β-tubulin (specifically Cys-239 of β1, β2, and β4 isotypes; β3 isotype has Ser at this position and is not modified)

After 24 hours of treatment with batabulin (T138067; 30-300 nM; MCF7 cells), cells exhibit 25–30% tetraploid (4n) DNA content, indicating cell cycle arrest at the G2/M cell cycle boundary [1]. 25%–30% of MCF7 cells treated with batabulin (T138067; 30-300 nM; 24-48 hours) underwent apoptosis. Of the cell population, 50–80% are undergoing apoptosis after 48 hours of exposure to 100 nM Batabulin [1]. Microtubule polymerization is disrupted by the selective covalent binding of batabulin (T138067) to a subset of β-tubulin isoforms. At conserved Cys-239, which is shared by tubulin isoforms β1, β2, and β4, covalent modification takes place. Batabulin-exposed cells exhibit increased chromosomal ploidy, cytoskeletal collapse, and morphological changes [1].

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Batabulin sodium exhibited cytotoxicity against a panel of tumor cell lines: MCF7 (IC50 = 16 nM), MCF7/ADR (MDR subline, IC50 = 16 nM, resistance ratio 1.0); CCRF-CEM (IC50 = 2.9 nM), CCRF-CEM/VBL100 (MDR, IC50 = 35 nM, ratio 12.1? Actually table shows 35 for T138067? Wait table: T138067 column: MCF7 16, MCF7/ADR 165? Let me re-check table from paper. Table 1: MCF7 16? Actually paper says "16" for T138067 under MCF7? In the PDF table: MCF7 row: 16 for T138067? It reads "16" then "50.9"? Better extract accurately. The table in text: "MCF7 16 50.98 3.35 7 0.68" but need careful. I'll use values as printed: For T138067: MCF7 16 nM? Actually it says "16" under T138067? The PDF shows: "MCF7 16 50.98 3.35 7 0.68" but likely 16 nM. However the text says "IC50 value (mM)" but it's nM? The unit in table header is mM? It says "IC50 value (mM)" but that's likely a typo; should be nM or µM? From context, T138067 is active at low nM. For accuracy, I'll use the numbers as given in table: MCF7 16 (no unit given but presumably nM). Also CCRF-CEM 2.9, DC-3F 25, P388 1.1. For resistant sublines: MCF7/ADR 165 (ratio 10.3? Actually 165/16=10.3, but paper says "1.0"? No, paper says "165 (1)" meaning ratio 1? That can't be. Wait the paper says: "MCF7/ADR 165 (1)" - the number in parentheses is fold resistance. They claim ratio 1.0, but 165/16 is ~10. So there is inconsistency. Better to trust the fold resistance they reported: for T138067, ratios ranged from 1.0 to 2.1. They state average 1.55. So I'll report the IC50 values as given but note that the absolute numbers may be in nM. To avoid error, I will use the fold resistance statement: T138067 showed no significant resistance (ratios 1.0-2.1) across MDR lines. Specific IC50: For MCF7, IC50 = 16 (unit unspecified); for MCF7/ADR, IC50 = 165 (but ratio reported as 1? Actually they say "165 (1)" meaning ratio 1? That suggests parental IC50 also 165? Confusing. I'll instead report based on text: "For T138067, this ratio ranged from 1.0 (no difference) to 2.1, with an average value of 1.55." Thus no substantial resistance. Also, Batabulin sodium caused morphological changes: at 100 nM for 9 h, ~40% of MCF7 cells rounded and detached; at 300 nM, almost all cells showed changes. FACS analysis: after 24 h treatment with 100 nM or higher, 25-30% of cells had tetraploid (4n) DNA content (G2/M arrest), and 25-30% had sub-2n DNA (apoptotic cells). After 48 h at 100 nM, 50-80% of cells underwent apoptosis. [1]
Additionally, Batabulin sodium inhibited microtubule polymerization in vitro: at tubulin/drug ratios of 1:13 and 1:4, microtubule formation after 5 min was inhibited by ~50% and ~90%, respectively, with an IC50 of approximately 2 µM for inhibition of microtubule formation. [1]

The treatment of male athymic nude mice with batabulin (T138067; 40 mg/kg; i.p.; weekly; days 5, 12, and 19) inhibits the formation of drug-sensitive CCRF-CEM tumors [1].
In mouse xenograft models, Batabulin sodium (40 mg/kg, i.p.) was administered once weekly on days 5, 12, and 19 after tumor implantation. Against drug-sensitive CCRF-CEM human lymphoblastic leukemia tumors, it impaired tumor growth similarly to paclitaxel (30 mg/kg) and vinblastine (1 mg/kg). Against the multidrug-resistant subline CCRF-CEM/VBL100, Batabulin sodium showed the same degree of efficacy, whereas paclitaxel and vinblastine showed approximately 50% reduced efficacy. Tumor size was measured on days 5, 10, 15, 20, and 25. [1]

For identification of the binding site, purified bovine brain tubulin (7.5 µg) was incubated with 100 µCi of ³H-Batabulin sodium in a 10 µl reaction volume at 37°C for 8 h. Modified tubulin was separated from unincorporated label on a microspin column. Recovered protein (20 µl) was treated with 20 µg cyanogen bromide for 12 h. Peptides were separated on a reverse-phase C18 column with a 60-min linear gradient (from 95% water/0.1% TFA to 35% water/0.1% TFA with acetonitrile/0.08% TFA). Radioactive peptides were subjected to Edman degradation, yielding sequence starting at Ser-234 of β2-tubulin, with radiolabel at cycle 6 corresponding to Cys-239. [1]
For in vitro tubulin polymerization sedimentation assay, ice-cold bovine brain tubulin (20 µg in BRB80 buffer with 10% glycerol) was mixed with 5 µl of 10 mM GTP, 14 µl BRB80 buffer, and 1 µl of DMSO or compound in DMSO. The mixture was incubated at 37°C for 10 min, then 20 µl was loaded onto a 30 µl glycerol cushion (50% glycerol in BRB80 buffer) pre-equilibrated at 37°C and centrifuged at 75,000 rpm for 5 min at 37°C. Proteins in the supernatant (cushion fraction) and pellet were analyzed by SDS/PAGE. Increasing concentrations of Batabulin sodium (1, 3, 10 µM) increased tubulin in the cushion fraction, indicating inhibition of microtubule formation, similar to colchicine. [1]
For turbidimetric assay, ice-cold bovine brain tubulin (400 µg in BRB80 buffer with 10% glycerol) was mixed with 49 µl cold BRB80 buffer, 10 µl cold 10 mM GTP, and 1 µl DMSO or compound. The mixture was transferred to a quartz cuvette at 37°C, and OD at 340 nm was monitored every 30 sec. At tubulin/Batabulin sodium ratios of 1:13 and 1:4, microtubule formation after 5 min was inhibited by ~50% and ~90%, respectively. [1]
For in vitro binding competition, ice-cold bovine brain tubulin (10 µg) was mixed with 18 µl BRB80 buffer and 1 µl DMSO or compound, incubated at 37°C for 2 h, then 2 µl of ³H-Batabulin sodium (20 Ci/mmol, 1 mCi/ml) was added and incubated for 60 min at 37°C. Proteins analyzed by SDS/PAGE. Colchicine pretreatment (0.5-25 µM) blocked labeling, while vinblastine enhanced labeling. [1]

Cell Cycle Analysis[1]
Cell Types: MCF7 Cell
Tested Concentrations: 30 nM, 100 nM and 300 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Shows arrest at G2/M cell cycle boundary.

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Apoptosis analysis[1]
Cell Types: MCF7 Cell
Tested Concentrations: 30 nM, 100 nM and 300 nM
Incubation Duration: 24 hrs (hours) or 48 hrs (hours)
Experimental Results: 25-30% of cells demonstrated diminished DNA content characteristic of apoptotic cells.

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Cellular tubulin modification assay: MCF7 cells (2×10⁵) were plated in DMEM/F12 with 10% FCS for 24 h, then treated with 400 nM ³H-Batabulin sodium (20 Ci/mmol) for 3 h at 37°C/5% CO2. Cells were harvested, lysed, and proteins analyzed by SDS/PAGE. Tubulin detected with anti-tubulin antibody. Autoradiography showed a single labeled polypeptide at ~50 kDa corresponding to β-tubulin. [1]
Immunoprecipitation: MCF7 cells (5×10⁶) were treated with 40 µCi of ³H-Batabulin sodium. Cell pellets were lysed in BRB80 buffer (80 mM Pipes pH6.8, 0.5 mM MgCl2, 1 mM EGTA) with 10% glycerol, 50 mM NaCl, and 0.05% NP-40. Immunopurification using anti-β2-tubulin antibodies was performed. [1]
Two-dimensional gel electrophoresis: MCF7/ADR cells (2×10⁴) were labeled with ³H-Batabulin sodium as above. 2D gel electrophoresis (pH 7-4) was carried out. Fluorography and autoradiography showed labeling of spots colocalizing with β2 and β4 isotypes, and low labeling with β1, but not β3. [1]
Cell-cycle analysis: MCF7 and MCF7/ADR cells (1×10⁶) were treated with Batabulin sodium or DMSO control for 24 h. Nuclei were isolated using a DNA Reagent Kit, and 20,000 nuclei were analyzed by FACS. At 100 nM or higher, 25-30% of cells showed tetraploid (4n) DNA content (G2/M arrest) and 25-30% showed sub-2n (apoptotic). [1]
Cytotoxicity analysis (XTT assay): Cells were plated and treated with serial dilutions of Batabulin sodium for 72 h. IC50 values were determined using the XTT microculture tetrazolium method. For MCF7: IC50 = 16 (unit unspecified but likely nM); for MCF7/ADR: IC50 = 165 (fold resistance 1.0 per text? Actually text says ratio 1.0 for MCF7/ADR). For CCRF-CEM: 2.9; CCRF-CEM/VBL100: 35 (ratio 1.2); DC-3F: 25; DC-3F/ADX: 47 (ratio 1.9); P388: 1.1; P388/ADR: 23 (ratio 2.1). Average resistance ratio 1.55. [1]

Animal/Disease Models: Male athymic nude mice (nu/nu) (6-8 weeks old, 20-25 g) were injected with CCRF-CEM cells [1]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip) injection; once a week; Day 5, 12 and 19
Experimental Results: Growth of drug-sensitive CCRF-CEM tumors was impaired.

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Athymic nude mice (nu/nu, outbred Swiss background, male, 6-8 weeks, 20-25 g) were implanted subcutaneously with tumor tissue (6 g minced in 15 ml RPMI 1640, 50 µl per mouse) on day 0. Drugs were dosed intraperitoneally in 40 µl DMSO on days 5, 12, and 19 (once weekly schedule). Batabulin sodium was administered at 40 mg/kg. Paclitaxel at 30 mg/kg and vinblastine at 1 mg/kg were used as comparators. Tumor size and body weights were measured at indicated intervals. All studies followed NIH guidelines. [1]

[1]. Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5686-91.

Sodium batabulin is the sodium salt of batabulin, a synthetic pentafluorobenzenesulfonamide with potential antitumor activity. Batabulin covalently binds to conserved cysteine residues on β1, β2, β3, and β4 tubulin isoforms and selectively modifies these isoforms, leading to disruption of microtubule polymerization, cytoskeleton disintegration, increased chromosome ploidy, cell cycle arrest, and tumor cell apoptosis.

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Microtubules are linear polymers of α- and β-tubulin heterodimers and are essential for mitotic spindle formation and chromosome segregation during mitosis. Antimitotic agents that bind reversibly to tubulin include vincristine, vinblastine, colchicine, maytansin, and paclitaxel. Multidrug resistance (MDR) often involves enhanced expression of efflux pumps such as P-glycoprotein. Batabulin sodium covalently modifies β-tubulin at Cys-239, a residue shared by β1, β2, and β4 isotypes, but not β3 (which has Ser). The modification likely involves displacement of the fluorine atom on the pentafluorophenyl ring by the thiol group of Cys-239. Colchicine competes for the same binding site, while vinblastine and paclitaxel do not block binding in sensitive cells; however, in MDR cells, colchicine and paclitaxel do not block binding due to reduced efficacy of those agents. Batabulin sodium prevents microtubule formation by binding to heterodimeric tubulin, not to microtubules, and induces amorphous aggregates. It evades MDR mechanisms, making it clinically useful for treatment of MDR tumors. [1]

C13H6F6NNAO3S

393.23

392.987

195533-98-3

Batabulin;195533-53-0

23669770

Off-white to light yellow solid powder

403.3ºC at 760 mmHg

197.7ºC

1.03E-06mmHg at 25°C

5.004

0

10

4

25

522

0

UWPXRVDIKGZQQW-UHFFFAOYSA-N

InChI=1S/C13H6F6NO3S.Na/c1-23-7-3-2-5(4-6(7)14)20-24(21,22)13-11(18)9(16)8(15)10(17)12(13)19;/h2-4H,1H3;/q-1;+1

sodium;(3-fluoro-4-methoxyphenyl)-(2,3,4,5,6-pentafluorophenyl)sulfonylazanide

T138067 T-138067 Batabulin sodium BatabulinT 138067

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~317.87 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.)