Melflufen (Melphalan flufenamide) HCl, a dipeptide precursor active molecule of Melphalan, is an alkylating agent. Melflufen HCl has anticancer effect against multiple myeloma cells and inhibits angiogenesis. Melflufen HCl causes irreversible DNA damage and cytotoxic effects on myeloma cells.

Physicochemical Properties

Molecular Formula	C24H31CL3FN3O3
Molecular Weight	534.878647089005
Exact Mass	533.141
CAS #	380449-54-7
Related CAS #	Melflufen;380449-51-4
PubChem CID	70675838
Appearance	Off-white to light yellow solid powder
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	14
Heavy Atom Count	34
Complexity	579
Defined Atom Stereocenter Count	2
SMILES	ClCCN(CCCl)C1C=CC(=CC=1)C[C@@H](C(N[C@H](C(=O)OCC)CC1C=CC(=CC=1)F)=O)N.Cl
InChi Key	ZCMWSKHHXLCVHI-VROPFNGYSA-N
InChi Code	InChI=1S/C24H30Cl2FN3O3.ClH/c1-2-33-24(32)22(16-18-3-7-19(27)8-4-18)29-23(31)21(28)15-17-5-9-20(10-6-17)30(13-11-25)14-12-26;/h3-10,21-22H,2,11-16,28H2,1H3,(H,29,31);1H/t21-,22-;/m0./s1
Chemical Name	ethyl (2S)-2-[[(2S)-2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate;hydrochloride
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: Please store this product in a sealed and protected environment, 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	In a concentration-dependent manner, Melflufen (Melphalan flufenamide) hydrochloride (0.5-10 μM; 24 hours) decreases the viability of MM.1S, INA-6, RPMI-8226, MM.1R, Dox-40, ARP-1, and ANBL-6 cells[1]. MM.1S cells undergo apoptosis when exposed to melflufen hydrochloride [1]. Another strong exosome secretion activator is melflufen hydrochloride [3].
ln Vivo	In a xenograft mouse model, melflufen (Melphalan flufenamide) hydrochloride (3 mg/kg; intravenously; twice weekly for two weeks) exhibits anti-MM action [1].
Cell Assay	Cell Viability Assay[1] Cell Types: Multiple myeloma cells: MM.1S, INA-6, RPMI-8226, MM.1R, Dox-40, ARP-1, ANBL-6 cells Tested Concentrations: 0.5, 1, 3, 5, 10 μM Incubation Duration: 24 hrs (hours) Experimental Results: A significant concentration-dependent decrease in viability of all cell lines was observed.
Animal Protocol	Animal/Disease Models: CB-17 SCID (severe combined immunodeficient) mouse (human plasmacytoma MM.1S xenograft mouse model)[1] Doses: 3 mg/kg Route of Administration: 1.v. ; twice-weekly for two weeks Experimental Results: Dramatically inhibited MM tumor growth and prolonged survival of mice.
References	[1]. In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells. Clin Cancer Res. 2013;19(11):3019-3031. [2]. A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells. Br J Haematol. 2016;174(3):397-409. [3]. Mechanisms associated with biogenesis of exosomes in cancer. Mol Cancer. 2019 Mar 30;18(1):52.
Additional Infomation	Melphalan Flufenamide Hydrochloride is the hydrochloride salt form of melphalan flufenamide, a peptide-drug conjugate composed of a peptide conjugated, via an aminopeptidase-targeting linkage, to the alkylating agent melphalan, with potential antineoplastic and anti-angiogenic activities. Upon administration, the highly lipophilic melphalan flufenamide penetrates cell membranes and enters cells. In aminopeptidase-positive tumor cells, melphalan flufenamide is hydrolyzed by peptidases to release the hydrophilic alkylating agent melphalan. This results in the specific release and accumulation of melphalan in aminopeptidase-positive tumor cells. Melphalan alkylates DNA at the N7 position of guanine residues and induces DNA intra- and inter-strand cross-linkages. This results in the inhibition of DNA and RNA synthesis and the induction of apoptosis, thereby inhibiting tumor cell proliferation. Peptidases are overexpressed by certain cancer cells. The administration of melphalan flufenamide allows for enhanced efficacy and reduced toxicity compared to melphalan. Drug Indication Pepaxti is indicated, in combination with dexamethasone, for the treatment of adult patients with multiple myeloma who have received at least three prior lines of therapies, whose disease is refractory to at least one proteasome inhibitor, one immunomodulatory agent, and one anti-CD38 monoclonal antibody, and who have demonstrated disease progression on or after the last therapy. For patients with a prior autologous stem cell transplantation, the time to progression should be at least 3 years from transplantation (see section 4. 4).

Solubility Data

Solubility (In Vitro)

DMSO : 33.33 mg/mL (62.31 mM) H2O : 1 mg/mL (1.87 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.67 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 25.0 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: ≥ 2.5 mg/mL (4.67 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 25.0 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: ≥ 2.5 mg/mL (4.67 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 1 mg/mL (1.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8696 mL	9.3479 mL	18.6958 mL
	5 mM	0.3739 mL	1.8696 mL	3.7392 mL
	10 mM	0.1870 mL	0.9348 mL	1.8696 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.