Synonym
Methotrexate 5-methyl ester; Methotrexate related compound H Methotrexate related compound H [USP];
IUPAC/Chemical Name
(S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)-5-methoxy-5-oxopentanoic acid
InChi Key
RYGFOBBTCUXHBB-AWEZNQCLSA-N
InChi Code
InChI=1S/C21H24N8O5/c1-29(10-12-9-24-18-16(25-12)17(22)27-21(23)28-18)13-5-3-11(4-6-13)19(31)26-14(20(32)33)7-8-15(30)34-2/h3-6,9,14H,7-8,10H2,1-2H3,(H,26,31)(H,32,33)(H4,22,23,24,27,28)/t14-/m0/s1
SMILES Code
O=C(OC)CC[C@@H](C(O)=O)NC(C1=CC=C(N(CC2=NC3=C(N)N=C(N)N=C3N=C2)C)C=C1)=O
Purity
>98% (or refer to the Certificate of Analysis)
Shipping Condition
Shipped under ambient temperature as non-hazardous chemical. This product is stable enough for a few weeks during ordinary shipping and time spent in Customs.
Storage Condition
Dry, dark and at 0 - 4 C for short term (days to weeks) or -20 C for long term (months to years).
Solubility
Soluble in DMSO
Shelf Life
>3 years if stored properly
Drug Formulation
This drug may be formulated in DMSO
Stock Solution Storage
0 - 4 C for short term (days to weeks), or -20 C for long term (months).
HS Tariff Code
2934.99.03.00
Biological target:
Methotrexate 1-methyl ester is an impurity of Methotrexate, a folic acid antagonist.
In vitro activity:
Methotrexate (0.1-10 mM) induces apoptosis of in vitro activated T cells from human peripheral blood. Methotrexate achieves clonal deletion of activated T cells in mixed lymphocyte reactions. Methotrexate can selectively delete activated peripheral blood T cells by a CD95-independent pathway. Methotrexate is taken up by cells via the reduced folate carrier and then is converted within the cells to polyglutamates. Methotrexate leads to diminished production of leukotriene B4 by neutrophils stimulated ex vivo. Methotrexate polyglutamates inhibit the enzyme aminoimidazolecarboxamidoadenosineribonucleotide (AICAR) transformylase more potently than the other enzymes involved in purine biosynthesis. Methotrexate is also known to suppress TNF activity by suppressing TNF-induced nuclear factor-κB activation in vitro, in part related to a reduction in the degradation and inactivation of an inhibitor of this factor, IκBα, and probably related to the release of adenosine. Methotrexate suppresses the production of both TNF and IFN-γ by T-cell-receptor-primed T lymphocytes from both healthy human donors and RA patients. Methotrexate treatment is associated with a significant decrease of TNF-α-positive CD4+ T cells, while the number of T cells expressing the anti-inflammatory cytokine IL-10 increased.
Reference: J Clin Invest. 1998 Jul 15;102(2):322-8. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC508890/
Arthritis Res. 2002;4(4):266-73. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC128935/
In vivo activity:
Methotrexate reduces thymus and spleen indices of mice. Methotrexate markedly decreases white blood cells, thymic and splenic lymphocytes at dose greater than or equal to 5 mg/kg. However, there is a significant difference between the treatment plus control group and the model group (p<0.01). The combination of grape seed proanthocyanidins and Siberian ginseng eleutherosides obviously diminishes the effects of Methotrexate exposure on indices of thymus and spleens in mice
Reference: Pharmacol Rep. 2006 Jul-Aug;58(4):473-92. http://if-pan.krakow.pl/pjp/pdf/2006/4_473.pdf
Preparing Stock Solutions
The following data is based on the
product
molecular weight
468.47
Batch specific molecular weights may vary
from batch to batch
due to the degree of hydration, which will
affect the solvent
volumes required to prepare stock solutions.
| Concentration / Solvent Volume / Mass |
1 mg |
5 mg |
10 mg |
| 1 mM |
1.15 mL |
5.76 mL |
11.51 mL |
| 5 mM |
0.23 mL |
1.15 mL |
2.3 mL |
| 10 mM |
0.12 mL |
0.58 mL |
1.15 mL |
| 50 mM |
0.02 mL |
0.12 mL |
0.23 mL |
Formulation protocol:
1. Karasik A, Váradi A, Szeri F. In vitro transport of methotrexate by Drosophila Multidrug Resistance-associated Protein. PLoS One. 2018 Oct 12;13(10):e0205657. doi: 10.1371/journal.pone.0205657. PMID: 30312334; PMCID: PMC6185855.
2. Yang J, Gao L, Yu P, Kosgey JC, Jia L, Fang Y, Xiong J, Zhang F. In vitro synergy of azole antifungals and methotrexate against Candida albicans. Life Sci. 2019 Oct 15;235:116827. doi: 10.1016/j.lfs.2019.116827. Epub 2019 Aug 31. PMID: 31479680.
3. Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH. Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis. Bioorg Med Chem. 2020 Jan 15;28(2):115247. doi: 10.1016/j.bmc.2019.115247. Epub 2019 Dec 6. PMID: 31843461.
4. Choi SJ, Oh JM, Chung HE, Hong SH, Kim IH, Choy JH. In vivo anticancer activity of methotrexate-loaded layered double hydroxide nanoparticles. Curr Pharm Des. 2013;19(41):7196-202. doi: 10.2174/138161281941131219123718. PMID: 23489199.
In vitro protocol:
1. Karasik A, Váradi A, Szeri F. In vitro transport of methotrexate by Drosophila Multidrug Resistance-associated Protein. PLoS One. 2018 Oct 12;13(10):e0205657. doi: 10.1371/journal.pone.0205657. PMID: 30312334; PMCID: PMC6185855.
2. Yang J, Gao L, Yu P, Kosgey JC, Jia L, Fang Y, Xiong J, Zhang F. In vitro synergy of azole antifungals and methotrexate against Candida albicans. Life Sci. 2019 Oct 15;235:116827. doi: 10.1016/j.lfs.2019.116827. Epub 2019 Aug 31. PMID: 31479680.
In vivo protocol:
1. Previtali V, Petrovic K, Peiró Cadahía J, Troelsen NS, Clausen MH. Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis. Bioorg Med Chem. 2020 Jan 15;28(2):115247. doi: 10.1016/j.bmc.2019.115247. Epub 2019 Dec 6. PMID: 31843461.
2. Choi SJ, Oh JM, Chung HE, Hong SH, Kim IH, Choy JH. In vivo anticancer activity of methotrexate-loaded layered double hydroxide nanoparticles. Curr Pharm Des. 2013;19(41):7196-202. doi: 10.2174/138161281941131219123718. PMID: 23489199.
1: Witt TL, Stapels SE, Matherly LH. Restoration of transport activity by co-expression of human reduced folate carrier half-molecules in transport-impaired K562 cells: localization of a substrate binding domain to transmembrane domains 7-12. J Biol Chem. 2004 Nov 5;279(45):46755-63. Epub 2004 Aug 26. PubMed PMID: 15337749.
2: Piper JR, DeGraw JI, Colwell WT, Johnson CA, Smith RL, Waud WR, Sirotnak FM. Analogues of methotrexate in rheumatoid arthritis. 2. Effects of 5-deazaaminopterin, 5,10-dideazaaminopterin, and analogues on type II collagen-induced arthritis in mice. J Med Chem. 1997 Jan 31;40(3):377-84. PubMed PMID: 9022805.
3: Piper JR, Ramamurthy B, Johnson CA, Otter GM, Sirotnak FM. Analogues of 10-deazaaminopterin and 5-alkyl-5,10-dideazaaminopterin with the 4-substituted 1-naphthoyl group in the place of 4-substituted benzoyl. J Med Chem. 1996 Jan 19;39(2):614-8. PubMed PMID: 8558535.
4: Piper JR, Johnson CA, Maddry JA, Malik ND, McGuire JJ, Otter GM, Sirotnak FM. Studies on analogues of classical antifolates bearing the naphthoyl group in place of benzoyl in the side chain. J Med Chem. 1993 Dec 24;36(26):4161-71. Erratum in: J Med Chem 1994 Jun 24;37(13):2120. PubMed PMID: 8277497.
5: Huennekens FM, Vitols KS, Pope LE, Fan J. Membrane transport of folate compounds. J Nutr Sci Vitaminol (Tokyo). 1992;Spec No:52-7. Review. PubMed PMID: 1297801.
6: Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Thorndike J, Sirotnak FM. Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase. J Med Chem. 1988 Nov;31(11):2164-9. PubMed PMID: 3184124.
7: Piper JR, McCaleb GS, Montgomery JA, Kisliuk RL, Gaumont Y, Sirotnak FM. Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid. J Med Chem. 1986 Jun;29(6):1080-7. PubMed PMID: 2423690.
