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| Targets |
DNA Alkylator
Phosphoramide mustard targets DNA as an alkylating agent. It forms cross-linked DNA adducts by covalently binding to guanine bases, preventing DNA strand separation during replication. This mechanism underlies its potential use in cancer chemotherapy, as it can selectively target rapidly dividing cells. The compound also induces the DNA damage repair response and causes cytotoxicity through DNA adduct formation. |
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| ln Vitro |
In order to prevent DNA strand separation during replication, phosphoramide mustard forms cross-linked DNA adducts, which are toxic[1]. In rat spontaneously immortalized granulosa cells (SIGCs), phosphoramide mustard (3-6 μM; 48 hours) decreases cell viability[1]. Ovarian DNA damage and DNA adduct formation are induced by mustard (3-6 μM; 24-48 hours)[1]. DNA damage responses (DDR) gene mRNA expression levels and DDR proteins are increased by phosphoramide mustard (3-6 μM; 24-48 hours)[1].
In vitro, phosphoramide mustard causes cytotoxicity through forming cross-linked DNA adducts that inhibit DNA strand separation during replication. It reduces cell viability in rat spontaneously immortalized granulosa cells (SIGCs) at concentrations of 3-6 μM over 48 hours. The compound induces DNA damage in ovarian granulosa cells and triggers the DNA damage repair response. It exhibits anticancer activity by inducing DNA damage and interfering with cell proliferation. |
| ln Vivo |
In rats, phosphorus mustard (2.1-20.7 mg/kg; ip; daily; for 5 days) suppresses the formation of subcutaneous tumors[2]. After intravenous treatment, phosphoramide mustard has terminal elimination half-lives (rat 15.1 min; rat 59.4 mg/kg)[2].
In vivo, phosphoramide mustard inhibits subcutaneous tumor growth in rats when administered intraperitoneally at doses of 2.1-20.7 mg/kg daily for 5 days. As the active metabolite of cyclophosphamide, it contributes to the antitumor efficacy of the parent drug. It also exhibits ovarian toxicity in animal models. The compound's alkylating activity leads to DNA cross-linking in rapidly dividing cells, including cancer cells and normal proliferating tissues. |
| Enzyme Assay |
For non-cellular enzyme/receptor binding assays, phosphoramide mustard is evaluated for its DNA alkylating activity. Purified DNA is incubated with the compound, and DNA adduct formation is analyzed using mass spectrometry or 32P-postlabeling techniques. DNA cross-linking is assessed using gel electrophoresis or HPLC-based assays. The compound's ability to form covalent bonds with nucleophilic sites on DNA is the primary mechanism of action. Alkylation of guanine bases is typically quantified using specific antibodies or chromatographic methods.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: SIGCs Tested Concentrations: 0.5 μM, 1 μM, 3 μM, 6 μM Incubation Duration: 48 hrs (hours) Experimental Results: decreased cell viability at concentrations of 3 μM and higher. RT-PCR[1] Cell Types: SIGCs Tested Concentrations: 3 μM, 6 μM Incubation Duration: 24 hrs (hours), 48 hrs (hours) Experimental Results: Increased DDR gene mRNA expression levels. Western Blot Analysis[1] Cell Types: SIGCs Tested Concentrations: 3 μM, 6 μM Incubation Duration: 24 hrs (hours), 48 hrs (hours) Experimental Results: Generally increased DDR proteins. In vitro cell culture experiments are performed using cancer cell lines (e.g., ovarian cancer cells, leukemia cells) and normal cells (e.g., granulosa cells). Cells are treated with phosphoramide mustard at concentrations ranging from 1 to 20 μM for 24-72 hours. Endpoints include cell viability (MTT assay), DNA damage (comet assay, γ-H2AX foci), apoptosis (Annexin V/PI, caspase activation), and cell cycle analysis (flow cytometry). The compound's cytotoxicity is assessed in both cancer cells and normal cells to evaluate selectivity. |
| Animal Protocol |
Animal/Disease Models: Rat, subcutaneously (sc) implanted Walker 256 carcinosarcoma tumor[2]
Doses: 2.1 mg/kg, 4.8 mg/kg, 10.4 mg /kg, 20.7 mg/kg Route of Administration: intraperitoneal (ip)injection, one time/day, for 5 days Experimental Results: Required to produce 50% inhibition of subcutaneous (sc)tumor growth with dose of 12 mg/kg. Animal/Disease Models: Rats[2] Doses: 59.4 mg /kg (pharmacokinetic/PK Analysis) Route of Administration: intravenous (iv)injection Experimental Results: T1/2 (15.1 min). In vivo animal studies use rodent models of cancer, typically xenograft or syngeneic tumor models. Phosphoramide mustard is administered intraperitoneally at doses of 2-20 mg/kg. In ovarian toxicity studies, female rats are treated and ovarian function is assessed by histology, hormone levels, and follicle counts. Tumor growth inhibition is measured by caliper measurements of subcutaneous tumors. Endpoints also include body weight, hematological parameters, and tissue histology for toxicity assessment. |
| ADME/Pharmacokinetics |
Phosphoramide mustard has a molecular formula of C4H11Cl2N2O2P and a molecular weight of 221.02 g/mol. It is a synthetic chemical compound that belongs to the class of alkylating agents. As the active metabolite of cyclophosphamide, its pharmacokinetics are influenced by the parent drug's metabolism. It is typically administered in a controlled manner due to its cytotoxic effects. The compound is soluble in aqueous solutions and is stable under recommended storage conditions.
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| Toxicity/Toxicokinetics |
Phosphoramide mustard is highly cytotoxic and genotoxic. Its pharmacological profile includes antitumor activity and potential side effects such as myelosuppression and gastrointestinal toxicity. It also exhibits ovarian toxicity. Safety precautions are essential when handling this compound, as it can pose risks to human health and the environment. It is classified as a hazardous substance and requires handling in a fume hood with appropriate protective equipment.
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| References |
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| Additional Infomation |
Phosphoramide mustard is a nitrogen mustard and phosphadiamide compound. It is one of many structure-related alkylating agents with antitumor activity. The prototype of this class of drugs is cyclophosphamide. Most phosphoramide mustard is administered in its active form, undergoing reductive activation under hypoxic conditions to generate cytotoxic metabolites. These drugs can alkylate and cross-link DNA, thereby inhibiting DNA replication. Phosphoramide mustard also has immunosuppressive, mutagenic, and teratogenic effects. (NCI04)
Phosphoramide mustard is also known as Asta 5317, Friedman acid, and N,N-Bis(2-chloroethyl)phosphorodiamidic acid. It is the active metabolite of cyclophosphamide, a widely used chemotherapeutic agent. The compound exemplifies the dual nature of chemotherapeutic agents, offering therapeutic benefits while necessitating careful management of toxicological properties. It is used in cancer research to study DNA repair mechanisms and the effectiveness of alkylating agents in therapeutic applications. It is for research use only and not for therapeutic applications. |
| Molecular Formula |
C4H11N2O2PCL2
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| Molecular Weight |
221.02
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| Exact Mass |
219.994
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| CAS # |
10159-53-2
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| Related CAS # |
Phosphoramide mustard cyclohexanamine;1566-15-0
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| PubChem CID |
96356
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.474g/cm3
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| Boiling Point |
363.5ºC at 760mmHg
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| Flash Point |
173.6ºC
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| Vapour Pressure |
2.84E-06mmHg at 25°C
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| Index of Refraction |
1.525
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| LogP |
1.525
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
11
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| Complexity |
151
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(CN(CCCl)P(=O)(N)O)Cl
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| InChi Key |
RJXQSIKBGKVNRT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H11Cl2N2O2P/c5-1-3-8(4-2-6)11(7,9)10/h1-4H2,(H3,7,9,10)
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| Chemical Name |
amino-[bis(2-chloroethyl)amino]phosphinic acid
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| Synonyms |
Phosphoramide mustard
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| HS Tariff Code |
2934.99.9001
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| 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)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
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| 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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