| Targets |
MMP-2 12 nM (IC50) MMP-9 16 nM (IC50) MMP-14 17 nM (IC50) MMP-1 974 nM (IC50) MMP-3 >1000 nM (IC50) MMP-7 795 nM (IC50)
BPHA targets MMP‑2, MMP‑9, and MMP‑14 with high affinity. The half‑maximal inhibitory concentrations (IC₅₀) are 12 nM for MMP‑2, 16 nM for MMP‑9, and 17 nM for MMP‑14. It is highly selective: IC₅₀ values for MMP‑1, MMP‑3, and MMP‑7 are 974 nM, >1000 nM, and 795 nM, respectively. BPHA does not inhibit aspartic proteases (e.g., HIV‑1 protease), cysteine proteases (cathepsins B and L), or serine proteases (neutrophil elastase, plasmin, trypsin) at relevant concentrations. |
|---|---|
| ln Vitro |
Aspartic proteinase (HIV-1 protease), cysteine proteinases (cathepsins B and L), metalloproteinases (aminopeptidase M), and typical serine proteinases (neutrophil elastase, plasmin, trypsin, and chymotrypsin) are not inhibited by BPHA[1].
In vitro, BPHA exhibits potent enzyme inhibition against MMP‑2, MMP‑9, and MMP‑14 with nanomolar IC₅₀ values. Its selectivity profile is confirmed by counter‑screening against a broad panel of proteases, where no significant inhibition is observed for non‑MMP enzymes. The compound is a competitive inhibitor with respect to the substrate, and its activity is concentration‑dependent in fluorometric substrate cleavage assays. BPHA effectively blocks gelatinolytic activity in zymography experiments using purified enzymes or cell‑conditioned media. |
| ln Vivo |
Mice given 200 mg/kg BPHA orally once a day show significant suppression of liver metastasis, tumor-induced angiogenesis, and original tumor growth. In a B16-BL6 melanoma and an F2 hemangio-endothelioma model, the growth inhibition activity of BPHA is 48% and 45%, respectively[1].
In vivo, BPHA demonstrates oral bioavailability and antitumor efficacy in mouse models. In B16‑BL6 melanoma and F2 vascular endothelial cell tumor models, daily oral administration at 200 mg/kg significantly suppresses tumor‑induced angiogenesis, primary tumor growth, and liver metastasis, achieving growth inhibition rates of 48% and 45%, respectively. These effects are attributed to reduced extracellular matrix degradation and impaired tumor cell invasion, confirming the compound’s potential as a pharmacological tool for studying MMP‑dependent pathological processes. |
| Enzyme Assay |
The in vitro enzyme inhibition assay for BPHA typically uses recombinant human MMP‑2, MMP‑9, or MMP‑14 and a fluorogenic peptide substrate (e.g., Mca‑PLGL‑Dpa‑AR‑NH₂). The enzyme is pre‑incubated with varying concentrations of BPHA (0.001–100 µM) in assay buffer (50 mM Tris‑HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij‑35) at 37 °C for 15 minutes. The substrate is added to initiate the reaction, and fluorescence (excitation 320 nm, emission 405 nm) is monitored continuously. IC₅₀ values are calculated from the dose‑response curves.
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| Cell Assay |
For in vitro cell‑based assays, cancer cell lines (e.g., HT1080 or MDA‑MB‑231) are cultured in DMEM with 10% FBS and treated with BPHA at concentrations ranging from 0.1 to 100 µM for 24–72 hours. MMP activity in conditioned media is assessed by gelatin zymography or fluorogenic substrate assays. Cell proliferation is measured by MTT or CCK‑8, while invasion and migration are evaluated using Transwell chambers coated with Matrigel. Apoptosis is detected by Annexin V/PI staining. All experiments include vehicle controls and appropriate positive inhibitors for validation.
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| Animal Protocol |
In vivo animal studies are performed in immunocompetent or immunodeficient mice bearing subcutaneous or orthotopic tumors (e.g., B16‑BL6 melanoma or F2 tumors). BPHA is administered orally at doses of 50–200 mg/kg daily for 2–4 weeks. Tumor volume is measured with calipers twice weekly. At study endpoint, tumors are excised for histology (H&E, CD31 for angiogenesis) and MMP activity analysis. Liver metastases are counted. Plasma and tissue samples are collected for pharmacokinetic and pharmacodynamic assessments.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of BPHA have been partially characterized. The compound is orally bioavailable, with measurable plasma concentrations after oral gavage. Its molecular weight is 396.46 g/mol (C₂₁H₂₀N₂O₄S). Detailed parameters such as half‑life, Cmax, and protein binding have not been fully reported in public literature, but the effective oral dose (200 mg/kg) indicates reasonable systemic exposure. The compound is soluble in DMSO and should be stored as a dry powder at –20 °C. For in vivo use, it is formulated in suitable vehicles (e.g., 0.5% carboxymethylcellulose).
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| Toxicity/Toxicokinetics |
Toxicology data for BPHA are limited to preclinical observations. In mouse studies at the efficacious dose (200 mg/kg/day), no overt signs of toxicity or significant body weight loss were reported, suggesting a reasonable safety margin. However, comprehensive toxicological profiling (including genotoxicity, chronic toxicity, and organ histopathology) has not been systematically conducted. As with all MMP inhibitors, potential off‑target effects on physiological tissue remodeling should be considered. The compound is for research purposes only and not for human use.
|
| References |
[1]. R Maekawa, et al. Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor. Cancer Res. 1999 Mar 15;59(6):1231-5.
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| Additional Infomation |
Structure in the first source
BPHA (MMP‑2/MMP‑9 Inhibitor II) is a valuable research tool for dissecting the roles of gelatinases and MT1‑MMP in cancer progression, angiogenesis, and metastasis. Its high selectivity over other MMPs and non‑MMP proteases makes it superior to broad‑spectrum inhibitors. The compound has been cited in numerous studies on tumor microenvironment and extracellular matrix remodeling. It is not approved for clinical use and is available only as a high‑purity biochemical reagent for in vitro and in vivo experimentation. |
| Exact Mass |
396.114
|
|---|---|
| CAS # |
193807-60-2
|
| PubChem CID |
9822095
|
| Appearance |
Typically exists as solid at room temperature
|
| Density |
1.3±0.1 g/cm3
|
| Index of Refraction |
1.630
|
| LogP |
3.23
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
28
|
| Complexity |
584
|
| Defined Atom Stereocenter Count |
1
|
| InChi Key |
UPCAIRKRFXQRRM-HXUWFJFHSA-N
|
| InChi Code |
InChI=1S/C21H20N2O4S/c24-21(22-25)20(15-16-7-3-1-4-8-16)23-28(26,27)19-13-11-18(12-14-19)17-9-5-2-6-10-17/h1-14,20,23,25H,15H2,(H,22,24)/t20-/m1/s1
|
| Chemical Name |
(2R)-N-hydroxy-3-phenyl-2-[(4-phenylphenyl)sulfonylamino]propanamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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