Plant tubulin (from Rosa sp. cv. Paul‘s scarlet): apparent affinity constant (Kapp) for tubulin-oryzalin complex formation = 1.19•10⁵ M⁻¹ (24°C, pH 7.1); inhibition constant (Ki) for rapid phase of taxol-induced MT polymerization = 2.59•10⁻⁶ M. [1]
Plant tubulin (from Chlamydomonas flagellar tubulin): binding constant (K) = 2.08•10⁵ M⁻¹. [1]
Leishmania infantum (intracellular amastigote form): IC50 = 16.4 μM (liposomal formulations F1 and F2). [2]

In Haemanthus katherinae endosperm cells, oryzalin at 0.01 μM reduced the migration rate of anaphase chromosomes to 0.4-0.8 μm•min⁻¹ from the control mean rate of 1.2 μm•min⁻¹; at 0.1 μM oryzalin halted anaphase chromosome migration immediately (≤30 s). Oryzalin (0.1 μM) depolymerized MTs and prevented polymerization of new MTs at all mitotic stages, with only a few short MTs remaining associated with the nucleus. The chromosome condensation cycle was unaffected. Metaphase and anaphase cells treated with 0.1 μM oryzalin (≤2 min) showed complete disassembly of non-kinetochore MTs and partial depolymerization of kinetochore MTs. Cytoplasmic MTs in telophase cells (phragmoplasts) were depolymerized by 0.1 μM oryzalin, but many MTs in the developing cell plate region were not completely depolymerized, even after 2 h at 1.0 μM oryzalin. [1]
In Xenopus leavis heart endothelial cells, oryzalin (10 μM, up to 10 h) had no effect on chromosome movements and condensation, and cells contained a normal distribution of cytoplasmic and mitotic MTs. [1]
In vitro polymerization of rose (Rosa sp. cv. Paul‘s scarlet) tubulin: oryzalin (1, 5, or 7 μM) with 10 μM tubulin and 40 μM taxol at 24°C increased lag-times and decreased rates and extents of turbidity development. Nearly 1:1 molar ratios of oryzalin and tubulin produced maximum inhibition of turbidity. Oryzalin (7 μM) had no effect on bovine brain tubulin polymerization. Preformed rose MTs (10 μM tubulin, 40 μM taxol, 1 h steady state) were partially depolymerized by 12.5 μM oryzalin (A400 decreased from 0.033 to 0.023 within 3 min). Electron microscopy showed fewer and shorter MTs and more amorphous materials at increasing oryzalin concentrations; at 7 μM oryzalin, only amorphous structures were observed. Sedimentation analysis showed approx. 50% inhibition of rose MT yield at 5 μM oryzalin, and 94% inhibition at 7 μM oryzalin. [1]
Oryzalin binding to rose tubulin: time- and pH-dependent. Maximum binding at pH 5.5-6.0. At 24°C, pH 7.1, binding reached a maximum after 2 h (r ≈ 0.08). Apparent binding constant Kapp = 1.19•10⁵ M⁻¹, estimated maximum binding stoichiometry = 0.14. [1]
Binding specificity: [¹⁴C]oryzalin binding to rose tubulin (r = 8.00×10⁻², 100%) was approx. 17-fold greater than to bovine brain tubulin (r = 0.45×10⁻², 6%). [1]
Haemolytic activity: Free oryzalin showed haemolytic activity with HC50 = 425 μM (approx. 20% haemolysis at 100 μM, 54% at 500 μM). Liposomal formulations F1 and F2 showed no significant haemolytic activity even at 500 μM (<6.1% haemolysis). [2]
Cytotoxicity on THP-1 cells: Free oryzalin showed cytotoxic effect with CC50 = 39 μM. Liposomal formulations F1 and F2 showed no cytotoxic effect at concentrations up to 50 μM (CC50 > 50 μM). [2]
Antileishmanial activity against intracellular L. infantum amastigotes: Free oryzalin IC50 = 24.2 μM; liposomal formulations F1 and F2 IC50 = 16.4 μM. [2]

In Haemanthus katherinae endosperm cells, perfusion with 0.01-1.0 μM oryzalin had no apparent effect on the chromosome cycle. Cells treated with 0.01 μM oryzalin in interphase, prophase, and metaphase proceeded to the next mitotic stage. Cells treated with 0.1 μM oryzalin in prophase or metaphase exhibited c-mitotic figures. Long-term observations (≥2 h) showed numerous cells with c-mitotic arrangements and restitution nuclei producing no cell plate. The inhibition of anaphase chromosome migration was not readily reversible (after 5 min treatment with 0.1 μM oryzalin followed by 1 h washing, chromosomes did not resume poleward movements). [1]
Immunogold staining of oryzalin-treated Haemanthus cells followed by washing showed MTs did not repolymerize, correlating with lack of resumption of anaphase chromosome migration. [1]

Turbidimetric measurement of taxol-induced MT polymerization: Rose tubulin (10 μM) was mixed with buffer containing taxol (40 μM) and oryzalin (1, 5, or 7 μM). Reactions were monitored at 24°C by turbidity at A400. An inhibition constant (Ki) for the rapid phase of polymerization was calculated from the steepest portion of turbidity curves using the method of Wilson et al. (1976), giving Ki = 2.59 μM. [1]
Sedimentation analysis of MT polymer yield: Samples polymerized in the presence or absence of oryzalin for 1 h were centrifuged, and pellets were assayed for protein to quantitate the effect of oryzalin on the mass of taxol-induced polymer. [1]
Oryzalin-binding measurements using DEAE-cellulose filter-disc method: Protein (3.5 μM in sucrose isolation buffer) was incubated with [¹⁴C]oryzalin at 24°C. At desired times, 100 μl aliquots were applied to DEAE-cellulose filter discs, slowly adsorbed, washed five times with 10 ml of cold isolation buffer. Discs were air-dried and counted in scintillation mixture. Blanks (without tubulin) were used for correction of non-specific binding. Binding was time- and pH-dependent. Scatchard analysis (Scatchard 1949) gave a line corresponding to a single class of binding sites with Kapp = 1.19•10⁵ M⁻¹ and estimated maximum binding stoichiometry of 0.14. [1]
Differential scanning calorimetry (DSC): Liposomal formulations (lipid concentration ca. 50 mM) were measured in aluminum pans at temperature range of 18-30°C at a heating rate of 0.5°C/min. Empty F1 liposomes (DMPC:DMPG 7:3) showed a typical transition temperature (Tt) at 23.5°C (enthalpy 34.9 J/g). Incorporation of ORZ abolished the Tt and reduced enthalpy to 3.49 J/g. [2]

Haemolytic activity assay: Human red blood cells were distributed in 96-well microplates (100 μl/well), and an equal volume of free or liposomal ORZ (concentrations between 500 and 6 μM) was added. After incubation at 37°C for 1 h, plates were centrifuged (800g, 10 min), supernatants were measured at 540 nm (reference 620 nm). Percentage of haemolytic activity = [(A - A₀)/(A_max - A₀)] × 100. HC50 was calculated using sigmoidal regression analysis. [2]
Cytotoxicity assay on THP-1 cells: THP-1 cells (1×10⁶ cells/mL) were incubated with different concentrations (50-0.4 μM) of liposomal and free ORZ for 72 h. Approximately 4×10⁶ cells were stained with propidium iodide and SYBR-14 using LIVE/DEAD viability kit. Stained cells were analysed by flow cytometry (excitation at 488 nm; green fluorescence at 545 nm for SYBR-14, red fluorescence at 645 nm for PI). At least 10,000 cells were analysed per sample. CC50 was calculated using sigmoidal regression analysis. [2]
Intracellular amastigote drug susceptibility assay: THP-1 cells were differentiated with 1 μM retinoic acid for 72 h at 37°C/5% CO₂. Cells were incubated overnight with L. infantum promastigotes at 4:1 parasite/cell ratio at 37°C/5% CO₂. Free promastigotes were removed by Histopaque 1077 centrifugation (1000g, 20 min). Infected cells (4×10⁵ cells/mL) were seeded in 24-well plates (200 μl/well) and incubated for 48 h with free ORZ or liposomal formulations at several dilutions (50-6 μM). After incubation, cells were fixed with methanol, stained with Giemsa, and the number of infected cells in 100 cells counted. IC50 was calculated using sigmoidal regression analysis. [2]
Chromosome doubling in haploid apple shoots: In experiment 3 (embedding in agar solution), shoots with intact axillary meristems were embedded in low-melting-point agar containing colchicine (0.025, 0.25, 1.25 mM) or oryzalin (5, 15, 30 μM). Survival rates: oryzalin 93.8-100%; colchicine 41.7-100%. Efficiencies (E = % doubled shoots × survival rate): oryzalin 75-100; colchicine 20.8-77.8. Mixoploid shoots (2n = 17/34) were obtained at 30 μM oryzalin. [3]

Pharmacokinetic and biodistribution studies in CD₁ mice (25-30 g): 200 μl of ORZ liposomal formulation (F1: DMPC:DMPG 7:3, 0.15 μm pore size, ORZ dose 0.15 μmol/mouse) containing traces of [¹⁴C]oryzalin (4.1×10⁶ cpm/mL) and [³H]-cholesterol (1.8×10⁶ cpm/mL) was administered via lateral tail vein. As comparison, free ORZ solution with traces of [¹⁴C]oryzalin (0.15 μmol ORZ injected) prepared in Tween 80:citrate buffer (40:60, v/v) was also evaluated. Groups of 5 animals were sacrificed at 30 min, 1, 2, 4, 6, and 24 h post-dosing. Blood samples were collected from orbital sinus into EDTA-containing tubes. Liver, spleen, heart, lungs, and kidneys were removed, rinsed with PBS, dried, weighed, and minced. Samples (0.05 g organ or 0.05 mL blood) were digested overnight at 60°C with perchloric acid (72%) and hydrogen peroxide (30%), neutralized with acetic acid, and counted for radioactivity. [2]
In vivo treatment of Haemanthus katherinae endosperm cells: Cell preparations were perfused with oryzalin-containing buffer (1% ethanol). Oryzalin concentrations tested: 0.01-1.0 μM. Treatment times varied. Cells were observed by differential interference phase microscopy. For reversibility studies, short treatment (5 min) with 0.1 μM oryzalin was followed by washing with oryzalin-free buffer for up to 1 h. [1]

Absorption, Distribution and Excretion
No significant absorption or translocation of glyphosate was observed in soybean plants or wheat grown as a rotation crop.

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Blood clearance in CD₁ mice after i.v. administration (0.15 μmol ORZ/mouse): Free [¹⁴C]oryzalin decreased rapidly to 17% ID at 30 min, with only residual amounts at 24 h. Liposomal ORZ (Lip-¹⁴C-ORZ) decreased to 58% ID at 30 min, remaining at 20% ID at 24 h. A small transient increase in free ORZ was observed at 4 h post-administration; a similar increase for liposomal ORZ occurred at 6 h. [2]
Biodistribution in CD₁ mice: At 2 h post-administration, liposomal ORZ (Lip-¹⁴C-ORZ) showed 9-fold higher levels in liver (14% ID/g) and 13-fold higher levels in spleen (21% ID/g) compared to free ORZ (approx. 4% ID/g in both organs). At 24 h, liposomal ORZ levels were still about 6% ID/g in liver and 5% ID/g in spleen; free ORZ only residual amounts. [2]
Gall bladder accumulation: Liposomal ORZ components (Lip-¹⁴C-ORZ and ³H-Lip) accumulated in gall bladder with two phases: increase between 30 min and 2 h, then gradual decrease. Free ORZ groups had empty gall bladders. [2]

In Haemanthus katherinae endosperm cells, oryzalin (0.1 μM) completely depolymerized MTs within 2 min but did not affect the chromosome condensation cycle. [1]
In Xenopus leavis heart endothelial cells, oryzalin (10 μM, up to 10 h) showed no toxicity (normal chromosome movements and condensation, normal MT distribution). [1]
Haemolytic activity: Free oryzalin HC50 = 425 μM. Liposomal formulations showed HC50 > 500 μM. [2]
Cytotoxicity on THP-1 cells: Free oryzalin CC50 = 39 μM. Liposomal formulations showed CC50 > 50 μM. [2]
In haploid apple shoots, survival rates after oryzalin treatment (5-30 μM, agar embedding method) were 93.8-100%; colchicine treatment (0.025-1.25 mM) gave survival rates of 41.7-100%. Oryzalin at 30 μM produced mixoploid shoots (2n = 17/34). [3]

[1]. Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and inhibits microtubule polymerization in vitro. Planta. 1987 Oct;172(2):252-64.

[2]. Formulation of oryzalin (ORZ) liposomes: in vitro studies and in vivo fate. Eur J Pharm Biopharm. 2012 Oct;82(2):281-90.

[3]. Oryzalin as an efficient agent for chromosome doubling of haploid apple shoots in vitro. Plant Breeding, 1994, 113(4): 343-346.

Oryzalin is a dinitroaniline herbicide (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) with molecular weight 346.36. It is a yellow-orange crystalline solid, soluble in water at 2.5 mg/L at pH 7/25°C, pKa of 8.6. Its herbicidal effect is due to antimitotic activity via binding to plant tubulins, depolymerizing microtubules, and preventing polymerization of new MTs. [1][2]
Oryzalin shows pharmacological differences between plant and animal tubulins. It binds selectively to plant tubulin with moderate affinity (Kapp = 1.19•10⁵ M⁻¹) and inhibits plant MT polymerization at low micromolar concentrations, but has no effect on animal tubulin polymerization or MT-dependent processes. The dinitroaniline-binding site on tubulin has been conserved during evolution of the plant kingdom. [1]
For leishmaniasis treatment, liposomal ORZ formulations (DMPC:DMPG 7:3 or 9:1) were developed using the dehydration-rehydration method. These formulations increased ORZ concentration in aqueous suspensions at least 150-fold without toxic solvents, reduced haemolytic activity and cytotoxicity, and significantly enhanced delivery to liver and spleen (9-13-fold higher accumulation than free ORZ). [2]
For plant breeding, oryzalin (5-15 μM) is more efficient than colchicine for chromosome doubling of haploid apple shoots in vitro, with higher survival rates (93.8-100% vs. 41.7-100%) and higher efficiencies (75-100 vs. 20.8-77.8). Oryzalin shows strong binding affinity to plant tubulins and at low micromolar concentrations can depolymerize microtubules and block cells in metaphase. [3]

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According to the U.S. Environmental Protection Agency (EPA), orizaline may be carcinogenic. Orizaline is a yellow-orange crystalline powder, non-corrosive, and used as a herbicide. Orizaline is a sulfonamide compound with the structure benzenesulfonamide, substituted with nitro groups at positions 3 and 5, and dipropylamino at position 4. It functions as a herbicide, agrochemical, and antimitotic agent. It is a sulfonamide compound, a C-nitro compound, an aromatic amine, and a tertiary amine compound. Orizaline is a pre-emergence surface-applied herbicide. Its mechanism of action is to disrupt (depolymerize) microtubules, thereby blocking anisotropic growth in plant cells. It is used to control annual grasses and broadleaf weeds around fruit trees, nut trees, vineyards, lawns, and ornamental plants by selectively affecting physiological growth processes. It can also be used as an alternative to colchicine for inducing seed ploidy. It has low water solubility, is not easily volatile, and based on its chemical properties, is not expected to seep into groundwater. However, groundwater monitoring programs have identified it as a contaminant. It typically does not persist in soil or aquatic systems. It has low toxicity to mammals but is a potential carcinogen. It is moderately toxic to birds, most aquatic organisms, bees, and earthworms.

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Mechanism of Action
It affects physiological growth processes associated with seed germination.

C12H18N4O6S

346.35952

346.095

19044-88-3

29393

Orange to red solid powder

1.2

514ºC at 760mmHg

141°C

264.6ºC

1.13E-10mmHg at 25°C

1.588

4.604

1

8

6

23

493

0

CCCN(CCC)C1=C(C=C(C=C1[N+](=O)[O-])S(=O)(=O)N)[N+](=O)[O-]

UNAHYJYOSSSJHH-UHFFFAOYSA-N

InChI=1S/C12H18N4O6S/c1-3-5-14(6-4-2)12-10(15(17)18)7-9(23(13,21)22)8-11(12)16(19)20/h7-8H,3-6H2,1-2H3,(H2,13,21,22)

4-(dipropylamino)-3,5-dinitrobenzenesulfonamide

ORYZALIN; 19044-88-3; Surflan; Dirimal; 4-(Dipropylamino)-3,5-dinitrobenzenesulfonamide;

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)

DMSO : ~250 mg/mL (~721.79 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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