Sulfaphenazole (RN-0308518; Firmazolo; Inamil; BRN 0308518), an antibiotic of the sulfonamide class, is a potent and specific inhibitor of CYP2C9 that can block atherogenic and pro-inflammatory effects of linoleic acid (increase in oxidative stress and activation of AP-1) mediated by CYP2C9. Treatment of intracerebral hemorrhage is often pointless, although considerable effort has been devoted to developing treatments for ischemic stroke. Sulfaphenazole (SPZ) has a different mechanism such as reactive oxygen species scavenging, in addition to the inhibition of superoxide production by cytochrome P450. Systemic SPZ treatment after intracerebral hemorrhage reduces striatal dysfunction, the elevation of lipid peroxidation, and brain edema in the rat. These results suggest that SPZ is a potentially effective therapeutic approach for intracerebral hemorrhage as the effect of SPZ was initiated for either 1 h or 3 d post-intracerebral hemorrhage.
Physicochemical Properties
| Molecular Formula | C15H14N4O2S | |
| Molecular Weight | 314.36 | |
| Exact Mass | 314.083 | |
| Elemental Analysis | C, 57.31; H, 4.49; N, 17.82; O, 10.18; S, 10.20 | |
| CAS # | 526-08-9 | |
| Related CAS # |
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| PubChem CID | 5335 | |
| Appearance | White to yellow solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 541.9±56.0 °C at 760 mmHg | |
| Melting Point | 179-183ºC | |
| Flash Point | 281.5±31.8 °C | |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C | |
| Index of Refraction | 1.684 | |
| LogP | 1.52 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 4 | |
| Heavy Atom Count | 22 | |
| Complexity | 451 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | S(C1C([H])=C([H])C(=C([H])C=1[H])N([H])[H])(N([H])C1=C([H])C([H])=NN1C1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O |
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| InChi Key | QWCJHSGMANYXCW-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C15H14N4O2S/c16-12-6-8-14(9-7-12)22(20,21)18-15-10-11-17-19(15)13-4-2-1-3-5-13/h1-11,18H,16H2 | |
| Chemical Name | 4-amino-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide | |
| Synonyms | BRN-0308518; BRN0308518; BRN 0308518; Sulfaphenazole; Firmazolo; sulfaphenazole; 526-08-9; Sulphaphenazole; Sulfabid; Sulfafenazol; Sulfaphenazon; 4-Amino-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide; Plisulfan; Inamil | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | CYP2C9 |
| ln Vitro | Sulfaphenazole (10 μM) reduces the number of light-induced necrotic and apoptotic cells by 44% and 33%, respectively, over the course of one hour[1]. |
| ln Vivo |
In diabetic mice, sulfaphenazole (5.13 mg/kg) administered intraperitoneally once a day for eight weeks restores endothelium-mediated relaxation. Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice[2]. Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion[3]. Effects of sulfaphenazole after collagenase-induced experimental intracerebral hemorrhage in rats[4]. |
| Enzyme Assay |
Enzymatic Assay of CYP2C9-GFP Fusion Protein [1] A cell-based luminogenic P450-Glo assay was used to detect the enzymatic activity of CYP2C9-GFP fusion protein in pCMV6-CYP2C9-GFP stably transfected 661W cells. The assay is based on CYP2C9, which catalyzes a selective, cell-permeable proluciferin substrate (luciferin-H) to generate luminogenic signal for detection. Endogenous cellular NADPH is provided as the electron donor for the reaction. Essentially, 661W cells were seeded on 96-well plates (5 × 103 cells/well) overnight in regular culture medium. The next day, the cells in some wells were pretreated with sulfaphenazole (10 μm) for 2 h at 37 °C. Substrate incubation, enzymatic assay, and luminescence detection all followed the manufacturer's protocol. Mock pCMV6-GFP stably transfected 661W cells and wild-type 661W cells were normal controls. Cell-free empty wells with luciferin-H were blank controls. Enzymatic activity was expressed as luminescence (relative light units)/h/5 × 103 cells after subtraction of background luminescence from blank controls. Reactive Oxygen Species and Lipid Peroxidation Assays [1] Cellular hydroxyl radical (•OH) and superoxide anion (O2⨪) were detected using the oxidation-sensitive fluorescent dyes chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (488-nm excitation/535-nm emission) and dihydroethidium (495-nm excitation/595-nm emission) (Invitrogen), respectively. Light-exposed 661W cells on 96-well plates for different durations (15 min to 2 h) with/without sulfaphenazole pretreatment and control cells in the dark were washed with PBS. The fluorescent dyes (1 μm for each) predissolved in PBS were then added into the cells with incubation at 37 °C for 30 min. After incubation, the cells were washed again with PBS. Fluorescence was recorded with a microplate reader. In some experiments, the cells were also examined with a fluorescence microscope to evaluate consistency of the results. |
| Cell Assay |
Secondary Validation of Sulfaphenazole [1] Confluent 661W cells on 96-well plates were treated with serially diluted (0.625–100 μm) sulfaphenazole at 37 °C for 2 h. Minocycline and l-sulforaphane were co-tested separately at equivalent concentrations at the same time. Cells were then exposed to light for 4–6 h after which cell viability was detected using CellTiter-Glo, which measures cellular ATP according to the manufacturer's protocol. Luminescence was detected with a microplate reader at an integration time of 0.25 s/well. |
| Animal Protocol |
Animal Model: Diabetic male mice (db/db strain)[2] Dosage: 5.13 mg/kg Administration: Intraperitoneal injections; daily; for 8 weeks Result: In diabetic mice, sulfaphenazole (5.13 mg/kg) administered intraperitoneally once a day for eight weeks restores endothelium-mediated relaxation[2]. Sulfaphenazole (SPZ) has a different mechanism such as reactive oxygen species scavenging, in addition to the inhibition of superoxide production by cytochrome P450. The present study investigated the properties of SPZ in collagenase-induced intracerebral hemorrhage rat brain damage. The results show that systemic SPZ treatment after intracerebral hemorrhage reduces striatal dysfunction, the elevation of lipid peroxidation, and brain edema in the rat. These results suggest that SPZ is a potentially effective therapeutic approach for intracerebral hemorrhage as the effect of SPZ was initiated for either 1 h or 3 d post-intracerebral hemorrhage.[4] |
| ADME/Pharmacokinetics |
Metabolism / Metabolites Hepatic. |
| Toxicity/Toxicokinetics |
rat LD50 subcutaneous 900 mg/kg Drugs in Japan, 6(387), 1982 rat LD50 intravenous 525 mg/kg Drugs in Japan, 6(387), 1982 mouse LD50 oral 3016 mg/kg Minerva Medica., 52(1789), 1961 mouse LD50 subcutaneous 660 mg/kg Drugs in Japan, 6(387), 1982 mouse LD50 intravenous 470 mg/kg Drugs in Japan, 6(387), 1982 |
| References |
[1]. Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors. J Biol Chem. 2014 Mar 21;289(12):8337-52. [2]. Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice. Vascul Pharmacol. 2008 Jan;48(1):1-8. [3]. Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion. Sci Rep. 2022 Jul 23;12(1):12622. [4]. Effects of sulfaphenazole after collagenase-induced experimental intracerebral hemorrhage in rats. Biol Pharm Bull.2012;35(10):1849-53. |
| Additional Infomation |
Sulfaphenazole is a sulfonamide that is sulfanilamide in which the sulfonamide nitrogen is substituted by a 1-phenyl-1H-pyrazol-5-yl group. It is a selective inhibitor of cytochrome P450 (CYP) 2C9 isozyme, and antibacterial agent. It has a role as an antibacterial drug, an EC 1.14.13.181 (13-deoxydaunorubicin hydroxylase) inhibitor, an EC 1.14.13.67 (quinine 3-monooxygenase) inhibitor and a P450 inhibitor. It is a substituted aniline, a sulfonamide, a member of pyrazoles, a primary amino compound and a sulfonamide antibiotic. Sulfaphenazole is a sulfonamide antibacterial. Sulfaphenazole is a long-acting sulfonamide antibiotic used in the treatment of leprosy. A sulfonilamide anti-infective agent. Drug Indication For the treatment bacterial infections. Mechanism of Action Sulfaphenazole is a sulfonamide antibacterial. In bacteria, antibacterial sulfonamides act as competitive inhibitors of the enzyme dihydropteroate synthetase (DHPS), an enzyme involved in folate synthesis. As such, the microorganism will be "starved" of folate and die. |
Solubility Data
| Solubility (In Vitro) |
DMSO : 63~100 mg/mL ( 200.4~318.11 mM ) Ethanol : ~20 mg/mL |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.62 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.62 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 20.8 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (6.62 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.08 mg/mL (6.62 mM) (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1811 mL | 15.9053 mL | 31.8107 mL | |
| 5 mM | 0.6362 mL | 3.1811 mL | 6.3621 mL | |
| 10 mM | 0.3181 mL | 1.5905 mL | 3.1811 mL |