A2AAR/HDAC-IN-1 (compound 14c) is an orally bioactive and balanced A2AAR/HDAC dual (bifunctional) inhibitor (antagonist) with Ki of 163.5 nM for A2AAR and IC50 of 145.3 nM for HDAC1. A2AAR/HDAC-IN-1 has anti-cancer effect.

Physicochemical Properties

Molecular Formula	C24H21N7O2
Molecular Weight	439.469243764877
Exact Mass	439.175
CAS #	2767560-51-8
PubChem CID	165412579
Appearance	Typically exists as solid at room temperature
LogP	2.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	33
Complexity	659
Defined Atom Stereocenter Count	0
InChi Key	BENMZDAJCRYRCC-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H21N7O2/c25-18-4-1-2-5-19(18)28-23(32)16-9-7-15(8-10-16)11-12-31-22-17(14-27-31)21(29-24(26)30-22)20-6-3-13-33-20/h1-10,13-14H,11-12,25H2,(H,28,32)(H2,26,29,30)
Chemical Name	4-[2-[6-amino-4-(furan-2-yl)pyrazolo[3,4-d]pyrimidin-1-yl]ethyl]-N-(2-aminophenyl)benzamide
HS Tariff Code	2934.99.9001
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)

Biological Activity

ln Vitro	The antiproliferative effect of A2AAR/HDAC-IN-1 (compound 14c) (0-100 μM, 72 hours) was demonstrated against the colon cancer cell lines CT26 and MC38 [1]. Histone acetylation is increased by A2AAR/HDAC-IN-1 (0–5 μM, 24 hours) [1]. A2AAR/HDAC-IN-1 exhibits a 3-fold increase in selectivity for A1AR and a negligible binding to A2BAR and A3AR [1].
ln Vivo	In a mouse MC38 xenograft model, A2AAR/HDAC-IN-1 (Compound 14c) (30 or 60 mg/kg, IP, twice daily for 9 days) demonstrated strong antitumor effects [1]. Weak tumor inhibitory effect is shown by A2AAR/HDAC-IN-1 (90 mg/kg, bid; 150 mg/kg, qd; oral gavage for 15 days) [1]. The pharmacokinetic profile of A2AAR/HDAC-IN-1 (5 mg/kg IV; 20 mg/kg PO or IP; once) is generally favorable [1].
Cell Assay	Cell proliferation experiment [1] Cell Types: colon cancer cell line (CT26 and MC38) Tested Concentrations: 0-100 μM Incubation Duration: 72 h Experimental Results: demonstrated good cytotoxicity in CT26 and MC38 cells, with GI50 values of 0.29 ± 0.29 ± 0.03 μM and 0.38 are ±0.03μM respectively. Western Blot Analysis[1] Cell Types: MC38 mouse colon cancer cells Tested Concentrations: 0, 0.1, 1 and 5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Histone H3 and H4 acetylation increased in a concentration-dependent manner.
Animal Protocol	Animal/Disease Models: Male ICR mouse (6-8 weeks, ~25 g body weight, MC38 colon cancer model) [1] Doses: 30 or 60 mg/kg Route of Administration: IP twice (two times) daily for 9 days Experimental Results: Significant The tumor growth inhibition dose was 30 mg/kg (ip, bid), and the tumor growth inhibition (TGI) rate was 68%. When the dose was 60 mg/kg, the TGI rate further increased to 85%. Animal/Disease Models: Male ICR mouse (6-8 weeks, weight about 25 grams, MC38 colon cancer model) [1] Doses: 90 mg/kg, bid; 150 mg/kg, one time/day Route of Administration: po (oral gavage), twice (two times) daily (90 mg/kg) or daily (150 mg/kg) for 15 days Experimental Results: Even at the dose of 90 mg/kg (po (po (oral gavage)) bid), the inhibition rate was 44%. Animal/Disease Models: Male ICR mice (6-8 weeks, approximately 25 g body weight) [1] Doses: 5 mg/kg (IV) and 20 mg/kg (PO, IP) Route of Administration: po (po (oral gavage)) IP or IV; Primary (pharmacokinetic/PK/PK Analysis) Experimental Results: pharmacokinetic/PK/PK parameters of A2AAR/HDAC-IN-1 in male ICR mice [1].
References	[1]. Dual-acting antitumor agents targeting the A2A adenosine receptor and histone deacetylases: Design and synthesis of 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine derivatives. Eur J Med Chem. 2022 Jun 5;236:114326.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2755 mL	11.3773 mL	22.7547 mL
	5 mM	0.4551 mL	2.2755 mL	4.5509 mL
	10 mM	0.2275 mL	1.1377 mL	2.2755 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.