Sapacitabine (CS-682; CYC-682) is a highly potent and orally bioactive pyrimidine analogue prodrug with potential anticancer activity. Sapacitabine is is converted by amidases into CNDAC (2'-Cyano-2'-deoxyarabinofuranosylcytosine), the deoxycytosine analogue, and phosphorylated into the active triphosphate form. Apoptosis and cell cycle arrest occur when CNDAC triphosphate, an analogue of deoxycytidine triphosphate, incorporates into DNA strands during replication and causes single-stranded DNA breaks during polymerization due to beta-elimination during the fidelity checkpoint process. It's possible that the unmetabolized prodrug has antitumor properties as well.

Physicochemical Properties

Molecular Formula	C26H42N4O5
Molecular Weight	490.63548
Exact Mass	490.315
Elemental Analysis	C, 63.65; H, 8.63; N, 11.42; O, 16.30
CAS #	151823-14-2
Related CAS #	151823-14-2
PubChem CID	153970
Appearance	White to off-white solid powder
Density	1.2±0.1 g/cm3
Index of Refraction	1.575
LogP	5.67
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	17
Heavy Atom Count	35
Complexity	775
Defined Atom Stereocenter Count	4
SMILES	O=C(N=C(NC(CCCCCCCCCCCCCCC)=O)C=C1)N1[C@H]2[C@@H](C#N)[C@H](O)[C@@H](CO)O2
InChi Key	LBGFKUUHOPIEMA-PEARBKPGSA-N
InChi Code	InChI=1S/C26H42N4O5/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-23(32)28-22-16-17-30(26(34)29-22)25-20(18-27)24(33)21(19-31)35-25/h16-17,20-21,24-25,31,33H,2-15,19H2,1H3,(H,28,29,32,34)/t20-,21+,24-,25+/m0/s1
Chemical Name	N-[1-[(2R,3S,4S,5R)-3-cyano-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidin-4-yl]hexadecanamide
Synonyms	CYC682; CYC-682; CYC 682; CS682; CYC682; CYC-682; Sapacitabine
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	nucleoside analog
ln Vitro	The amounts of sapacitabine needed to attain an IC50 vary, ranging from 3±0.6 μM for the colon cancer cell line HCT116 to 67±14 μM for the breast cancer cell line MDA-MB-435. According to a cell cycle analysis, 41% of cells treated with sapacitabine are arrested in the G2/M phase and 35% are arrested in the late-S phase. Sapacitabine (IC50 = 20±6 μM) is sensitive to L1210 cells expressing deoxycytidine kinase (dCK). When docetaxel is administered prior to Sapacitabine in both cell lines, synergistic effects (CI<1) are noted in the combinations of the two drugs[1].
ln Vivo	At Day 14, the group treated with sapacitabine (5 mg/kg) plus vorinostat (33 mg/kg) had a mean tumour volume of 245 mm3 and a tumour growth inhibition (TGI) of 92%, while the group treated with sapacitabine (15 mg/kg) plus vorinostat (33 mg/kg) had a mean tumour volume of 107 mm3 and a TGI of 112%(2).
Cell Assay	In this study, a panel of cancer cell lines from the colon (HT29, HCT116, COLO205, HCC2998), breast (MCF7, MDA-MB-435), lung (HOP62, HOP92), and ovarian (OVCAR3, IGROV1) is used. Using flow cytometry, the cell cycle stage and percentage of apoptotic cells are evaluated. In short, cells are seeded into 25 cm3 flasks and either left untreated or subjected to different levels of sapacitabine treatment. Adherent and non-adherent cells are harvested at the designated intervals, cleaned with PBS, preserved in 70% ethanol, and kept at 4°C until needed. After being rehydrated in PBS, the cells are incubated for 20 minutes at room temperature (25°C) with 250 μg/mL RNAse A and Triton X-100, and for another 20 minutes at 4°C in the dark with 50 μg/mL propidium iodide. FACS Calibur is used to analyze the cell cycle distribution and percentage of apoptotic cells, both of which are measured using a flow cytometer[1].
Animal Protocol	Subcutaneous injections of 1×107 MV4-11 cells resuspended in 50% Matrigel are administered subcutaneously to female (nu/nu) mice at a single flank injection site. Animals are paired according to tumor size into treatment groups (no more than six mice per group) once tumor volumes reach 126 to 256 mm3 (16 days after implantation), with an average tumor size of about 190 mm3. The formula for measuring tumors is volume (mm3) = width2 (mm)×length (mm)×0.5. Dosing begins the same day as distribution to the treatment groups. Sapacitabine is given orally once daily (5 or 15 mg/kg) for 4 days, followed by a 3-day break before another 4 days of treatment[2].
References	[1]. Antiproliferative effects of sapacitabine (CYC682), a novel 2'-deoxycytidine-derivative, in human cancer cells. Br J Cancer. 2007 Sep 3;97(5):628-36. [2]. Combination of sapacitabine and HDAC inhibitors stimulates cell death in AML and other tumour types. Br J Cancer. 2010 Oct 26;103(9):1391-9.
Additional Infomation	Sapacitabine is a nucleoside analogue resulting from the formal condensation of the carboxy group of hexadecanoic acid with the amino group of CNDAC. It is the prodrug of CNDAC and is currently in clinical development for the treatment of acute myeloid leukemia (AML). It has a role as an antimetabolite, an antineoplastic agent, a prodrug and a DNA synthesis inhibitor. It is a nucleoside analogue, a nitrile and a secondary carboxamide. It is functionally related to a hexadecanoic acid and a CNDAC. Sapacitabine is an orally bioavailable pyrimidine analogue prodrug with potential antineoplastic activity. Sapacitabine is hydrolyzed by amidases to the deoxycytosine analogue CNDAC (2'-Cyano-2'-deoxyarabinofuranosylcytosine), which is then phosphorylated into the active triphosphate form. As an analogue of deoxycytidine triphosphate, CNDAC triphosphate incorporates into DNA strands during replication, resulting in single-stranded DNA breaks during polymerization due to beta-elimination during the fidelity checkpoint process; cell cycle arrest in the G2 phase and apoptosis ensue. The unmetabolized prodrug may exhibit antineoplastic activity as well. Drug Indication Investigated for use/treatment in solid tumors, cutaneous t-cell lymphoma, myelodysplastic syndrome, and leukemia (lymphoid). Mechanism of Action Sapacitabine appears to act through a dual mechanism. It interferes with DNA synthesis by causing single-strand DNA breaks and also induces arrest of cell cycle progression mainly at G2/M-Phase. Both sapacitabine and CNDAC, its major metabolite or a substance into which the drugs converts after ingestion by patients, have demonstrated potent anti-tumor activity in preclinical studies.

Solubility Data

Solubility (In Vitro)

DMSO: ~33.3 mg/mL (~67.9 mM)

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	2.0382 mL	10.1908 mL	20.3815 mL
	5 mM	0.4076 mL	2.0382 mL	4.0763 mL
	10 mM	0.2038 mL	1.0191 mL	2.0382 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.