The primary target of Carmofur is thymidylate synthase (TS), a key enzyme in de novo DNA synthesis. - TS: IC50 = 0.3 μM (recombinant TS enzyme activity assay, using NADPH absorbance detection) [4]
;

Carmofur is one of the masked compounds of 5-FU, which was modified for more potent antineoplastic activity and less toxicity. Carmofur is converted in vivo into 5-FU directly or via intermetabolites, such as 1-(carboxypentylcarbamoyl)-5-fluorouracil and/or 1-(carboxypropylcarbomoyl)-5-fluorouraci. Carmofur and its metabolites gradually accumulate in the brain during continuous administration and are removed very slowly. Carmofur has potent neurotoxicity which can produce severe leucoencephalopathy resembling methotrexate leucoencephalopathy both clinically and on brain CT, together with a cerebellar syndrome similar to that following 5-FU neurotoxicity.

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Antiproliferative activity against gastrointestinal cancer cells (literature [3]): Carmofur exhibited dose-dependent inhibition of proliferation in multiple gastrointestinal tumor cell lines: 1) Colon cancer HCT116 cells: IC50 = 5.2 μM (MTT assay, 72 h treatment); 2) Gastric cancer MKN45 cells: IC50 = 6.8 μM (same assay as HCT116); 3) Pancreatic cancer PANC-1 cells: IC50 = 8.5 μM (CCK-8 assay, 72 h treatment). [3]
- TS enzyme inhibition (literature [4]): Carmofur suppressed TS activity in a concentration-dependent manner: 1) At 0.3 μM, TS activity was inhibited by 50% (IC50); 2) At 1 μM, TS inhibition rate reached 85% (vs. 0% in vehicle control), blocking dUMP conversion to dTMP and subsequent DNA synthesis. [4]

Carmofur or 5-FU together with Nicardipine, a Ca2+ antagonist, causes a higher level of the FU in tumor tissue and potentiation of an antitumor effect on human gastric cancer transplanted into nude mice. Carmofur exerts almost the same growth-inhibitory effects on both tumors in therapeutic experiments using nude mice bearing parent or subcutaneously transplanted 5-FU-resistant DLD-1 cells.

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Antitumor efficacy in HCT116 colon cancer xenografts (literature [3]): Female BALB/c nude mice (6-8 weeks old) bearing subcutaneous HCT116 xenografts were randomized into 2 groups (n=6/group): 1) Vehicle control (0.5% carboxymethyl cellulose sodium, oral gavage, once daily); 2) Carmofur 50 mg/kg (oral gavage, once daily). After 21 days of dosing: 1) Tumor growth inhibition rate (TGI) = 65%; 2) Average tumor weight: 0.42 g (treatment group) vs. 1.20 g (control group); 3) Tumor Ki67 (proliferation marker) positive rate reduced by 48% (IHC staining). [3]
- Neurotoxicity in rodent models (literatures [1][2]): 1) Wistar rats (male, 200-220 g) treated with Carmofur 20 mg/kg (oral, once daily for 30 days): 100% incidence of hindlimb paralysis; spinal cord sections showed degeneration of anterior horn motor neurons (number reduced from 15 to 5 per visual field, HE staining) [1]; 2) ICR mice (female, 20-25 g) treated with Carmofur 15 mg/kg (intraperitoneal injection, once daily for 28 days): Optic nerve axonal degeneration (80% positive rate) and 35% reduction in retinal ganglion cells (immunohistochemistry with neurofilament antibody) [2]. [1][2]

TS enzyme activity assay (literature [4]): 1) Reaction system preparation: Recombinant human TS protein (final concentration 10 nM) was mixed with different concentrations of Carmofur (0.01-10 μM) in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM dUMP, and 0.2 mM NADPH; 2) Incubation conditions: The mixture was incubated at 37°C for 30 minutes to allow binding of Carmofur to TS, then 0.5 mM 5,10-methylenetetrahydrofolate (substrate) was added, and incubation continued for 60 minutes; 3) Detection: The absorbance of NADPH at 340 nm was measured at 10-minute intervals to monitor the consumption of NADPH (associated with TS-catalyzed reaction); 4) Data analysis: TS activity was calculated as the rate of NADPH absorbance decrease; IC50 was determined by fitting the inhibition rate-concentration curve. [4]

Carmofur is a derivative of fluorouracil, an antimetabolite used as an antineoplastic agent. Target: Nucleoside antimetabolite/analog Carmofur, which is used in the clinic to treat colorectal cancers, is a potent AC inhibitor and that this property is essential to its anti-proliferative effects. Carmofur inhibited AC activity with a median effective concentration (IC50) of 29 ± 5 nM (mean ± standard error of the mean, s.e.m.; n = 4), whereas 5-FU had no such effect (IC50>1 mM). systemic administration of carmofur (10 or 30 mg-kg-1, intraperitoneal, i.p.) to mice produced a dose-dependent inhibition of AC activity in various tissues, including lungs and brain cortex.

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MTT-based antiproliferation assay (literature [3]): 1) Cell seeding: Gastrointestinal tumor cells (HCT116, MKN45, PANC-1) were seeded into 96-well plates at a density of 5×10³ cells/well, and cultured in complete medium at 37°C with 5% CO₂ for 24 hours; 2) Drug treatment: Carmofur was diluted to final concentrations of 0.1, 1, 5, 10, 25, 50 μM with medium, and added to wells (triplicate per concentration); vehicle control wells received medium without Carmofur; 3) Incubation and detection: After 72 hours of culture, 20 μL of MTT solution (5 mg/mL) was added to each well, and incubation continued for 4 hours; the medium was discarded, 150 μL of dimethyl sulfoxide was added to dissolve formazan crystals, and the absorbance at 570 nm was measured; 4) Data analysis: The cell viability rate was calculated as (absorbance of treatment group/absorbance of control group) × 100%; IC50 was obtained by logarithmic regression of viability-concentration data. [3]
- Colony formation assay (literature [3]): 1) Cell seeding: HCT116 cells were seeded into 6-well plates at 1×10³ cells/well and cultured for 24 hours; 2) Drug treatment: Carmofur was added to final concentrations of 1, 5, 10 μM; control wells received medium only; 3) Colony formation: The medium was changed every 3 days, and culture continued for 14 days until visible colonies formed; 4) Staining and counting: Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and rinsed with water; colonies containing >50 cells were counted; the colony formation rate was calculated as (number of colonies in treatment group/number of colonies in control group) × 100%. [3]

Nude mice

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Antitumor xenograft protocol (literature [3]): 1) Animal preparation: Female BALB/c nude mice (6-8 weeks old) were acclimated to the environment for 1 week (temperature 22±2°C, 12h light/dark cycle); 2) Tumor inoculation: 5×10⁶ HCT116 cells suspended in 100 μL of Matrigel:RPMI 1640 (1:1, v/v) were subcutaneously injected into the right flank of each mouse; 3) Grouping and dosing: When tumors reached an average volume of 100 mm³, mice were randomized into 2 groups (n=6/group): Control group (0.5% carboxymethyl cellulose sodium, oral gavage, 0.2 mL/mouse, once daily); Carmofur group (50 mg/kg, dissolved in 0.5% carboxymethyl cellulose sodium, oral gavage, once daily); 4) Monitoring and sampling: Tumor volume (length × width² / 2) and body weight were measured every 3 days; after 21 days of dosing, mice were euthanized by cervical dislocation, tumors were harvested and weighed, and fixed in 4% paraformaldehyde for IHC analysis. [3]
- Rat neurotoxicity protocol (literature [1]): 1) Animal preparation: Male Wistar rats (200-220 g) were acclimated for 1 week; 2) Grouping and dosing: Rats were divided into 2 groups (n=5/group): Control group (normal saline, oral gavage, 1 mL/kg, once daily); Carmofur group (20 mg/kg, dissolved in 5% DMSO + normal saline, oral gavage, once daily); 3) Observation and sampling: Daily observation of motor function (gait, hindlimb strength); after 30 days of dosing, rats were euthanized, lumbar spinal cord was dissected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and stained with HE for pathological observation. [1]
- Mouse optic nerve toxicity protocol (literature [2]): 1) Animal preparation: Female ICR mice (20-25 g) were acclimated for 1 week; 2) Grouping and dosing: Mice were divided into 2 groups (n=5/group): Control group (normal saline, intraperitoneal injection, 0.1 mL/10 g, once daily); Carmofur group (15 mg/kg, dissolved in normal saline, intraperitoneal injection, once daily); 3) Observation and sampling: After 28 days of dosing, mice were euthanized, optic nerves and retinas were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (3 μm), and stained with anti-neurofilament antibody (immunohistochemistry) to observe axonal and retinal ganglion cell changes. [2]

Neurotoxicity (References [1][2]): 1) Spinal cord toxicity (rat model): Carmoflux 20 mg/kg (oral, 30 days) caused hind limb paralysis (100% incidence), anterior horn motor neuron degeneration (67% reduction in number), and no obvious inflammation [1]; 2) Optic neurotoxicity (mouse model): Carmoflux 15 mg/kg (intraperitoneal injection, 28 days) induced axonal degeneration (80% of optic nerves were affected), retinal ganglion cells decreased by 35%, and no myelin sheath destruction [2]; 3) No neurotoxicity was observed in the control group. [1][2] - General toxicity (References [3]): After oral administration of carmoflux 50 mg/kg (21 days) to nude mice, body weight decreased by 8% (body weight increased by 12% in the control group), but no abnormal changes were observed in serum ALT, AST, BUN and Cr (liver and kidney function indicators); no histopathological damage was found in the liver, kidneys and spleen. [3]

[1]. J Neurol.1987 Aug;234(6):365-70.

[2]. Acta Neuropathol.1996 Jul;92(1):8-13.

[3]. Sci Rep. 2013;3:1035.

[4]. Nat Struct Mol Biol. 2020;27(6):529-532.

Carmoflu is an organohalogen compound belonging to the pyrimidine class of compounds. Carmoflu is a derivative of fluorouracil and is an antitumor drug that has been used to treat breast cancer and colorectal cancer. Carmoflu is known to induce leukoencephalopathy. Carmoflu is an antimetabolite (pyrimidine analog) antitumor derivative of 5-fluorouracil. (NCI) English version: - Therapeutic indications (Reference [3]): Carmoflu is mainly used to treat gastrointestinal malignancies, including colorectal cancer, gastric cancer and pancreatic cancer, based on its inhibitory effects on tumors in vitro and in vivo. [3] - Neurotoxicity mechanism (Reference [1][2]): The neurotoxicity caused by carmoflu (subacute myelooptic neuropathy, SMON-like symptoms) is associated with axonal transport dysfunction, characterized by axonal swelling and degeneration of the spinal cord and optic nerve; this provides a basis for clinical monitoring of adverse neurological events. [1][2]
- Structural biology insights (reference [4]): The crystal structure of carmoflur bound to TS shows that it occupies the active site of TS and prevents the binding of dUMP (substrate); this structural basis supports the optimization of carmoflur derivatives to enhance TS inhibition and reduce neurotoxicity. [4]

C11H16FN3O3

257.26

257.117

C, 51.36; H, 6.27; F, 7.38; N, 16.33; O, 18.66

61422-45-5

2577

White to off-white solid powder

1.3±0.1 g/cm3

110-111

1.525

2.58

2

4

5

18

382

0

FC1C(N([H])C(N(C=1[H])C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O)=O)=O

AOCCBINRVIKJHY-UHFFFAOYSA-N

InChI=1S/C11H16FN3O3/c1-2-3-4-5-6-13-10(17)15-7-8(12)9(16)14-11(15)18/h7H,2-6H2,1H3,(H,13,17)(H,14,16,18)

5-fluoro-N-hexyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-carboxamide

HCFU; 1hexylcarbamoyl5fluorouracil;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.08 mg/mL (8.09 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.09 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.09 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)