AS-99 is a first-in-class, potent and specific ASH1L histone methyltransferase inhibitor (IC50= 0.79 µM, Kd= 0.89 µM) with anti-leukemia activity. AS-99 blocks cell proliferation/growth in vivo, causes apoptosis and differentiation, downregulates MLL fusion target genes, and alleviates leukemia.

Physicochemical Properties

Molecular Formula	C27H30F3N5O3S2
Molecular Weight	593.68
Exact Mass	593.174
CAS #	2323623-93-2
Related CAS #	AS-99 TFA;AS-99
PubChem CID	138630366
Appearance	Typically exists as solid at room temperature
LogP	2.9
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	7
Heavy Atom Count	40
Complexity	1040
Defined Atom Stereocenter Count	0
InChi Key	VPQCHIWQHIOPQS-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H30F3N5O3S2/c1-33-14-20(15-33)26(36)32-13-17-5-6-22-23(18-3-2-4-19(12-18)25(31)39)16-35(24(22)11-17)21-7-9-34(10-8-21)40(37,38)27(28,29)30/h2-6,11-12,16,20-21H,7-10,13-15H2,1H3,(H2,31,39)(H,32,36)
Chemical Name	N-[[3-(3-carbamothioylphenyl)-1-[1-(trifluoromethylsulfonyl)piperidin-4-yl]indol-6-yl]methyl]-1-methylazetidine-3-carboxamide
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

Targets	0.79 µM (ASH1L histone methyltransferase)[1]
ln Vitro	AS-99 is examined in relation to a panel of 20 histone methyltransferases, which comprises SETD2, NSD1, NSD2, and NSD3. For all studied histone methyltransferases, NO substantial inhibition is seen at 50 µM of AS-99, suggesting > 100-fold selectivity towards ASH1L[1]. AS-99 exhibits negligible or no effects at 10 µM or greater doses on the proliferation of leukemia cells without MLL1 translocations, such as SET2 and K562[1]. Quantification of Annexin V positive cells[1] indicates that AS-99 (1 -8 µM; 7 days) similarly promotes apoptosis in MLL leukemia cells, but not in K562 cells. Transcriptional programs driven by MLL fusion are suppressed by AS-99[1].Comparing AS-99-treated cells to DMSO-treated cells, less H3K36me2 peaks are observed[1].
ln Vivo	In mice, leukemia burden is decreased by AS-99 (30 mg/kg; ip; qd, administered for 14 consecutive days)[1]. The in vivo investigations conducted on mice using AS-99 show that the drug has an acceptable half-life (~5–6 h), a favorable exposure in plasma upon iv and ip injection (AUC = 9701 hr* ng/mL and 10,699 hr* ng/mL, respectively), and a Cmax >10 µM[1].
Cell Assay	RT -PCR[1] Cell Types: MOLM13 cells Tested Concentrations: 2-6 µM Incubation Duration: 7 days Experimental Results: Led to a dose-dependent downregulation of canonical MLL fusion target genes required for leukemogenesis including MEF2C, DLX2, FLT3, and HOXA9.
Animal Protocol	Animal/Disease Models: 8- to 10-week old female NSG mice (bearing MV4;11 cells)[1] Doses: 30 mg/kg Route of Administration: Ip; qd, treated for 14 days Experimental Results: diminished the leukemia burden in the xenotransplantation mouse model of MLL leukemia without affecting blood counts in normal mice.
References	[1]. Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity. Nat Commun. 2021 May 14;12(1):2792.

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	1.6844 mL	8.4220 mL	16.8441 mL
	5 mM	0.3369 mL	1.6844 mL	3.3688 mL
	10 mM	0.1684 mL	0.8422 mL	1.6844 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.