Colchicine metabolite; tubulin

Effective scavenging of reactive radicals and low reactivity of generated secondary antioxidant radicals towards vital intracellular components are two critical requirements for a chain-breaking antioxidant. Tubulin-binding properties aside, colchicine metabolites remain largely untested for other possible biological activities, including antioxidant activity. Mourelle et al. [Life Sci. 45 (1989) 891] proposed that colchiceine (EIN) acts as an antioxidant and protective agent against lipid peroxidation in a rat model of liver injury. Since EIN as well as two other colchicine metabolites, 2-demethylcolchicine (2DM) and 3-demethylcolchicine (3DM), possess a hydroxy-group on their carbon ring that could participate in radical scavenging, we tested whether they can act as chain-breaking antioxidants. Using our fluorescence-HPLC assay with metabolically incorporated oxidation-sensitive cis-parinaric acid (PnA) we studied the effects of colchicine metabolites on peroxidation of different classes of membrane phospholipids in HL-60 cells. None of the colchicine metabolites in concentrations ranging from 10(-6) to 10(-4) M was able to protect phospholipids against peroxidation induced by either azo-initiators of peroxyl radicals or via myeloperoxidase (MPO)-catalyzed reactions in the presence of hydrogen peroxide. However, the metabolites did exhibit dose-dependent depletion of glutathione, resembling the behavior of etoposide, a hindered phenol with antioxidant properties against lipid peroxidation. Electron spin resonance (ESR) experiments demonstrated that in a catalytic system containing horseradish peroxidase (HRP)/H(2)O(2), colchicine metabolites undergo one-electron oxidation to form phenoxyl radicals that, in turn, cause ESR-detectable ascorbate radicals by oxidation of ascorbate. Phenoxyl radicals of colchicine metabolites formed through MPO-catalyzed H(2)O(2)-dependent reactions in HL-60 cells and via HRP/H(2)O(2) in model systems can also oxidize GSH. Thus, colchicine metabolites possess the prerequisites of many antioxidants, i.e. a nucleophilic hydroxy-group on a carbon ring and the ability to scavenge reactive radicals and form a secondary radical. However, the latter retain high reactivity towards critical biomolecules in cells such as lipids, thiols, ascorbate, thereby, rendering colchicine metabolites effective radical scavengers but not effective chain-breaking antioxidants. [1]

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The effects of colchicine and its analogs on the carrageenin-induced footpad edema in rats were investigated. The anti-inflammatory effects of colchicine analogs were measured at 3 and 5 hr after the carrageenin injection. Colchicine, 1-demethylcolchicine and 3-demethylcolchicine markedly inhibited the carrageenin edema whereas 2-demethylcolchicine was much less active. Thiocolchicinoids, having a thiomethyl group at C-10 instead of a methoxy group, were considerably less potent. These results suggest that the presence of methoxy groups at C-2 and C-10 in colchicine is necessary to maintain anti-inflammatory activity. Inactivity of deacetylcolchicine indicates that substitution of the amino group at C-7 with electron withdrawing groups is also important. Significant inhibition of carrageenin edema and strong binding to tubulin in vitro were manifested by colchicine, 3-demethylcolchicine, N-butyryldeacetylcolchicine and colchifoline. On the other hand, N-carbethoxydeacetylcolchicine which did bind well to tubulin, did not show much effect on the carrageenin edema. These results suggest that the anti-inflammatory action of colchicinoids may not be regulated through the microtubule system [2].

Lipid peroxidation experiments [1]
Two milliliter of cell suspension (1×106 cells per ml) were transferred to glass tubes with screw-caps and were pre-incubated for 15 min with one of the following agents: PMC, phenol, etoposide, 2DM, 3-demethylcolchicine (3DM), COL, EIN, or NDE. Concentration of colchicine and its metabolites varied from 10−6 to 10−4 M. Control samples were treated with the vehicle DMSO. Pre-incubated cells were exposed to three different oxidants: a lipid-soluble azo-initiator of peroxyl radicals, AMVN (0.5 mM for 2 h at 37 °C), a water-soluble azo-initiators, AAPH (50 mM for 30 min at 37 °C), and H2O2 (25 μM, added every 15 min during 1 h incubation at 37 °C).

mouse LD50 intraperitoneal 570 ug/kg Journal of Medicinal Chemistry., 24(636), 1981

[1]. Anti-/pro-oxidant effects of phenolic compounds in cells: are colchicine metabolites chain-breaking antioxidants?. Toxicology. 2002;177(1):105-117.

[2]. Separation of tubulin-binding and anti-inflammatory activity in colchicine analogs and congeners. Life Sci. 1987;40(1):35-39.

3-Desmethylcolchicine has been reported in Colchicum arenarium, Colchicum autumnale, and other organisms with data available.

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The colchicine metabolites tested contain electron-donating hydroxy-groups that can readily undergo one-electron oxidation by MPO/H2O2 to their respective phenoxyl radicals. Our results clearly demonstrated that even at high concentrations (up to 100 μM), colchicine metabolites were unable to substantially inhibit MPO/H2O2-catalyzed phospholipid peroxidation in HL-60 cells (in contrast to a vitamin E homolog, PMC, or the phenolic antitumor drug etoposide). Neither did colchicine metabolites enhance phospholipid peroxidation-the effect readily observed in H2O2-treated HL-60 upon exposure to phenol. These results suggest that phenoxyl radicals formed from colchicine homologs are likely to be less reactive than the radicals generated from phenol but reactive enough to directly attack phospholipids and cause their peroxidation. To obtain further evidence for peroxidase-catalyzed production of phenoxyl radicals from colchicine metabolites, we performed model experiments using HRP/H2O2-catalytic system to detect radical formation by one-electron oxidation of ascorbate and ESR monitoring of the ascorbate radicals. We found that colchicine metabolites with hydroxy-groups were indeed oxidized to their phenoxyl radicals ESR-detectable by their subsequent reactions with ascorbate as ascorbate radicals. We further established that phenoxyl radials generated by peroxidases (MPO in HL-60 cells or HRP in model system) from colchicine homologs were able to react with thiols and cause GSH oxidation. This high reactivity of phenoxyl radicals of colchicine metabolites is similar to that of phenoxyl radicals of etoposide or phenol. In contrast, the vitamin E homolog, PMC, protected GSH against peroxidase-catalyzed oxidation suggesting that its phenoxyl radicals are not reactive towards thiols (Kagan et al., 1999). Taken together, the results demonstrate that even though all colchicine metabolites are capable of forming phenoxyl radicals by reaction with more reactive free radical species, they remain sufficiently reactive to cause not only consumption of glutathione and ascorbate but also oxidation of phospholipids. Thus, they are not chain-breaking antioxidants. This finding rules out the hypothesis that EIN, and probably other colchicine metabolites, can prevent lipid peroxidation via direct antioxidant action. It does not, however, rule out the possibility that they can protect against lipid peroxidation by other means such as modulation of neutrophil ‘oxidative burst’ associated with inflammation. [1]

C21H23NO6

385.41

357.158

7336-33-6

299664

White to yellow solid powder

1.31g/cm3

768.3ºC at 760 mmHg

163-166ºC

418.5ºC

1.61

3.157

2

6

4

28

724

1

CC(=O)N[C@H]1CCC2=CC(=C(C(=C2C3=CC=C(C(=O)C=C13)OC)OC)OC)O

JRRUSQGIRBEMRN-HNNXBMFYSA-N

InChI=1S/C21H23NO6/c1-11(23)22-15-7-5-12-9-17(25)20(27-3)21(28-4)19(12)13-6-8-18(26-2)16(24)10-14(13)15/h6,8-10,15,25H,5,7H2,1-4H3,(H,22,23)/t15-/m0/s1

N-[(7S)-3-hydroxy-1,2,10-trimethoxy-9-oxo-6,7-dihydro-5H-benzo[a]heptalen-7-yl]acetamide

3-Desmethylcolchicine; 7336-33-6; 3-Demethylcolchicine; 3-Demethyl Colchicine; (-)-3-demethylcolchicine; O3-Demethylcolchicine; 3-O-Demethylcolchicine; Desmethylcolchicine;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: 100 mg/mL (259.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.49 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.49 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.49 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.

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