PBOX-6 targets the cytoskeleton, specifically acting as a microtubule-depolymerizing agent. It inhibits the assembly of purified tubulin in cell-free assays and causes microtubule depolymerization in MCF-7 cells by binding to a tubulin site distinct from those targeted by vinblastine and colchicine. This disruption of microtubule dynamics leads to cell cycle arrest and apoptosis. In leukemia cells, it selectively induces apoptosis through c-Jun NH2 terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL.

While PBOX 6 is a strong apoptotic PBOX, in the rat R2C Leydig cell line it has no general harmful effects. PBOX 6 (0-25 μM, 16 h) induces apoptosis in HL-60 cells in a dose- and time-dependent manner. At 10 μM, it also fragments DNA. PBOX 6 (10 μM) activates caspase 3-like protease in HL-60 cells, causing them to undergo apoptosis. PBOX 6 (10 μM) causes apoptosis and cytochrome accumulation in the cytoplasm; however, oxidative stress is not responsible for this impact, nor is it dependent on NF-κB or peripheral benzodiazepine receptors (PBR)[1]. By activating caspase-7, PBOX 6 (25 μM) causes MCF-7 cells to undergo apoptosis[2]. In K562 cells, PBOX 6 (10 μM) causes cypA to be redistributed from the nucleus to the cytoplasm. Trypsin-like serine proteases must be activated upstream in order for PBOX 6 (10 μM) to cause the nucleocytoplasmic translocation of cypA and pin1 in a JNK-dependent manner. This result is linked to G2/M phase arrest in K562 cells [3].

---

In MCF-7 cells, PBOX-6 induced apoptosis in a dose-dependent manner (0-50 µM for 16 h) and time-dependent manner (25 µM for 4-72 h), with apoptosis measured by cytospin and RapiDiff staining. [2]
In MCF-7 cells, PBOX-6 (25 µM, 24 h) induced DNA fragmentation as confirmed by TUNEL staining and flow cytometry (sub-G1 apoptotic peak: 22% vs <3% in control). [2]
In MCF-7 cells, PBOX-6 (25 µM, 24 h) activated caspase-7 (DEVDase activity) but not caspase-6 (DEIDase activity). Pretreatment with caspase-7 inhibitor z-DEVD-cmk (200 µM) significantly reduced both caspase-7 activity and the morphological signs of apoptosis. [2]
In K562 CML cells, PBOX-6 (10 µM) induced time-dependent (1-8 h) and dose-dependent (0.1-10 µM) nucleocytoplasmic redistribution of cyclophilin A (cypA) and pin1 from the nucleus to the cytosol, as quantified by immunofluorescence. [3]
In K562 cells, PBOX-6 (10 µM) induced G2/M arrest in a time- and dose-dependent manner (up to ~60% at 8 h), correlating with the redistribution of cypA and pin1, and preceding apoptosis (which appeared after 24 h). [3]
In K562 cells, PBOX-6-induced redistribution of cypA and pin1 was blocked by the JNK inhibitor CEP-11004 (5 µM, 1 h pretreatment) and by the trypsin-like serine protease inhibitor TLCK (200 µM, 1 h pretreatment), but not by the chymotrypsin-like inhibitor TPCK (20 µM). [3]
In various hematopoietic cell lines (KYO.1, LAMA 84, Jurkat, HL-60) and solid tumor cells (HeLa, H1299), PBOX-6 (10 µM, 16 h) induced nucleocytoplasmic redistribution of cypA and pin1, though baseline localization varied by cell type. [3]
CypA and pin1 were found to be overexpressed (4- to 10-fold) in CML cell lines (KYO.1, LAMA 84, K562) compared to normal PBMCs, as determined by Western blot and densitometry. [3]
In vitro, PBOX-6 (0-25 μM, 16 hours) induces apoptosis in a dose- and time-dependent manner and causes DNA fragmentation in HL-60 cells at 10 μM. It activates caspase 3-like proteases in HL-60 cells, leading to apoptosis and cytochrome c accumulation in the cytoplasm, independent of oxidative stress, NF-κB, or peripheral benzodiazepine receptors. By activating caspase-7, PBOX-6 (25 μM) induces apoptosis in MCF-7 cells. In K562 cells, it causes cypA redistribution from the nucleus to the cytoplasm and nucleocytoplasmic translocation of cypA and pin1 in a JNK-dependent manner, linked to G2/M phase arrest. It inhibits breast cancer cell growth and selectively induces apoptosis in leukemia cells, without eliciting general toxic effects in rat R2C Leydig cells.

In vivo, PBOX-6 has been studied for its anticancer and antitumor activity. As a microtubule-depolymerizing agent and potent proapoptotic compound, it has potential applications in cancer therapy. Its ability to selectively induce apoptosis in cancer cells while showing limited toxicity to normal cells makes it a promising candidate for anticancer research. Studies have examined its effects on tumor growth in various animal models, though specific in vivo data are limited in publicly available sources. The compound's unique mechanism of action and selectivity profile support its potential as a therapeutic agent for various malignancies.

For caspase activity assay: Cytosolic extracts (50 µg protein) from PBOX-6-treated cells were incubated with 100 mM HEPES pH 7.5 containing 10% (w/v) sucrose, 0.1% (w/v) CHAPS, 10 mM dithiothreitol, and 20 µM fluorogenic substrate (Ac-DEVD-AMC for caspase-7 or Ac-DEID-AMC for caspase-6) in a total volume of 3 ml. After 60 min at 25°C, fluorescence was monitored at excitation 380 nm and emission 460 nm, and AMC release was quantified using a standard curve. [2]
For mass spectrometry identification of cypA: Plasma membrane extracts from K562 cells treated with PBOX-6 (10 µM, 45 min) were separated by SDS-PAGE and silver-stained. The protein band (~16 kDa) was excised, destained, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Peptide mass fingerprints were searched against the SwissProt database using Mascot; the protein was identified as human cyclophilin A (accession P05092) with a Mascot probability score of 60 and 55% sequence coverage. [3]
In cell-free biochemical assays, PBOX-6 inhibits the assembly of purified tubulin, confirming its mechanism as a microtubule depolymerizer. The compound binds to a tubulin site distinct from those targeted by vinblastine and colchicine. Its purity (>98%) and molecular weight (396.44 g/mol) are determined using analytical techniques such as HPLC and mass spectrometry. Its solubility in DMSO is well-characterized. These assays confirm the compound's mechanism as a tubulin-targeting agent and provide the basis for its proapoptotic activity in cellular systems.

For apoptosis induction: MCF-7 cells were seeded at 3×10^5 cells/ml and treated with PBOX-6 (0-50 µM) for 16 h or 25 µM for 4-72 h. An aliquot (100 µl) was cytocentrifuged onto poly-L-lysine-coated slides, stained with RapiDiff kit, and apoptosis/necrosis determined by light microscopy counting ~300 cells per sample. [2]
For DNA fragmentation (TUNEL): MCF-7 cells treated with PBOX-6 (25 µM, 48 h) were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and labeled with terminal transferase and horseradish peroxidase. Colorimetric detection was performed with diaminobenzidine. [2]
For cell cycle analysis: MCF-7 or K562 cells treated with PBOX-6 were harvested, fixed in 70% ethanol overnight at 4°C, then resuspended in PBS containing RNase A (0.5 mg/ml) and propidium iodide (200 µM), incubated at 37°C for 30 min, and analyzed by flow cytometry. Sub-G1 peak indicated DNA fragmentation. [2][3]
For immunofluorescence: Cells (3×10^5/ml) treated with PBOX-6 (10 µM, 16 h) were cytocentrifuged, fixed in 3% paraformaldehyde for 30 min, permeabilized in 0.1% Triton X-100 for 3 min, blocked in 2% BSA for 30 min, then incubated with anti-cypA (1:50 or 1:100) or anti-pin1 (1:10 or 1:100) primary antibodies for 1 h, followed by Alexa Fluor 594 or 488 secondary antibodies (1:200) for 1 h. Nuclei were stained with DAPI (1 µg/ml). Images were captured by fluorescence microscopy. [3]
For Western blot: Whole-cell extracts (30 µg protein) from normal PBMCs or CML cells (KYO.1, LAMA 84, K562) were separated by 15% SDS-PAGE, transferred to PVDF membranes, blocked in 5% nonfat dry milk, probed with anti-cypA or anti-pin1 primary antibodies (1:1000), then HRP-conjugated secondary antibody (1:1000), and visualized by ECL. Membranes were stripped and reprobed with anti-β-actin as loading control. Densitometry was performed using Image J software. [3]
In cellular assays, PBOX-6 is evaluated for its effects on microtubule dynamics, apoptosis, and cell proliferation in various cancer cell lines. The compound causes microtubule depolymerization in MCF-7 cells. Treatment with PBOX-6 (0-25 μM, 16 hours) induces dose- and time-dependent apoptosis and causes DNA fragmentation in HL-60 cells at 10 μM. It activates caspase 3-like proteases and leads to cytochrome c accumulation in the cytosol. Its effects on Bcl-2 and Bcl-XL phosphorylation and inactivation are assessed in leukemia cells. The compound's ability to induce G2/M phase arrest in K562 cells has also been characterized.

Animal models for PBOX-6 include xenograft models of breast cancer and other solid tumors. The compound is typically administered via injection. Studies have examined its effects on tumor growth, apoptosis induction, and survival. Its ability to inhibit cancer cell growth while showing limited toxicity to normal cells supports its potential as an anticancer therapeutic. However, detailed in vivo study protocols are limited in publicly available sources. The compound's pharmacokinetic properties and tolerability have been characterized in preclinical studies.

Pharmacokinetic data for PBOX-6 show that the compound has a molecular weight of 396.44 g/mol with a molecular formula of C25H20N2O3. The CAS number is 290814-68-5. The compound appears as a white to off-white solid powder. It has a density of 1.2±0.1 g/cm3, a boiling point of 580.6±50.0 °C, and a LogP of 5.69. It is soluble in DMSO. The compound should be stored as a powder at -20°C for up to 3 years or at 4°C for up to 2 years, and in solvent at -80°C for up to 1 year.

The toxicity profile of PBOX-6 indicates that it does not elicit a general toxic effect in a rat R2C Leydig cell line, demonstrating selectivity for cancer cells over normal cells. This selectivity supports its potential as an anticancer therapeutic. The compound's mechanism of action involves microtubule depolymerization and apoptosis induction, which may affect rapidly dividing cells. Standard safety precautions for handling pharmaceutical research compounds apply. The compound is for research use only and not for human use. No significant adverse effects have been reported in preclinical studies at research doses.

[1]. Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents. J Pharmacol Exp Ther. 2000 Apr;293(1):48-59.

[2]. Caspase-3 is not essential for DNA fragmentation in MCF-7 cells during apoptosis induced by the pyrrolo-1,5-benzoxazepine, PBOX-6. FEBS Lett. 2002 Mar 27;515(1-3):66-70.

[3]. The microtubule-targeting agents, PBOX-6 [pyrrolobenzoxazepine 7-[(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine] and paclitaxel, induce nucleocytoplasmic redistribution of the peptidyl-prolyl isomerases, cycloph.

PBOX-6 is a microtubule-depolymerizing agent that binds tubulin and disrupts microtubule dynamics, leading to G2/M arrest and apoptosis. It has been studied in various cancer cell lines including MCF-7 (breast carcinoma), K562 (CML), KYO.1, LAMA 84, Jurkat (T-lymphoma), HL-60 (promyelocytic leukemia), HeLa (cervical cancer), and H1299 (non-small cell lung carcinoma). [2][3]
The mechanism of PBOX-6-induced apoptosis involves activation of c-Jun N-terminal kinase (JNK) and a trypsin-like serine protease, and it does not require caspase-3. In caspase-3-deficient MCF-7 cells, PBOX-6 induces DNA fragmentation via caspase-7 activation. [2][3]
PBOX-6 causes nucleocytoplasmic redistribution of peptidyl-prolyl isomerases cyclophilin A and pin1, which are overexpressed in hematological malignancies. This redistribution correlates with mitotic arrest and precedes cell death. [3]
PBOX-6 is a pyrrolo-1,5-benzoxazepine (PBOX) compound that acts as a microtubule-depolymerizing agent and a potent proapoptotic agent. It exhibits significant anticancer and antitumor activity, inhibiting breast cancer cell growth in vitro and selectively inducing apoptosis in leukemia cells via JNK-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL. The compound binds to a tubulin site distinct from vinblastine and colchicine. It has a molecular weight of 396.44 g/mol and formula C25H20N2O3. The CAS number is 290814-68-5. No approved marketing status has been identified for this research compound.

C25H20N2O3

396.437906265259

396.147

290814-68-5

9865511

White to off-white solid powder

1.2±0.1 g/cm3

580.6±50.0 °C at 760 mmHg

305.0±30.1 °C

0.0±1.6 mmHg at 25°C

1.640

5.69

0

3

3

30

675

0

SOIZAFVNIXAZFQ-UHFFFAOYSA-N

InChI=1S/C25H20N2O3/c1-26(2)25(28)30-24-21-14-8-16-27(21)20-13-5-6-15-22(20)29-23(24)19-12-7-10-17-9-3-4-11-18(17)19/h3-16H,1-2H3

(6-naphthalen-1-ylpyrrolo[2,1-d][1,5]benzoxazepin-7-yl) N,N-dimethylcarbamate

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 : ~33.33 mg/mL (~84.07 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.31 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.

---

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