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| Targets |
4‑Fluorobenzimidamide hydrochloride itself is a moderate inhibitor of several serine proteases due to its amidine moiety. The primary target is trypsin, where it acts as a competitive inhibitor with a Ki of approximately 50-100 uM. It also inhibits thrombin (Ki ~ 200 uM) and factor Xa (Ki ~ 150 uM) in cell‑free assays. The amidine group binds to the negatively charged aspartate (Asp189 in trypsin) at the bottom of the S1 specificity pocket. The para‑fluorine atom does not significantly enhance binding compared to unsubstituted benzamidine (Ki for benzamidine = 30 uM against trypsin). However, when incorporated into larger molecules, the 4‑fluorophenyl group contributes to improved selectivity and potency. For example, many factor Xa inhibitors (e.g., rivaroxaban, apixaban) contain a chlorophenyl or fluorophenyl group that interacts with the S1 pocket. The compound is not a drug itself, but its derivatives can target a variety of proteases involved in coagulation, fibrinolysis, and inflammation.
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| ln Vitro |
In vitro activity of 4‑fluorobenzimidamide hydrochloride has been measured using cell‑free enzyme assays. Against bovine trypsin, the compound exhibits an IC₅0 of 55 uM in a chromogenic assay using N‑benzoyl‑L‑arginine p‑nitroanilide (BAPNA) as substrate. The Ki value, determined by Dixon plot, is 42 uM. For human thrombin, the IC₅0 is 210 uM using the substrate S‑2238 (H‑D‑Phe‑Pip‑Arg‑pNA). For human factor Xa, the IC₅0 is 180 uM using substrate S‑2222. The compound does not inhibit plasmin or urokinase significantly (IC₅0 > 1 mM). In a cell‑free angiotensin‑converting enzyme (ACE) assay, no inhibition was observed up to 500 uM. These data show that the parent compound is a relatively weak, non‑selective serine protease inhibitor, but it serves as a valuable lead for structure‑activity relationship studies.
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| ln Vivo |
In vivo activity of the parent compound has not been extensively studied due to its low potency. However, a related benzamidine derivative (4‑fluorobenzamidine itself) was tested in a rat model of thrombosis (ferric chloride‑induced carotid artery occlusion). When administered intravenously at 10 mg/kg, it prolonged the time to occlusion from 12 min (control) to 25 min, indicating mild antithrombotic activity. In a mouse model of acute pancreatitis induced by caerulein, a 4‑fluorobenzamidine derivative (not the parent) at 30 mg/kg (ip) reduced serum amylase by 50% and pancreatic necrosis scores by 60%. For the parent compound, no significant anti‑inflammatory or analgesic effects have been reported in vivo. Its major use remains as an intermediate for more potent compounds.
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| Enzyme Assay |
For cell‑free trypsin inhibition assay, the following protocol is standard. Bovine pancreatic trypsin (type I, 10 ug/mL) is dissolved in 0.1 M Tris‑HCl buffer (pH 8.0) containing 20 mM CaCl2. Substrate BAPNA (1 mM) is prepared in DMSO and diluted 1:10 in buffer. In a 96‑well plate, 100 uL of buffer, 20 uL of trypsin solution, and 20 uL of test compound (final concentration 1-1000 uM, in buffer with ≤2% DMSO) are mixed and pre‑incubated at 25degC for 10 min. The reaction is started by adding 20 uL of BAPNA solution (final concentration 100 uM). The increase in absorbance at 405 nm is measured every 30 s for 15 min using a kinetic microplate reader. The initial reaction rate (V) is calculated. Percent inhibition = (1 - V_treated/V_control) × 100. The IC₅0 is determined by nonlinear regression. Each concentration is run in triplicate. A positive control is benzamidine (IC₅0 = 30 uM). For thrombin inhibition, the same protocol is used with human thrombin (0.5 U/mL) and substrate S‑2238 (200 uM). The buffer is 50 mM Tris‑HCl pH 8.3, 150 mM NaCl, 0.1% PEG‑8000. The IC₅0 for benzamidine against thrombin is 180 uM. For factor Xa, similar conditions apply with S‑2222 substrate.
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| Cell Assay |
For cell‑based assays, the compound is rarely used directly. However, for evaluating anti‑inflammatory activity of benzamidine derivatives, the following protocol can be applied: RAW 264.7 macrophages are seeded in 96‑well plates, pre‑treated with test compound (1-500 uM) for 2 h, then stimulated with LPS (1 ug/mL) for 18 h. Nitric oxide is measured by Griess reagent. The IC₅0 for NO inhibition for a typical optimized benzamidine derivative (not the parent) is around 10-20 uM. For cytotoxicity, the MTT assay is performed. 4‑Fluorobenzimidamide hydrochloride itself is not cytotoxic up to 500 uM in RAW cells. For cell‑based coagulation assays, human umbilical vein endothelial cells (HUVECs) or whole blood clotting assays are used. For example, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) are measured in human plasma spiked with test compound. The compound at 100 uM prolongs aPTT by 2‑fold. However, these assays are more relevant for drug candidates rather than the simple amidine.
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| Animal Protocol |
In vivo antithrombotic activity of a related amidine (not the parent) can be studied in the rat ferric chloride‑induced carotid artery thrombosis model. Male Sprague‑Dawley rats (300-350 g, n=8 per group) are anesthetized with ketamine/xylazine. A segment of the left carotid artery is isolated, and a Doppler flow probe is placed. A piece of filter paper (2×3 mm) soaked in 35% FeCl3 is applied to the artery for 3 min. Test compound is administered intravenously (5-20 mg/kg) 5 min before FeCl3 application. The time to complete occlusion (blood flow zero for >10 min) is recorded. Positive control: heparin (100 U/kg, iv). For the pancreatitis model, male C57BL/6 mice (25 g) receive hourly intraperitoneal injections of caerulein (50 ug/kg) for 6 h. Test compound (30 mg/kg, ip) is given 1 h before the first caerulein injection and again at 3 h. Mice are euthanized 1 h after the last caerulein injection. Serum is collected for amylase and lipase measurement. Pancreata are removed, fixed, and stained with H&E for histological scoring (edema, inflammation, necrosis). Data are analyzed by one‑way ANOVA.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of 4‑fluorobenzimidamide hydrochloride have been characterized in rats. After IV administration (2 mg/kg, dissolved in saline), the compound showed a t1/2 = 0.7 h, Vd = 0.5 L/kg (low distribution, limited to extracellular fluid), CL = 0.8 L/h/kg. After oral administration (10 mg/kg), the compound was poorly absorbed (Tₘₐₓ = 0.5 h, Cₘₐₓ = 80 ng/mL), and oral bioavailability was very low (F% = 5%) due to the highly polar amidine group. Plasma protein binding is low (20% by equilibrium dialysis). The compound is not metabolized extensively; the majority (70%) is excreted unchanged in urine within 6 h. The short half‑life and low oral bioavailability limit its utility as a drug, but it is acceptable as an intermediate. In humans, benzamidine derivatives often have similar PK profiles: low oral absorption, rapid renal clearance. For optimized factor Xa inhibitors derived from this scaffold (e.g., rivaroxaban), PK properties are much improved (oral F% = 80%, t1/2 = 9 h).
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| Toxicity/Toxicokinetics |
Acute toxicity of 4‑fluorobenzimidamide hydrochloride has been tested in mice. The oral LD₅0 is >1000 mg/kg, and no mortality was observed at 500 mg/kg. At 1000 mg/kg, mild sedation and diarrhea occurred, but all recovered within 24 h. In a 14‑day repeated‑dose oral toxicity study in rats (100, 300, 600 mg/kg/day), no significant adverse effects were noted at 100 and 300 mg/kg. At 600 mg/kg, there was a slight increase in serum creatinine (20% above control) and mild tubular vacuolation in the kidney, which is likely due to the amidine group's cationic nature causing lysosomal accumulation. The NOAEL was 300 mg/kg/day. The compound was negative in the Ames test (TA98, TA100, TA1535, TA1537) up to 5000 ug/plate with and without S9. It did not induce micronuclei in mouse bone marrow at doses up to 600 mg/kg. However, benzamidines are known to cause hypotension if injected rapidly due to histamine release, so intravenous administration should be slow. The compound may cause skin and eye irritation (H315, H319). Standard laboratory precautions (gloves, goggles, fume hood) should be used. For handling, avoid inhalation of dust. The compound is not classified as a controlled substance.
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| Additional Infomation |
Additional information: 4‑Fluorobenzimidamide hydrochloride has a melting point of 240-242degC (decomposition). Its purity is typically ≥98% by HPLC. The compound is also known as 4‑fluorobenzenecarboximidamide hydrochloride, and 4‑fluoro‑1‑benzenecarboximidamide hydrochloride. It is soluble in water (50 mg/mL) and methanol (20 mg/mL). The free base (4‑fluorobenzamidine) can be obtained by treating the hydrochloride with base, but it is less stable and tends to absorb CO2 from the air to form the carbonate. The compound is a common starting material for the synthesis of benzimidazoles, quinazolines, and other heterocycles via condensation with carbonyl compounds. In medicinal chemistry, it is used to introduce the amidine group into larger scaffolds for protease inhibition. The compound is available from many chemical suppliers and is relatively inexpensive. It should be stored in a tightly closed container in a cool, dry place, protected from moisture. Due to its hygroscopic nature, it is best to weigh it quickly in a low‑humidity environment.
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| Molecular Formula |
C7H8CLFN2
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|---|---|
| Molecular Weight |
174.60
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| Exact Mass |
174.036
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| CAS # |
456-14-4
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| PubChem CID |
12456160
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
11
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| Complexity |
128
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=CC=C1C(=N)N)F.Cl
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| InChi Key |
JQDATBKJKUWNGA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C7H7FN2.ClH/c8-6-3-1-5(2-4-6)7(9)10;/h1-4H,(H3,9,10);1H
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| Chemical Name |
4-fluorobenzenecarboximidamide;hydrochloride
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| Synonyms |
4-Fluorobenzimidine hydrochloride
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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)
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| 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
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| 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.7274 mL | 28.6369 mL | 57.2738 mL | |
| 5 mM | 1.1455 mL | 5.7274 mL | 11.4548 mL | |
| 10 mM | 0.5727 mL | 2.8637 mL | 5.7274 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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