| Size | Price | Stock | Qty |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| 500mg |
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| Other Sizes |
| Targets |
Cl-amidine targets protein arginine deiminases (PADs), which are enzymes that catalyze the calcium-dependent conversion of arginine residues in proteins to citrulline. It inhibits all active PAD isozymes, with IC50 values of 0.8 μM for PAD1, 6.2 μM for PAD3, and 5.9 μM for PAD4. The inhibition is irreversible, as it covalently modifies the active site cysteine residue.
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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].
In vitro, Cl-amidine demonstrates potent PAD inhibitory activity. It causes dose-dependent apoptosis in TK6 lymphoblastoid cells and HT29 colon cancer cells at concentrations of 0-50 μg/mL. It can induce miR-16, causing cell cycle arrest. It also blocks histone 3 citrullination and neutrophil extracellular trap (NET) formation. Its enzymatic activity is dependent on the active site cysteine, which is covalently modified by the compound, rendering PADs permanently inactive. |
| 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, Cl-amidine has shown efficacy in preclinical models. In a DSS colitis mouse model, it prevents and cures colitis at a dose of 75 mg/kg administered intraperitoneally once daily. It significantly lowers histological scores in a dose-dependent manner (5, 25, 75 mg/kg, oral gavage, once daily). It improves survival in septic mice by blocking NET formation. These studies highlight its potential for treating inflammatory and autoimmune diseases. |
| Enzyme Assay |
The in vitro enzyme assay for Cl-amidine involves measuring its ability to inhibit PAD activity. The enzyme is incubated with a peptide substrate (e.g., benzoyl-L-arginine ethyl ester or a citrulline-specific substrate) in the presence of varying concentrations of the compound. The production of citrulline is measured using a colorimetric method or by HPLC, and the IC50 is calculated from the inhibition curve. The kinetic parameters (kinact/KI) can also be determined.
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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 cell culture studies for Cl-amidine utilize TK6 lymphoblastoid cells and HT29 colon cancer cells. Cells are treated with the compound at concentrations of 0, 5, 10, 15, 20, 25, 50 μg/mL for 24 hours. Apoptosis is assessed by flow cytometry using Annexin V/PI staining. Cell cycle analysis is performed by propidium iodide staining. The expression of citrullinated proteins is analyzed by Western blotting. |
| 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 for Cl-amidine are performed in the DSS colitis mouse model. C57BL/6 mice (8-12 weeks old) are administered the compound at doses of 5, 25, or 75 mg/kg via oral gavage or intraperitoneal injection once daily. Disease severity is assessed by measuring body weight, stool consistency, and bleeding, and by histological scoring of colon tissues. PAD activity and protein citrullination are measured in colon tissues. |
| ADME/Pharmacokinetics |
Cl-amidine has a molecular formula of C14H19ClN4O2 and a molecular weight of 310.78 g/mol. It is soluble in DMSO. It is typically stored as a powder at -20°C for long-term stability. For in vivo studies, it can be formulated in suitable vehicles such as PBS or a solution of DMSO and saline. Its stability is maintained under recommended storage conditions.
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| Toxicity/Toxicokinetics |
Toxicological data for Cl-amidine indicate that it is generally well-tolerated in preclinical studies at therapeutic doses. In vitro, it causes dose-dependent apoptosis in cancer cell lines, suggesting potential cytotoxic effects at higher concentrations. In vivo, it has been shown to be safe at doses up to 75 mg/kg in mice. As a research compound, it is not intended for human use.
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| References |
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| Additional Infomation |
Cl-amidine belongs to the benzene family of compounds.
Cl-amidine is a research compound with no clinical approval. It is a valuable tool for studying PAD biology and the role of citrullination in various diseases. Its ability to inhibit NET formation and improve survival in septic mice makes it a compound of interest for research into sepsis and other inflammatory diseases. It is also used to explore the therapeutic potential of PAD inhibitors in cancer. |
| Molecular Formula |
C14H19N4O2CL
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|---|---|
| Molecular Weight |
310.779
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| Exact Mass |
310.119
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| CAS # |
913723-61-2
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| Related CAS # |
Cl-amidine hydrochloride;1373232-26-8;D-Cl-amidine hydrochloride;Cl-amidine TFA;1043444-18-3;D-Cl-amidine;1404060-15-6
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| PubChem CID |
24970878
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
0.518
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
21
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| Complexity |
381
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C(N[C@H](C(N)=O)CCCNC(CCl)=N)C1=CC=CC=C1
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| InChi Key |
BPWATVWOHQZVRP-NSHDSACASA-N
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| InChi Code |
InChI=1S/C14H19ClN4O2/c15-9-12(16)18-8-4-7-11(13(17)20)19-14(21)10-5-2-1-3-6-10/h1-3,5-6,11H,4,7-9H2,(H2,16,18)(H2,17,20)(H,19,21)/t11-/m0/s1
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| Chemical Name |
N-[(2S)-1-amino-5-[(1-amino-2-chloroethylidene)amino]-1-oxopentan-2-yl]benzamide
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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 | 3.2177 mL | 16.0886 mL | 32.1771 mL | |
| 5 mM | 0.6435 mL | 3.2177 mL | 6.4354 mL | |
| 10 mM | 0.3218 mL | 1.6089 mL | 3.2177 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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