| Size | Price | Stock | Qty |
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| 5mg |
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| 500mg | |||
| 1g | |||
| Other Sizes |
| Targets |
Cl-amidine TFA targets peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes that catalyze the post-translational conversion of arginine residues to citrulline (citrullination). The compound inhibits PAD1, PAD3, and PAD4 with IC50 values of 0.8 μM, 6.2 μM, and 5.9 μM, respectively. PAD4 is the most extensively studied isoform, with an IC50 of 5.9±0.3 μM. By inhibiting PADs, Cl-amidine TFA blocks protein citrullination, which plays critical roles in inflammation, autoimmune diseases, and cancer. The compound’s mechanism involves covalent modification of the active site cysteine residue of PAD enzymes.
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| ln Vitro |
Cl-amidine is a bioavailable haloacetamidine-based drug that inhibits all active PAD isozymes with nearly identical efficacy (kinact/KI=13,000 M-1·min-1 for PAD4) [1]. TK6 lymphoblastoid cells and HT29 colon cancer cells underwent dose-dependent apoptosis in response to Cl-amidine (0, 5, 10, 15, 20, 25, 50 μg/mL, 24 h). It's interesting to note that Cl-amidine-induced apoptosis does not fully kill the colon cancer cell line HT29 [2]. The enzymatic activity of PADs is dependent on the active site cysteine, which is covalently modified by clonidine, rendering PADs permanently inactive [4].
Cl-amidine TFA exhibits potent in vitro activity as a PAD inhibitor. It inhibits all active PAD isozymes with nearly identical efficacy (kinact/KI=13,000 M-1·min-1 for PAD4). The compound causes apoptosis in cancer cells and tumor cells. In cellular assays, Cl-amidine TFA prevents histone 3 citrullination and neutrophil extracellular trap (NET) formation. It has been shown to suppress colitis in mouse models and improve survival in murine sepsis models. These in vitro activities confirm its potential as a therapeutic agent for inflammatory and autoimmune diseases, as well as cancer. |
| ln Vivo |
Cl-amidine (75 mg/kg, intraperitoneal injection once daily) can prevent and cure colitis in mice produced by DSS [2]. Histological scores can be significantly lowered in a dose-dependent manner with clonidine (5, 25, 75 mg/kg, oral gavage, once daily) [2].
In vivo activity of Cl-amidine TFA has been demonstrated in animal models of inflammatory diseases. The compound suppresses colitis in mice, indicating efficacy in gastrointestinal inflammation. In a murine sepsis model, Cl-amidine TFA improves survival, suggesting protective effects against systemic inflammation. The compound is orally active, making it suitable for convenient administration in preclinical studies. Its ability to prevent histone 3 citrullination and NET formation in vivo supports its mechanism of action and therapeutic potential. Further studies have explored its effects in various inflammatory and autoimmune disease models. |
| Enzyme Assay |
In vitro enzyme assays for Cl-amidine TFA involve measuring its inhibition of PAD enzyme activity. These assays typically use recombinant PAD1, PAD3, or PAD4 and a fluorescent or colorimetric substrate. The enzyme is incubated with the substrate and varying concentrations of the compound. The decrease in substrate conversion is measured to determine the IC50 values. The inhibition kinetics (kinact/KI) are calculated to characterize the mechanism of covalent inhibition. These assays confirm the compound’s potency and selectivity for PAD isozymes.
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| Cell Assay |
Apoptosis analysis[2].
Cell Types: TK6 lymphoblastoid cells and HT29 colon cancer cells. Tested Concentrations: 0, 5, 10, 15, 20, 25, 50 μg/mL. Incubation Duration: 24 hrs (hours). Experimental Results: The induction of apoptosis was dose-dependent. In vitro cellular assays for Cl-amidine TFA are conducted in various cell types, including cancer cells and immune cells. Cells are treated with the compound at various concentrations, and protein citrullination is measured by Western blot using anti-citrulline antibodies. Apoptosis is assessed by Annexin V staining, caspase activity assays, or PARP cleavage. Neutrophil extracellular trap (NET) formation is measured in neutrophils. Cytokine production and inflammatory markers are assessed by ELISA or qPCR. These assays characterize the compound’s cellular effects and its mechanism of action as a PAD inhibitor. |
| Animal Protocol |
Animal/Disease Models: C57BL/6 mice (8-12 weeks old, DSS colitis mouse model) [2].
Doses: 75 mg/kg. Management: IP one time/day. Experimental Results: Inhibition of PAD activity in vivo, protein citrullination, and PAD levels in the colon. Animal/Disease Models: C57BL/6 mice (8-12 weeks old, DSS colitis mouse model) [2]. Doses: 5, 25, 75 mg/kg. Route of Administration: po (oral gavage), one time/day. Experimental Results: Resultant in Dramatically lower histological scores. In vivo animal experiments with Cl-amidine TFA are conducted in rodent models of inflammatory diseases. Colitis models are used to assess the compound’s efficacy in gastrointestinal inflammation. Sepsis models are used to evaluate survival benefits. The compound is administered orally or by injection at varying doses. Endpoints include disease severity scores, histological analysis of tissues, and measurement of inflammatory markers. Protein citrullination is assessed in tissues to confirm target engagement. These studies define the compound’s in vivo efficacy and its potential for treating inflammatory and autoimmune diseases. |
| ADME/Pharmacokinetics |
Pharmacokinetic data for Cl-amidine TFA indicate that the compound is orally active and bioavailable. It has a molecular weight of 424.80 and is soluble in DMSO. The compound’s absorption, distribution, metabolism, and excretion (ADME) properties have been characterized in preclinical studies. Its oral bioavailability supports convenient administration in animal models. The compound is typically stored at -20°C and is stable under recommended conditions. Further detailed PK studies may be available in the literature.
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| Toxicity/Toxicokinetics |
Cl-amidine TFA has been evaluated for safety in preclinical studies. The compound is generally well-tolerated at therapeutic doses. In animal models, it has been administered without significant adverse effects reported at efficacious doses. However, comprehensive toxicological evaluations have not been extensively published. The compound is intended for research use only and is not approved for human therapeutic use. Standard laboratory safety precautions should be followed when handling this compound. Further toxicity studies would be required to support any potential clinical development.
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| References |
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| Additional Infomation |
Cl-amidine TFA is a potent, orally active peptidylarginine deiminase (PAD) inhibitor. It inhibits PAD1, PAD3, and PAD4 with IC50 values of 0.8 μM, 6.2 μM, and 5.9 μM, respectively. The compound causes apoptosis in cancer cells and prevents histone 3 citrullination and NET formation. It has shown efficacy in suppressing colitis and improving survival in sepsis models. Cl-amidine TFA is also known as (2S)-5-(2-Chloroethanimidamido)-2-(phenylformamido)pentanamide trifluoroacetic acid salt. It is available in high purity (98%) for research applications in inflammation, immunology, and cancer biology.
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| Molecular Formula |
C₁₆H₂₀CLF₃N₄O₄
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|---|---|
| Molecular Weight |
424.80
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| Exact Mass |
424.112
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| CAS # |
1043444-18-3
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| Related CAS # |
Cl-amidine hydrochloride;1373232-26-8;D-Cl-amidine hydrochloride;Cl-amidine;913723-61-2
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| PubChem CID |
57402549
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| Appearance |
Typically exists as solid at room temperature
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
28
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| Complexity |
465
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| Defined Atom Stereocenter Count |
1
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| SMILES |
ClC/C(/N)=N\CCC[C@@H](C(N)=O)NC(C1C=CC=CC=1)=O.FC(C(=O)O)(F)F
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| InChi Key |
WUSNMVYWOLUWDD-MERQFXBCSA-N
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| InChi Code |
InChI=1S/C14H19ClN4O2.C2HF3O2/c15-9-12(16)18-8-4-7-11(13(17)20)19-14(21)10-5-2-1-3-6-10;3-2(4,5)1(6)7/h1-3,5-6,11H,4,7-9H2,(H2,16,18)(H2,17,20)(H,19,21);(H,6,7)/t11-;/m0./s1
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| Chemical Name |
N-[(2S)-1-amino-5-[(1-amino-2-chloroethylidene)amino]-1-oxopentan-2-yl]benzamide;2,2,2-trifluoroacetic acid
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| Synonyms |
Clamidine TFA Cl amidine TFA
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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 | 2.3540 mL | 11.7702 mL | 23.5405 mL | |
| 5 mM | 0.4708 mL | 2.3540 mL | 4.7081 mL | |
| 10 mM | 0.2354 mL | 1.1770 mL | 2.3540 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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