GEM-5 is a gemcitabine-based conjugate containing a HIF-1α inhibitor (YC-1) (IC50 = 30 nM). GEM-5 can significantly down-regulate the expression of HIF-1α and up-regulate the expression of the tumor suppressor p53. GEM-5 causes apoptosis in A2780 cells and inhibits tumor growth.

Physicochemical Properties

Molecular Formula	C32H29F2N5O8
Molecular Weight	649.60
Exact Mass	649.198
CAS #	2233543-49-0
PubChem CID	163322349
Appearance	Typically exists as solid at room temperature
LogP	2
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	13
Heavy Atom Count	47
Complexity	1220
Defined Atom Stereocenter Count	3
SMILES	C1=CC=C(C=C1)CN2C3=CC=CC=C3C(=N2)C4=CC=C(O4)COC(=O)CCC(=O)OC[C@@H]5[C@H](C([C@@H](O5)N6C=CC(=NC6=O)N)(F)F)O
InChi Key	HJFGXFTZAGKEDF-HGLPBTONSA-N
InChi Code	InChI=1S/C32H29F2N5O8/c33-32(34)29(42)24(47-30(32)38-15-14-25(35)36-31(38)43)18-45-27(41)13-12-26(40)44-17-20-10-11-23(46-20)28-21-8-4-5-9-22(21)39(37-28)16-19-6-2-1-3-7-19/h1-11,14-15,24,29-30,42H,12-13,16-18H2,(H2,35,36,43)/t24-,29-,30-/m1/s1
Chemical Name	1-O-[[(2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-4,4-difluoro-3-hydroxyoxolan-2-yl]methyl] 4-O-[[5-(1-benzylindazol-3-yl)furan-2-yl]methyl] butanedioate
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	GEM-5 (0-80 μM; 12 hours) has an IC50 of 0.03 μM and shows good anti-tumor action against A2780 cells in hypoxic settings [1]. A2780 cells' apoptotic population rose to 52.67% in normoxic settings and to 80.89% in hypoxic conditions when exposed to GEM-5 (0.5 μM) for 72 hours [1]. GEM-5 (0.5 μM; 24 hours) stops the cell cycle in S phase (63.02% in normoxic and 72.64% in hypoxic circumstances) [1]. In hypoxic settings, GEM-5 (0.1 and 1 μM; 24 h) decreases HIF-1α levels and raises p53 levels in a dose-dependent manner [1].
ln Vivo	GEM-5 (125 or 271 mg/kg; tail vein injection, once weekly for 4 weeks) substantially reduces tumor growth in the A2780 xenograft mice model and demonstrates low toxicity [1].
Cell Assay	Apoptosis analysis Cell Types: A2780 cells [1] Tested Concentrations: 0.5 μM Incubation Duration: 72 hrs (hours) Experimental Results: The cell apoptosis rate increased to 52.67% under normoxic conditions, and the cell apoptosis rate increased to 80.89% under hypoxic conditions. Cell cycle analysis Cell Types: A2780 cells [1] Tested Concentrations: 0.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: The cell cycle was arrested in S phase (63.02% under normoxic conditions and 72.64% under hypoxic conditions). Western Blot Analysis Cell Types: A2780 cells [1] Tested Concentrations: 0.1 and 1 μM Incubation Duration: 24 hrs (hours) Experimental Results: diminished HIF-1α levels and increased p53 levels in a dose-dependent manner
Animal Protocol	Animal/Disease Models: GEM-5 (125 or 271 mg/kg; tail vein injection, once a week for 4 weeks) can effectively inhibit tumor growth in the A2780 xenograft mouse model and exhibit low toxicity [1]. Doses: 125 or 271 mg/kg Route of Administration: Tail vein injection, once a week for 4 weeks Experimental Results: Effectively inhibited tumor growth in the A2780 xenograft mouse model with low toxicity.
References	[1]. A gemcitabine-based conjugate with enhanced antitumor efficacy by suppressing HIF-1α expression under hypoxia. Bioorg Med Chem. 2021;41:116214.

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	1.5394 mL	7.6970 mL	15.3941 mL
	5 mM	0.3079 mL	1.5394 mL	3.0788 mL
	10 mM	0.1539 mL	0.7697 mL	1.5394 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.