutes. Then, an excess (5×10⁻⁷ M) of unlabeled PTH(1-34) or M-PTH(1-15) was added, either alone (control) or in the presence of AH-3960 (1×10⁻⁵ M). At various times thereafter, aliquots were withdrawn, vacuum filtered, and counted for bound radioactivity. AH-3960 did not cause a significant change in the dissociation rate of either radioligand. [1] |
| Cell Assay |
HEK-293-derived GS-22A cells, which stably express the luciferase-based Glosensor cAMP reporter, were used. These cells were further transiently transfected with a plasmid encoding the PTHR1. Cells were pretreated with luciferin for 20 minutes, then treated with varying concentrations of AH-3960. Luminescence arising from intracellular cAMP binding to the Glosensor reporter was measured in real-time at 2-minute intervals. Ligand dose-response curves were generated using the data obtained at the peak response time, typically by 20 minutes after ligand addition. [1]
GS-22A-derived cells stably expressing the PTHR1 (GP-2.3 cells) or the PTHR2 (G2R-5 cells) were also used. Cells were treated with AH-3960 over a dose range of 1 μM to 300 μM, and cAMP-dependent luminescence was recorded. AH-3960 induced clear and dose-dependent increases in cAMP in GP-2.3 cells but was much less active in G2R-5 cells. [1] Time-course analyses were performed in GS-22A cells transiently transfected to express various constitutively active PTHR1 mutants (PTHR1-H223R, PTHR1-T410P, PTHR1-HR/TP). Cells were preloaded with luciferin for 20 minutes and then treated with AH-3960 (1×10⁻⁵ M). AH-3960 exhibited agonist activity on each single mutant receptor and was the only ligand to induce an appreciable increase in cAMP at the double-mutant receptor. [1] |
| References |
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| Additional Infomation |
AH-3960 is a non-peptide small molecule ligand for the PTHR1. It was previously identified to act as an agonist on the PTHR1. It exhibits agonist activity on the wild-type PTHR1 with a potency that is several orders of magnitude weaker than that of PTH(1-34) (EC50s = ~1×10⁻⁴ M vs 1×10⁻⁷ M). The study confirms that AH-3960 mediates its agonist actions by interacting with the PTHR1 TMD region. [1]
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| Molecular Formula |
C13H22N4O3
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|---|---|
| Molecular Weight |
282.34
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| Exact Mass |
282.169
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| CAS # |
862907-48-0
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| PubChem CID |
135508376
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| Appearance |
White to light yellow solid powder
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| LogP |
1.782
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
20
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| Complexity |
445
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCCCN1C(=C(C(=O)N(C1=O)CCCC)C(=N)N)O
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| InChi Key |
VOYADQIFGGIKAT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H22N4O3/c1-3-5-7-16-11(18)9(10(14)15)12(19)17(13(16)20)8-6-4-2/h18H,3-8H2,1-2H3,(H3,14,15)
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| Chemical Name |
1,3-Dibutyl-5-(diaminomethylidene)-1,3-diazinane-2,4,6-trione
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| Synonyms |
AH 3960 AH3960 AH-3960
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 100 mg/mL (~354.18 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5418 mL | 17.7091 mL | 35.4183 mL | |
| 5 mM | 0.7084 mL | 3.5418 mL | 7.0837 mL | |
| 10 mM | 0.3542 mL | 1.7709 mL | 3.5418 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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