Palifosfamide (also known as Isophosphoramide mustard; IPM; ZIO201), a synthetic mustard compound and the active metabolite of ifosfamide, is a novel DNA alkylator with antitumor activity. Palifosfamide, an active metabolite of ifosfamide, cross-links DNA irreversibly through GC base pairs, forming irreversible 7-atom inter-strand cross-links that inhibit DNA replication and cause cell death. Palifosfamide is covalently linked to the amino acid lysine for stability. This agent does not metabolize to acrolein or chloroacetaldehyde, which are metabolites linked to bladder and central nervous system toxicities, unlike ifosfamide. Additionally, palifosfamide may be able to overcome the tumor resistance associated with ifosfamide because it does not require activation by aldehyde dehydrogenase.

Physicochemical Properties

Molecular Formula	C₄H₁₁CL₂N₂O₂P
Molecular Weight	221.02
Exact Mass	219.993
Elemental Analysis	C, 28.08; H, 6.48; Cl, 20.72; N, 12.28; O, 23.38; P, 9.05
CAS #	31645-39-3
Related CAS #	31645-39-3 (free acid); 1070409-31-2 (tris salt)
PubChem CID	100427
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	341.5±52.0 °C at 760 mmHg
Melting Point	106-107ºC
Flash Point	160.4±30.7 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.498
LogP	-1.05
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	11
Complexity	134
Defined Atom Stereocenter Count	0
SMILES	O=P(NCCCl)(NCCCl)O
InChi Key	BKCJZNIZRWYHBN-UHFFFAOYSA-N
InChi Code	InChI=1S/C4H11Cl2N2O2P/c5-1-3-7-11(9,10)8-4-2-6/h1-4H2,(H3,7,8,9,10)
Chemical Name	bis(2-chloroethylamino)phosphinic acid
Synonyms	NSC 297900; NSC-297900; NSC297900; ZIO201; ZIO-201; ZIO 201; IPAM; isophosphoramide mustard
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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
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	Palifosfamide lysine (ZIO-201) is a stable form of the drug. In vitro, palifosfamide lysine exhibits wide activity in sarcoma lines. Except for OS222 (IC50=31.5 μM), the IC50 for most cell lines ranges from 2.25 to 6.75 μM[1].
ln Vivo	In the OS31, OS33, and RMS xenografts, as well as in the control group, tumor growth inhibition is observed. This leads to a statistically significant difference in event-free survival. It has been observed that the OS31 xenograft exhibits differential gene expression of ALDH3A1 but not ALDH1A1[1]. Mice treated with stabilized palifosfamide exhibit over 80% growth suppression of MX-1 tumors, with 17% exhibiting full antitumor responses. In mice, antitumor activity is equivalent through both routes, and oral bioavailability in rats ranges from 48-73% of parenteral administration. Complete tumor regression is achieved in 62-75% of mice when palifosfamide-tris and docetaxelor doxorubicin are administered according to optimal regimens[2].
Cell Assay	Phosphate buffered saline (PBS) is used to dissolve palifosfamide. Approximately 500 cells per well in 100 μL of media are plated in 96-well microtiter plates. Cells are treated with increasing concentrations of palifosfamide lysine in separate plates either as a single day treatment or three consecutive days of treatment, with fresh drug added each day, following a 24-hour incubation period at 37°C. The plates are incubated with 5% CO2 at 37°C for 72 hours. After 72 hours, each well receives 250 μg of MTT, and it is incubated for 6 hours at 37°C. After mitochondria of viable cells convert MTT to formazine crystals, the crystals are dissolved in 100 μL of dimethyl sulfoxide. The wavelength of optical density is 595 nm[2].
Animal Protocol	Mice: The study uses female CB17 SCID mice. Mice are randomized into treatment and control groups (5-8 mice/group) for each tumor line once the tumors have grown to a size of 50–150 mm3. For six weeks, 150 mg/kg of cyclophosphamide is given intraperitoneally once a week. Serial tumor volumes are measured over the next six weeks after palifosfamide lysine is injected intravenously for three days in a row at the highest tolerated dose of 100 mg/kg. The experiment concludes with the sacrifice of the mice[2].
References	[1]. Preclinical activity of palifosfamide lysine (ZIO-201) in pediatric sarcomas including oxazaphosphorine-resistant osteosarcoma. Cancer Chemother Pharmacol. 2009 Sep;64(4):733-40. [2]. Anticancer activity of stabilized palifosfamide in vivo: schedule effects, oral bioavailability, and enhanced activity with docetaxel and doxorubicin. Anticancer Drugs. 2012 Feb;23(2):173-84.
Additional Infomation	Isophosphamide mustard is a phosphorodiamide. Palifosfamide (ZIO-201) is a proprietary stabilized metabolite of ifosfamide. Ifosfamide has been shown to be effective in high doses in treating testicular cancer, sarcoma and lymphoma. Palifosfamide is a synthetic mustard compound with potential antineoplastic activity. An active metabolite of ifosfamide covalently linked to the amino acid lysine for stability, palifosfamide irreversibly alkylates and cross-links DNA through GC base pairs, resulting in irreparable 7-atom inter-strand cross-links; inhibition of DNA replication and cell death follow. Unlike ifosfamide, this agent is not metabolized to acrolein or chloroacetaldehyde, metabolites associated with bladder and CNS toxicities. In addition, because palifosfamide does not require activation by aldehyde dehydrogenase, it may overcome the tumor resistance seen with ifosfamide. Drug Indication Investigated for use/treatment in cancer/tumors (unspecified), lymphoma (unspecified), and sarcoma. Mechanism of Action After metabolic activation, palifosfamide alkylates or binds with many intracellular molecular structures, including nucleic acids. The cytotoxic action is primarily due to cross-linking of strands of DNA and RNA, as well as inhibition of protein synthesis.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 40 mg/mL Water: ~12 mg/mL Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3 mg/mL (13.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 3 mg/mL (13.57 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 3 mg/mL (13.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 10 mg/mL (45.24 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.5245 mL	22.6224 mL	45.2448 mL
	5 mM	0.9049 mL	4.5245 mL	9.0490 mL
	10 mM	0.4524 mL	2.2622 mL	4.5245 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.