7-Hydroxydichloromethotrexate is a novel and potent methotrexate metabolite

Physicochemical Properties

Molecular Formula	C20H20CL2N8O6
Molecular Weight	539.33
Exact Mass	538.088
Elemental Analysis	C, 44.54; H, 3.74; Cl, 13.15; N, 20.78; O, 17.80
CAS #	751-75-7
PubChem CID	5748153
Appearance	Typically exists as solid at room temperature
LogP	1.954
Hydrogen Bond Donor Count	6
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	9
Heavy Atom Count	36
Complexity	897
Defined Atom Stereocenter Count	1
SMILES	CN(CC1=NC2=C(N=C(N=C2NC1=O)N)N)C3=C(C=C(C=C3Cl)C(=O)NC(CCC(=O)O)C(=O)O)Cl
InChi Key	OMIDGYJIJHQJTO-JTQLQIEISA-N
InChi Code	InChI=1S/C20H20Cl2N8O6/c1-30(6-11-18(34)28-16-13(25-11)15(23)27-20(24)29-16)14-8(21)4-7(5-9(14)22)17(33)26-10(19(35)36)2-3-12(31)32/h4-5,10H,2-3,6H2,1H3,(H,26,33)(H,31,32)(H,35,36)(H5,23,24,27,28,29,34)/t10-/m0/s1
Chemical Name	(3,5-dichloro-4-(((2,4-diamino-7-oxo-1,7-dihydropteridin-6-yl)methyl)(methyl)amino)benzoyl)-L-glutamic acid
Synonyms	7-Hydroxydichloromethotrexate; 7 Hdcm; 7-Hydroxydichloromethotrexate; 751-75-7; 7-Hdcm; (2S)-2-[[3,5-dichloro-4-[(2,4-diamino-7-oxo-8H-pteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid; DTXSID50226071; (3,5-dichloro-4-(((2,4-diamino-7-oxo-1,7-dihydropteridin-6-yl)methyl)(methyl)amino)benzoyl)-L-glutamic acid; L-Glutamic acid, N-(3,5-dichloro-4-(((2,4-diamino-1,7-dihydro-7-oxo-6-pteridinyl)methyl)methylamino)benzoyl)-; DTXCID50148562; 7Hdcm; 7-Hdcm
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	MTX/methotrexate analog; DHFR/dihydrofolate reductase; anticancer
ln Vitro	Plasma levels of methotrexate, dichloromethotrexate, and their 7-hydroxy metabolites can be determined quickly and accurately by direct analysis in a high pressure liquid chromatographic system. This system utilizes a precolumn of CO:Pell ODS resin to protect the analytical mu-Bondapak C18 column. The precolumn also provides a bed for extraction of drugs from the plasma proteins which are trapped in it. Elution with a paired ion solvent provides good recovery of methotrexate from the plasma and excellent separation on the column. The primary advantages of this system are its speed and simplicity combined with its ability to determine the plasma level of parent drug and its metabolite simultaneously[1].
References	[1]. A Direct analysis of methotrexate, dichloromethotrexate and their 7-hydroxy metabolites in plasma by high pressure liquid chromatography, Clinica Chimica Acta, 1980 Oct 23;107(1-2):67-72.

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	1.8542 mL	9.2708 mL	18.5415 mL
	5 mM	0.3708 mL	1.8542 mL	3.7083 mL
	10 mM	0.1854 mL	0.9271 mL	1.8542 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.