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Chemical Structure| 59-05-2 Chemical Structure| 59-05-2

Structure of Methotrexate
CAS No.: 59-05-2

Chemical Structure| 59-05-2

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Methotrexate, an antifolate compound, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH and can interfere with the growth of certain cells of the body, especially cells that reproduce quickly, such as cancer cells, bone marrow cells, and skin cells.

Synonyms: Amethopterin; CL14377; NCI-C04671

4.5 *For Research Use Only !

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Product Citations

Product Citations

Verónica Urdaneta-Páez ; Randy Hamchand ; Karen Anthony ; Jason Crawford ; Alan G. Sutherland ; Barbara I. Kazmierczak

Abstract: Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In the first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate) riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and -proficient strain using high-resolution liquid chromatography-mass spectrometry (LC-MS). This simple yet powerful method, optimized for high-throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multidrug-resistant Gram-negative pathogen. IMPORTANCE: Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multidrug-resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside or enter the cell and are exported by efflux systems. This approach enables the development of rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.

Keywords: Pseudomonas aeruginosa ; antimicrobial resistance ; drug efflux

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Alternative Products

Product Details of Methotrexate

CAS No. :59-05-2
Formula : C20H22N8O5
M.W : 454.44
SMILES Code : O=C(O)[C@@H](NC(C1=CC=C(N(CC2=NC3=C(N)N=C(N)N=C3N=C2)C)C=C1)=O)CCC(O)=O
Synonyms :
Amethopterin; CL14377; NCI-C04671
MDL No. :MFCD00064370
InChI Key :FBOZXECLQNJBKD-ZDUSSCGKSA-N
Pubchem ID :126941

Safety of Methotrexate

GHS Pictogram:
Signal Word:Danger
Hazard Statements:H301-H341-H360-H372
Precautionary Statements:P202-P260-P264-P280-P301+P310-P405
Class:6.1
UN#:1544
Packing Group:

Isoform Comparison

Biological Activity

Target
  • DHFR

    hDHFR, IC50:24 nM

In Vitro:

Cell Line
Concentration Treated Time Description References
Ramos cells 20 μM 72 hours To investigate the effect of FTCD depletion on methotrexate sensitivity, results showed that FTCD depletion significantly reduced the sensitivity of Ramos cells to methotrexate Nature. 2018 Jul;559(7715):632-636
HEL cells 5 μM 3 days To investigate the effect of FTCD depletion on methotrexate sensitivity, results showed that FTCD depletion significantly reduced the sensitivity of HEL cells to methotrexate Nature. 2018 Jul;559(7715):632-636
BHK-CD16a 10 μM Used for culturing BHK-CD16a cells to study ADCC activity of CD19 antibodies Front Immunol. 2022 Aug 31;13:957874.
BT-474 cells 0.1 µM 72 h MTX treatment increases the intracellular concentration of the metabolite AICAR, resulting in AMPK activation. Methotrexate-induced AMPK activation leads to decreased one-carbon metabolism gene expression and cellular proliferation as well as increased global bioenergetic capacity. Sci Rep. 2020 May 12;10(1):7838.
mouse embryonic fibroblasts (MEFs) 0.1 µM 72 h MTX caused a strong increase (~30 fold) in endogenous AICAR levels in breast cancer cells and mouse embryonic fibroblasts (MEFs) Sci Rep. 2020 May 12;10(1):7838.
A549 cells 1 μM To investigate the effects of MTX on Th17 cell differentiation, results showed that MTX-treated A549 cells co-cultured with PBMCs significantly increased the frequency of Th17 cells. EBioMedicine. 2024 Oct;108:105339.
Molt4 cells 90 nM To evaluate the synergy between methotrexate and SHIN2, results showed that methotrexate increased sensitivity to SHIN2 Leukemia. 2021 Feb;35(2):377-388.

In Vivo:

Species
Animal Model
Administration Dosage Frequency Description References
C57BL/6 nude mice Acute lymphoblastic leukemia mouse model Intraperitoneal injection 80 mg/kg ginger extract and 5 mg/kg MTX Five times a week for ginger extract and once a week for MTX, lasting two months To evaluate the anti-leukemic activity of ginger extract alone or in combination with MTX on ALL mouse models, results showed that combined treatment significantly increased survival rate and reduced damages in brain and liver tissues. J Cell Mol Med. 2021 May 3;25(13):6148–60
DBA/1J mice Collagen-induced arthritis (CIA) mouse model Intraperitoneal injection 23 mg/kg Thrice a week for 38 days To evaluate the effect of MTX on arthritis in CIA mice, the results showed that MTX significantly alleviated arthritis symptoms in CIA mice. Clin Transl Med. 2022 Dec;12(12):e1148.
DBA/1J female mice Bovine collagen-induced arthritis (CIA) model Intragastric administration 0.92 mg/kg Twice a week for 30 days To evaluate the therapeutic effect of Methotrexate on arthritis in CIA mice, the results showed that Methotrexate was effective in controlling inflammation but had no obvious benefits in regulating weight loss and mechanical allodynia Front Pharmacol. 2021 May 28;12:693777
DBA/1 mice Collagen-induced arthritis (CIA) model Oral gavage 2 mg/kg Every 3 days for 20 days To evaluate the therapeutic effect of MTX on CIA model mice, the results showed that MTX significantly alleviated arthritis symptoms and footpad swelling. Front Immunol. 2018 Nov 27;9:2762
SKG mice RA-ILD model Intraperitoneal injection 7.5 mg/kg Twice weekly for 12 weeks To investigate the effects of MTX on pulmonary inflammation in the RA-ILD mouse model, results showed that MTX exacerbated pulmonary inflammation, increased immune cell infiltration, and fibrosis. EBioMedicine. 2024 Oct;108:105339.
Mice NOTCH1-driven mouse primary T-ALL model Intraperitoneal injection 10 mg/kg Administered every 5 days for 4 weeks To evaluate the synergy between methotrexate and SHIN2, results showed that the combination significantly extended mouse survival Leukemia. 2021 Feb;35(2):377-388.

Clinical Trial:

NCT Number Conditions Phases Recruitment Completion Date Locations
NCT01404429 Rheumatoid Arthritis PHASE4 COMPLETED 2025-07-12 New OPD Block, Rheumatology Cl... More >>inic, Level 3, Chandigarh, 160012, India Less <<
NCT01314417 Non-infectious Intermediate an... More >>d Posterior Uveitis Less << PHASE1|PHASE2 TERMINATED 2025-01-13 National Institutes of Health ... More >>Clinical Center, 9000 Rockville Pike, Bethesda, Maryland, 20892, United States Less <<
NCT02777736 Diffuse Large B-cell Lymphoma PHASE3 UNKNOWN 2025-12-19 University Hospital Brno-Bohun... More >>ice, Brno, 62500, Czech Republic|University Hospital Hradec Králové, Hradec Králové, 50005, Czech Republic|University Hospital Ostrava, Ostrava, 708 52, Czech Republic|University Hospital Pilsen, Pilsen, 304 60, Czech Republic|University Hospital Kralovske Vinohrady, Prague, 100 34, Czech Republic|General University Hospital Prague, Prague, 12808, Czech Republic Less <<
NCT02076997 Cancer COMPLETED 2019-05-10 Texas Children's Hospital, Hou... More >>ston, Texas, 77030, United States Less <<
NCT01187264 Psoriasis PHASE4 COMPLETED 2025-09-09 Postgraduate Institute of Medi... More >>cal Education and Research, Chandigarh, U.T, 160012, India Less <<
NCT03757364 Nail Psoriasis COMPLETED 2018-09-30 University of Warmia and Mazur... More >>y, Olsztyn, Warminsko-mazurskie, 10-900, Poland Less <<
NCT00006168 Liver Cirrhosis, Biliary PHASE3 COMPLETED 2025-03-04 Keck School of Medicine at U.S... More >>.C., Los Angeles, California, 90033, United States|U California Medical Center, San Francisco, California, 94143, United States|Yale University School of Medicine, New Haven, Connecticut, 06520-8019, United States|Emory University School of Medicine, Atlanta, Georgia, 30322, United States|Saint Louis University, St. Louis, Missouri, 63104, United States|University of Nebraska Medical Center, Omaha, Nebraska, 68198-3285, United States|Cleveland Clinic, Cleveland, Ohio, 44195, United States|Oregon Health Sciences University, Portland, Oregon, 97201, United States|Albert Einstein Medical Center, Philadelphia, Pennsylvania, 19141, United States|UT Southwestern Medical Center at Dallas, Dallas, Texas, 75235-9151, United States|Medical College of Virginia, Richmond, Virginia, 23298-0711, United States|University of Washington Medical Center, Seattle, Washington, 98195, United States Less <<
NCT06289205 Proliferative Vitreoretinopath... More >>y Less << PHASE1|PHASE2 RECRUITING 2025-05-25 Sahiwal Medical College & Teac... More >>hing Hospital, Sahiwal, Pakistan, Sahiwal, Punjab, 57000, Pakistan Less <<
NCT06300333 Ectopic Pregnancy NOT_YET_RECRUITING 2025-03-25 -
NCT04677868 Acute GVH Disease PHASE2 UNKNOWN 2025-06-22 Peking University Institute of... More >> Hematology,Beijing, Beijing, Beijing, 100044, China Less <<
NCT05315128 Non-melanoma Skin Cancers PHASE2 COMPLETED 2021-12-06 Zagazig university, Zagazig, S... More >>harkia, 44519, Egypt Less <<
NCT05353829 Rheumatoid Arthritis PHASE4 ACTIVE_NOT_RECRUITING 2026-12-31 Region S?rmland, Eskilstuna, S... More >>weden|Region Dalarna, Falun, Sweden|Region G?vleborg, G?vle, Sweden|Region Norrbotten, Lule?, Sweden|V?stra G?talandsregionen, Sk?vde, Sweden|Akademiskt specialistcentrum, Stockholm, Sweden|Danderyds sjukhus, Stockholm, Sweden|Karolinska sjukhuset, Stockholm, Sweden|Region V?sternorrland, Sundsvall, Sweden|Region V?sterbotten, Ume?, Sweden|Reumatologisk klinik, V?ster?s, V?ster?s, Sweden|Reumatologsektionen ?rebro universitetssjukhus, ?re, Sweden|Region J?mtland H?rjedalen, ?stersund, Sweden Less <<
NCT02553018 Arthritis, Rheumatoid PHASE3 COMPLETED 2025-08-17 SARAUX, Brest, France|HUDRY, P... More >>aris, France Less <<
NCT04379219 Cesarean Scar Pregnancy PHASE4 UNKNOWN 2025-12-20 Mansoura University, Mansoura,... More >> Dakahliya, Egypt Less <<
NCT03535519 Rheumatoid Arthritis UNKNOWN 2020-08-31 Hospital General Universitario... More >> Gregorio Mara?ón, Madrid, 28007, Spain Less <<
NCT02248792 Severe Plaque Type Psoriasis PHASE4 UNKNOWN - Narayana Med College and Hospi... More >>tal, Nellore, AP, 524003, India Less <<
NCT05208034 Ectopic Pregnancy COMPLETED 2021-07-30 Nishtar Medical University Hos... More >>pital, Multān, Pu, 66000, Pakistan Less <<
NCT00952016 Advanced Colorectal Cancer PHASE2 TERMINATED 2025-09-14 The Royal Marsden NHS Foundati... More >>on Trust, Sutton, Surrey, SM2 5PT, United Kingdom Less <<
NCT02897011 Rheumatoid Arthritis|Influenza... More >>|Methotrexate Less << UNKNOWN 2025-08-17 Seoul National Univ. Bundang H... More >>ospital, Bundang, Gyeonggi-do, 463-870, Korea, Republic of|Seoul National University Hospital, Seoul, 110-744, Korea, Republic of|SMG-SNU Boramae Medical Center, Seoul, 156-707, Korea, Republic of Less <<
NCT02074319 Schizophrenia|Schizoaffective ... More >>Disorder|Schizophreniform Disorder Less << PHASE1 COMPLETED 2025-08-15 Abasi Shaheed Hospital, Karach... More >>i, Sindh, 72000, Pakistan|Civil hospital Karachi, Karachi, Sindh, 72000, Pakistan|Institute of Behavioural Sciences, Karachi, Sindh, 72000, Pakistan|Karwn e Hayat, Karachi, Sindh, 72000, Pakistan Less <<
NCT00791531 Malaria PHASE1 COMPLETED 2025-08-09 Kenya Medical Research Institu... More >>te, Center for Clinical Research, Nairobi, 00200, Kenya Less <<
NCT06001125 Arthritis|Arthralgia PHASE2 RECRUITING 2025-11-29 Cross Cancer Institute, Edmont... More >>on, Canada Less <<
NCT06649136 Rheumatoid Arthritis PHASE4 RECRUITING 2026-09-30 Medical University of Vienna, ... More >>Vienna, 1090, Austria Less <<
NCT04830878 Proliferative Vitreoretinopath... More >>y Less << PHASE1 WITHDRAWN 2025-04-24 Byers Eye Institute at Stanfor... More >>d University and Lucille Packard Children Hospital, Palo Alto, California, 94303, United States Less <<
NCT00298012 Spondylarthropathies|Spondylit... More >>is, Ankylosing Less << PHASE4 WITHDRAWN - Rheumatism Foundation Hospital... More >>, Heinola, Finland|Helsinki University Central Hospital, Helsinki, Finland|Central Finland Central Hospital, Jyv?skyl?, Finland|Kuopio University Hospital, Kuopio, Finland|Tampere University Hospital, Tampere, Finland Less <<
NCT04482543 Proliferative Vitreoretinopath... More >>y Less << PHASE2|PHASE3 UNKNOWN 2021-09-01 Ophthalmic Research Center, Te... More >>hran, Iran, Islamic Republic of Less <<
NCT02097173 Psoriasis PHASE1 COMPLETED - Clinical Trials of Texas, Inc.... More >>, San Antonio, Texas, 78229, United States Less <<
NCT01860690 Ectopic Pregnancy PHASE4 UNKNOWN 2025-02-14 Dr. Zvi Klein, Kefar-saba, Isr... More >>ael Less <<
NCT04958538 Acute Graft Versus Host Diseas... More >>e Less << PHASE2 UNKNOWN 2023-01-30 Peking University Institute of... More >> Hematology,, Beijing, Beijing, 100044, China Less <<
NCT00243750 Ankylosing Spondylitis PHASE2 UNKNOWN 2025-11-05 Charité Campus Benjamin-Frankl... More >>in, Rheumatology, Berlin, 12200, Germany Less <<
NCT04360785 Spondyloarthritis COMPLETED 2023-02-27 Hopital Pellegrin, Bordeaux, F... More >>rance|Hopital Ambroise Paré, Boulogne-Billancourt, France|Hopital La Cavale Blanche, Brest, France|CHU Trousseau, Chambray-lès-Tours, France|CH Sud Francilien, Corbeil-Essonnes, France|Hopital Mondor, Créteil, France|Kremlin Bicetre hospital, Le Kremlin-Bicêtre, France|CH Le Mans, Le Mans, France|Hopital Saint Philibert, Lille, France|CHU Nantes, Nantes, France|CHR Orléans, Orléans, France|Hopital Bichat, Paris, France|Hopital Cochin, Paris, France|Hopital la Pitié Salpétrière, Paris, France|Hopital Saint Antoine, Paris, France|CH Pontoise, Pontoise, France|CHU Rouen, Rouen, France Less <<
NCT00035074 Inflammatory Bowel Disease COMPLETED - Childrens Hospital of Philadel... More >>phia, Philadelphia, Pennsylvania, 19104, United States Less <<
NCT02715947 Psoriasis Vulgaris PHASE2 UNKNOWN 2025-12-17 Xiangya Hospital Central South... More >> University, Changsha, Hunan, 410008, China Less <<
NCT00194272 Ectopic Pregnancy PHASE3 COMPLETED 2025-06-07 University of Southern Califor... More >>nia Women's and Children's Hospital, Los Angeles, California, 90033, United States|University of Miami, Miami, Florida, 33101, United States|University of Pennsylvania Reproductive Research Unit, Philadelphia, Pennsylvania, 19104, United States Less <<
NCT01393405 Ulcerative Colitis PHASE2 COMPLETED 2025-04-17 University of Southern Califor... More >>nia, Los Angeles, California, 90033, United States|University of Colorado, Denver, Colorado, 80045, United States|Shafran Gastroenterology, Winter Park, Florida, 32789, United States|Atlanta Gastroenterology, Atlanta, Georgia, 30342, United States|Northwestern University, Chicago, Illinois, 60611, United States|The University of Chicago, Chicago, Illinois, 60637, United States|Indiana University IU Health, Indianapolis, Indiana, 46202, United States|University of Iowa, Iowa City, Iowa, 52242, United States|University of Kentucky, Lexington, Kentucky, 40536, United States|University of Louisville, Louisville, Kentucky, 40202, United States|University of Maryland, Baltimore, Maryland, 21201, United States|Metropolitan Gastroenterology Group, PC, Chevy Chase Clinical Research, Chevy Chase, Maryland, 20815, United States|Massachusetts General Hospital, Boston, Massachusetts, 02114, United States|Henry Ford Health System, Novi, Michigan, 48377, United States|Minnesota Gastroenterology, Plymouth, Minnesota, 55446, United States|Mayo Clinic, Rochester, Minnesota, 55905, United States|Dartmouth College, Lebanon, New Hampshire, 03756, United States|Mt Sinai School of Medicine, New York, New York, 10029, United States|Asheville Gastroenterology Associates, Asheville, North Carolina, 28801, United States|University of North Carolina, Chapel Hill, North Carolina, 27599, United States|Charlotte Gastroenterology & Hepatology, Charlotte, North Carolina, 28207, United States|Case Western Reserve University, Cleveland, Ohio, 44106, United States|Great Lakes Gastroenterology, Mentor, Ohio, 44060, United States|University of Pennsylvania, Philadelphia, Pennsylvania, 19104, United States|Penn State University, State College, Pennsylvania, 17033, United States|Medical University of South Carolina, Charleston, South Carolina, 29425, United States|Baylor University, Houston, Texas, 77030, United States|University of Utah, Salt Lake City, Utah, 84132, United States|Harborview Medical Center, Seattle, Washington, 98104, United States|University of Washington, Seattle, Washington, 98195, United States|University of Wisconsin, Madison, Wisconsin, 53792, United States Less <<
NCT03327116 Atopic Dermatitis COMPLETED 2018-12-01 Hospital das Clínicas of the U... More >>niversity of S?o Paulo Medical School, Department of Dermatology, S?o Paulo, SP, 05403900, Brazil Less <<
NCT05054426 Diffuse Large B Cell Lymphoma|... More >>Central Nervous System Lymphoma Less << PHASE3 RECRUITING 2025-10-08 Peking union medical college h... More >>ospital, Beijing, Beijing, 100005, China Less <<
NCT02383901 Osteosarcoma COMPLETED 2025-05-16 Semmelweis Egyetem II. Sz. Gye... More >>rmekklinika, Budapest, 1094, Hungary|Oslo University Hospital, Oslo, N-0424, Norway|Intytut Matki Dziecka Klinika Chirurgii Onkologicznej Dzieci i M?odzie?y, Warszawa, 01 211, Poland|Sk?ne University Hospital, Lund, 221 85, Sweden|Karolinska University Hospital, Sweden, Stockholm, SE-17176, Sweden Less <<
NCT00656591 Child Mortality UNKNOWN 2025-12-08 International Child Health, IC... More >>H, London, WC1N 1EH, United Kingdom Less <<
NCT03531073 Psoriatic Arthritis COMPLETED 2024-12-31 Oxford University Hospitals NH... More >>S Trust, Oxford, Oxfordshire, OX3 7LD, United Kingdom Less <<

Protocol

Bio Calculators
Preparing Stock Solutions 1mg 5mg 10mg

1 mM

5 mM

10 mM

2.20mL

0.44mL

0.22mL

11.00mL

2.20mL

1.10mL

22.01mL

4.40mL

2.20mL

References

 

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