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6-Diazo-5-oxo-L-norleucine

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6-Diazo-5-oxo-L-norleucine
Legal status
Legal status
Identifiers
  • (5S)-5-Amino-1-diazonio-6-hydroxy-6-oxohex-1-en-2-olate[1]
CAS Number
PubChem CID
ChemSpider
UNII
ChEBI
ChEMBL
CompTox Dashboard (EPA)
ECHA InfoCard100.150.017 Edit this at Wikidata
Chemical and physical data
FormulaC6H9N3O3
Molar mass171.156 g·mol−1
3D model (JSmol)
  • O=C(CC[C@H](N)C(O)=O)\C=[N+]=[N-]
  • InChI=1S/C6H9N3O3/c7-5(6(11)12)2-1-4(10)3-9-8/h3,5H,1-2,7H2,(H,11,12)/t5-/m0/s1 X markN
  • Key:YCWQAMGASJSUIP-YFKPBYRVSA-N X markN
 X markNcheckY (what is this?)  (verify)

6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist, which was isolated originally from Streptomyces in a sample of Peruvian soil. This diazo compound is biosynthesized from lysine by three enzymes in bacteria.[2] It is one of the most famous non-proteinogenic amino acids, and was characterized in 1956 by Henry W Dion et al.,[3] who suggested a possible use in cancer therapy. This antitumoral efficacy was confirmed in different animal models.[4] DON was tested as chemotherapeutic agent in different clinical studies, but was never approved, due to adverse side effects causing gastrointestinal toxicities.[5]

In 2019, DON was shown to kill tumor cells while reversing disease symptoms and improve overall survival in late-stage experimental glioblastoma in mice, when combined with calorie-restricted ketogenic diet.[6]

History

[edit]

In 1957, the first-in-human study of DON within 63 patients with advanced, inoperable, solid tumours was published by Magill et al. Multiple varieties of low doses (0.2-1.1 mg/kg daily), and differing administration routes (oral, intramuscular, intravascular) were used to test the pharmacokinetics of DON.[5][7] The most common adverse side effects were mucositis (83%), diarrhea (48%), and nausea/vomiting (30%), with only 7/63 patients responding to treatment.[5]

Multiple studies on a variety of cancers continued to take place between 1957 and 1962 on a variety of cancers and age groups, including 71 pediatric patients with acute leukemia.[5][8] Complete remission was seen within 42% of leukiemia patients, when DON (daily dose of 0.25 mg/kg) was used in combination with a common leukiemia chemotherapy agent, 6-mercaptopurine (6-MP), which was better than 6-MP being used alone.[5][8]

In the 1980s-2000s, DON therapy was given in higher doses (25-100mg/kg in mice every 4 days), which showed to be promising in nude mouse models that were transplanted with human tumours. Since higher doses more intermittently was well tolerated, and improved tumour regression, this regimen was employed within further clinical trials.[5]

In the 1980s, five phase I dose escalation studies were carried out. DON was administered intravenously in doses ranging from 50 mg/m2/dose to 600 mg/m2/dose (1.35-16.2 mg/kg~), two to three times weekly, every three to four weeks. No responses to the therapy were seen within any adult group, and the pediatric patients displayed stable disease, or partial responses to treatment with DON.[5] Some patients also experienced blood abnormalities (leukopenia/thrombocytopenia), but this was in patients with underlying metastases, or diseases involving the bone marrow.[5]

Phase II Trials concluded that DON was ineffective at high doses, and too toxic, and was therefore abandoned.[5] It showed mixed results within a variance of cancers, with 24 out of 44 patients with advanced colorectal cancer experiencing progression regardless of dosage, no responses seen in 41 patients with advanced sarcomas, or 23 patients with advanced lung cancers, and only 1 out of 17 patients with advanced refractory solid tumours showing a partial response.[5]

Recent studies have shown that utilizing molecules to develop inactive forms of DON (known as prodrugs) until it reaches the site of a tumour, could be an effective anticancer therapy option. A focus is seen particularly on brain tumours (such as glioblastomas) as they tend to have an amplification of MYC or MYCN oncogenes, which promote glutamine catabolism and drive cancer cell survival, within an environment of increased energy demand.[5][9]

The prodrug of DON, DRP-104, has shown significant promise against tumours, whilst remaining inactive within healthy gastrointestinal tissues, reducing its gastrointestinal toxicity. It has significantly better tolerance than DON, and has been shown to enhance the effects of a subset of T cells (CD8+, 'cytotoxic') that help kill tumour cells.[10]

Chemistry

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DON is a water-soluble yellowish powder, which can be dissolved also in aqueous solutions of methanol, acetone or ethanol, but dissolution in absolute alcohols is difficult. Solutions of at least 50 μM DON in 0.9% NaCl are lightly yellowish. The crystalline form appears as yellowish greenish needles. The specific rotation is [α]26D +21° (c = 5.4% in H2O). In phosphate buffer, pH 7 are the ultraviolet absorption maxima at 274 nm (E1%1 cm. 683) and 244 nm (E1%1 cm 376).[3][11]

Biochemistry

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DON is used as an inhibitor of different glutamine-utilizing enzymes. Due to its similarity to glutamine, it can enter catalytic centres of these enzymes and inhibits them by covalent binding, or more precisely, by alkylation.[12][13] The following table gives a survey of DON targets.

Selection of enzymes inhibited by DON
EnzymeMetabolic pathwayReferences
Carbamoyl phosphate synthase (CAD)Pyrimidine-De-Novo-Synthesis[12][14]
Cytidine triphosphate (CTP) synthase (CTPS)Pyrimidine-De-Novo-Synthesis[12][14]
Formylglycinamide ribonucleotide amidotransferase (FGAR)Purine-De-Novo-Synthesis[12][15]
Guanosine monophosphate synthetase (GMPS)Purine-De-Novo-Synthesis[12][16]
PRPP amidotransferasePurine-De-Novo-Synthesis[12][16]
Glutaminase (GLS1)Glutamate Synthesis(Glutaminolysis)

Tricarboxylic acid (TCA) Cycle

 [5][12][16]
NAD+ synthetase (NADSYN)Coenzyme of the electron transport chain[12][17]
Asparagine synthetase (ASNS)Amino Acid Synthesis[5][12][18]
Glutamine synthetase (GS) Amino Acid Synthesis [5]
Glutamine:Fructose-6-phosphate amidotransferase (GFAT) Hexosamine synthesis [5]

Pharmacology

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Glutaminase (GLS1) is overexpressed in various cancer cells, and is associated with poor prognosis, promotion of epithelial to mesenchymal transition (EMT), and drug resistance.[19]

DON's primary target is GLS1, an enzyme involved in the conversion of glutamine to glutamate. DON undergoes a two-step process of inhibition which results in the irreversible binding to the target enzyme, and the subsequent inactivation. The first step of this process is the initial binding to the glutamine active site on the enzyme, which results in the formation of a reactive species, that alkylates a nucleophilic residue in the enzymes active site. This alkylation forms a covalent adduct, in which the enzyme becomes permanently inactive.[5]

Glutamate is involved in the generation of intermediates within the TCA cycle as a carbon source, as well as maintaining cellular redox homeostasis. Cancer cells depend on further cellular proliferation, which makes inhibition of glutamine and glutamate desirable as a therapeutic.[19]

DON is also inhibitor of many enzymes of nucleotide synthesis, resulting in cellular cytotoxicity, and cell death as a result.[5]

See also

[edit]
  • JHU-083, a prodrug of 6-diazo-5-oxo-L-norleucine

References

[edit]
  1. ↑ "CID 5359375". PubChem. United States National Library of Medicine.
  2. ↑ Kawai S, Sugaya Y, Hagihara R, Tomita H, Katsuyama Y, Ohnishi Y (April 2021). "Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6-Diazo-5-oxo-l-norleucine and One Molecule of Alanine". Angewandte Chemie. 60 (18): 10319–10325. doi:10.1002/anie.202100462. PMID 33624374. S2CID 232039107.
  3. 1 2 Dion HW, Fusari SA, Jakubowski ZL, Zora JG, Bartz QR, et al. (1954). 6-diazo-5-oxo-L-norleucine, A new tumor inhibitory substance. II: Isolation and Characterization. Antibiotics and Chemotherapy. Vol. 78. pp. 3075–3077.
  4. ↑ Yoshioka K, Takehara H, Okada A, Komi N (June 1992). "Glutamine antagonist with diet deficient in glutamine and aspartate reduce tumor growth". The Tokushima Journal of Experimental Medicine. 39 (1–2): 69–76. PMID 1412455.
  5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Lemberg KM, Vornov JJ, Rais R, Slusher BS (September 2018). "We're Not "DON" Yet: Optimal Dosing and Prodrug Delivery of 6-Diazo-5-oxo-L-norleucine". Molecular Cancer Therapeutics. 17 (9): 1824–1832. doi:10.1158/1535-7163.MCT-17-1148. PMC 6130910. PMID 30181331.
  6. ↑ Mukherjee P, Augur ZM, Li M, Hill C, Greenwood B, Domin MA, et al. (29 May 2019). "Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma". Communications Biology. 2 (1) 200. doi:10.1038/s42003-019-0455-x. PMC 6541653. PMID 31149644.
  7. ↑ Magill GB, Myers WP, Reilly HC, Putnam RC, Magill JW, Sykes MP, et al. (1957). "Pharmacological and initial therapeutic observations on 6-diazo-5-oxo-1-norleucine (DON) in human neoplastic disease". Cancer. 10 (6): 1138–1150. doi:10.1002/1097-0142(195711/12)10:6<1138::AID-CNCR2820100608>3.0.CO;2-K. PMID 13489662. Archived from the original on 2025-07-06.
  8. 1 2 Sullivan MP, Beatty EC, Hyman CB, Murphy ML, Pierce MI, Severo NC (May 1962). "A comparison of the effectiveness of standard dose 6-mercaptopurine, combination 6-mercaptopurine and DON, and high-loading 6-mercaptopurine therapies in treatment of the acute leukemias of childhood: results of a coperative study". Cancer Chemotherapy Reports. 18: 83–95. PMID 13918321.
  9. ↑ Kumar MA, Baba SK, Khan IR, Khan MS, Husain FM, Ahmad S, et al. (July 2025). "Glutamine Metabolism: Molecular Regulation, Biological Functions, and Diseases". MedComm. 6 (7) e70120. doi:10.1002/mco2.70120. PMC 12188105. PMID 40567251.
  10. ↑ Rais R, Lemberg KM, Tenora L, Arwood ML, Pal A, Alt J, et al. (November 2022). "Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug". Science Advances. 8 (46) eabq5925. Bibcode:2022SciA....8.5925R. doi:10.1126/sciadv.abq5925. PMC 9668306. PMID 36383674.
  11. ↑ DeWald HA, Moore AM (August 1958). "6-Diazo-5-oxo-L-norleucine, a New Tumor-inhibitory Substance.1a Preparation of L-, D- and DL-Forms1b". Journal of the American Chemical Society. 80 (15): 3941–3945. Bibcode:1958JAChS..80.3941D. doi:10.1021/ja01548a036.
  12. 1 2 3 4 5 6 7 8 9 Pinkus LM (1977). "Glutamine binding sites". Affinity labeling. Methods in Enzymology. Vol. 46. pp. 414–427. doi:10.1016/S0076-6879(77)46049-X. ISBN 978-0-12-181946-0. PMID 909432.
  13. ↑ Ortlund E, Lacount MW, Lewinski K, Lebioda L (February 2000). "Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu". Biochemistry. 39 (6): 1199–1204. doi:10.1021/bi991797d. PMID 10684596.
  14. 1 2 Eidinoff ML, Knoll JE, Marano B, Cheong L (1 January 1958). "Pyrimidine Studies: I. Effect of DON (6-Diazo-5-oxo-l-norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines". Cancer Research. 18 (1): 105–109.
  15. ↑ Levenberg B, Melnick I, Buchanan JM (March 1957). "Biosynthesis of the purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de novo". The Journal of Biological Chemistry. 225 (1): 163–176. doi:10.1016/S0021-9258(18)64919-1. PMID 13416227.
  16. 1 2 3 Ahluwalia GS, Grem JL, Hao Z, Cooney DA (1990). "Metabolism and action of amino acid analog anti-cancer agents". Pharmacology & Therapeutics. 46 (2): 243–271. doi:10.1016/0163-7258(90)90094-I. PMID 2108451.
  17. ↑ Barclay RK, Phillipps MA (February 1966). "Effects of 6-diazo-5-oxol-norleucine and other tumor inhibitors on the biosynthesis of nicotinamide adenine dinucleotide in mice". Cancer Research. 26 (2): 282–286. PMID 4285554.
  18. ↑ Rosenbluth RJ, Cooney DA, Jayaram HN, Milman HA, Homan ER (August 1976). "DON, CONV and DONV-II. Inhibition of L-'asparagine synthetase in vivo". Biochemical Pharmacology. 25 (16): 1851–1858. doi:10.1016/0006-2952(76)90189-1. PMID 9091.
  19. 1 2 Jin J, Byun JK, Choi YK, Park KG (April 2023). "Targeting glutamine metabolism as a therapeutic strategy for cancer". Experimental & Molecular Medicine. 55 (4): 706–715. doi:10.1038/s12276-023-00971-9. PMC 10167356. PMID 37009798.
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