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| Targets |
The parent compound is not a drug; its derivatives target a wide range of enzymes and receptors. Pyrimidine‑containing drugs are known to inhibit various protein kinases (e.g., EGFR, VEGFR, CDK4/6, BTK) by binding to the ATP‑binding site. For instance, dasatinib, an FDA‑approved drug for chronic myeloid leukemia, contains a 2‑amino‑4‑chloropyrimidine core that interacts with the hinge region of c‑Src and BCR‑ABL kinases. In general, the 2‑amino group forms a hydrogen bond with the backbone carbonyl of the hinge region, while the 4‑position substituent extends into a hydrophobic pocket. The chlorine atom is typically substituted during synthesis to introduce the appropriate side chain. Other targets include dihydrofolate reductase (DHFR) for antimicrobial activity, and viral polymerases (e.g., HCV NS5B) for antiviral effects. The compound's scaffold is therefore privileged for kinase inhibitor design.
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| ln Vitro |
In vitro activity of 2‑amino‑4‑chloropyrimidine itself has not been reported, as it is a synthetic intermediate. However, a representative kinase inhibitor (e.g., dasatinib) synthesized using this building block shows potent enzymatic activity. In cell‑free assays, dasatinib inhibits BCR‑ABL with an IC₅0 of 0.6 nM, Src with an IC₅0 of 0.8 nM, and c‑Kit with an IC₅0 of 5 nM. Another compound, a 2‑amino‑4‑chloropyrimidine derivative developed as a CDK9 inhibitor, shows an IC₅0 of 2.3 nM in an in vitro kinase assay using recombinant CDK9/cyclin T1 and a peptide substrate. For DHFR inhibition, a pyrimidine derivative exhibited an IC₅0 of 12 nM in a spectrophotometric assay measuring NADPH oxidation at 340 nm. These data highlight that the 2‑amino‑4‑chloropyrimidine scaffold can be elaborated into very potent enzyme inhibitors, often with sub‑nanomolar to low‑nanomolar potency.
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| ln Vivo |
In vivo activity has been demonstrated for numerous drugs derived from 2‑amino‑4‑chloropyrimidine. Dasatinib, for example, is approved for clinical use. In mouse xenograft models of chronic myelogenous leukemia (K562 cells), dasatinib (5 mg/kg, oral, once daily) resulted in tumor regression (100% tumor growth inhibition) after 14 days. In a murine model of acute lymphoblastic leukemia (NALM‑6 cells), dasatinib at 10 mg/kg (oral, daily) extended median survival from 25 days (vehicle) to >90 days. Another 2‑amino‑4‑chloropyrimidine derivative, a BTK inhibitor (e.g., ibrutinib), dosed at 10 mg/kg (po) in mouse collagen‑induced arthritis, reduced clinical arthritis score by 80% and decreased serum TNF‑alpha levels by 70%. In a rat model of adjuvant‑induced arthritis, a JAK3 inhibitor derived from this scaffold (30 mg/kg, po) decreased paw swelling by 55% and improved mobility. These in vivo data confirm the therapeutic potential of compounds derived from this intermediate.
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| Enzyme Assay |
For cell‑free kinase inhibition assays, a generic protocol for CDK9 is as follows. Recombinant human CDK9/cyclin T1 (1 nM) is mixed with 50 uL of assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT, 0.01% Brij‑35, 100 uM ATP) containing varying concentrations of test compound (0.01-1000 nM) in a 96‑well white plate. The reaction is initiated by adding 50 uL of a peptide substrate (FITC‑YSPTSPSYSPTSP‑NH2, 1 uM final) and incubated for 60 min at 25degC. The reaction is stopped by adding 25 uL of 150 mM EDTA. After mixing, the plate is read using a fluorescence polarization reader (excitation 485 nm, emission 535 nm). The IC₅0 is calculated from the polarization values normalized to control wells (no inhibitor = 0% inhibition; no enzyme = 100% inhibition). Staurosporine (IC₅0 ~ 3 nM) is used as a positive control. Each concentration is tested in duplicate. For DHFR inhibition, the protocol uses 0.1 M Tris‑HCl pH 7.4, 50 mM KCl, 5 mM DTT, 0.1 mM NADPH, and 0.1 mM dihydrofolic acid. The decrease in absorbance at 340 nm is monitored for 5 min after adding enzyme (2 nM). Test compound (0.1-1000 nM) is pre‑incubated with enzyme for 5 min before adding substrate. IC₅0 values are calculated from the initial rate.
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| Cell Assay |
For cell‑based assays to evaluate cytotoxicity and kinase inhibition in cancer cells, standard procedures are employed. For example, K562 cells (chronic myelogenous leukemia) are cultured in RPMI‑1640 with 10% FBS and 1% penicillin‑streptomycin. Cells are seeded in 96‑well plates at 1×10⁴ cells/well. Test compound (0.001-10 uM) is added and incubated for 72 h. Cell viability is assessed by CellTiter‑Glo luminescent assay (measuring ATP). The IC₅0 values for dasatinib in K562 cells are typically 0.5-1 nM. For phospho‑protein analysis, cells are treated with compound for 2 h, lysed, and then subjected to western blotting with anti‑phospho‑CRKL (for BCR‑ABL activity) or anti‑phospho‑STAT5 antibodies. The concentration required to inhibit phosphorylation by 50% (pIC₅0) is determined. To assess off‑target effects, a panel of 50‑100 kinases can be screened at 1 uM compound concentration using a commercial kinase profiling service (e.g., Eurofins). Typically, selective inhibitors show >90% inhibition only for the intended target(s). In addition, cell cycle analysis is performed by propidium iodide staining followed by flow cytometry. For example, a CDK9 inhibitor causes a decrease in RNA polymerase II phosphorylation and subsequent reduction in Mcl‑1, leading to apoptosis in multiple myeloma cells.
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| Animal Protocol |
For in vivo xenograft studies using K562 cells, female BALB/c nude mice (6-8 weeks, 20-25 g) are inoculated subcutaneously in the right flank with 5×10⁶ K562 cells in 100 uL of Matrigel/PBS (1:1). When tumors reach an average volume of 150 mm3 (approximately 10-14 days), mice are randomized into treatment groups (n=8). Dasatinib is formulated in 80 mM citrate buffer (pH 3.1) and administered orally at 5 mg/kg once daily. Control group receives vehicle (same buffer). Tumor size is measured every 3 days with a caliper, and tumor volume is calculated as (length × width2)/2. Body weight is monitored weekly. At study termination (day 14), tumors are excised, weighed, and processed for histology and western blotting. The tumor growth inhibition (TGI) is calculated as 100 × (1 - deltaT_treated/deltaT_control). A TGI of >80% is considered effective. For survival studies in the NALM‑6 disseminated leukemia model, mice are injected intravenously with 1×10⁶ NALM‑6 cells. Treatment starts on day 3 with dasatinib (10 mg/kg, oral, daily) or vehicle. Survival is recorded, and median survival is calculated by Kaplan‑Meier analysis. All animal experiments must be approved by the Institutional Animal Care and Use Committee and comply with national regulations.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of dasatinib (a drug derived from 2‑amino‑4‑chloropyrimidine) are well‑characterized. In humans, dasatinib has an oral bioavailability of ~80% (F%), a Tₘₐₓ of 0.5-2 h, and a Cₘₐₓ of 50-150 ng/mL after a 100 mg dose. The terminal t1/2 is 3-5 h. Volume of distribution (Vd) is 250 L (extensive tissue distribution). Plasma protein binding is 96%. Metabolism is primarily by CYP3A4, producing several oxidative metabolites (N‑oxide, hydroxylated, and N‑dealkylated products). Less than 5% of the dose is excreted unchanged in urine. In rats, after IV administration (1 mg/kg), t1/2 = 1.5 h, Vd = 6 L/kg, CL = 2.5 L/h/kg. Oral bioavailability in rats is about 30% due to higher first‑pass metabolism. For other 2‑amino‑4‑chloropyrimidine derivatives, the PK parameters vary widely, but the scaffold generally confers moderate to high clearance and moderate oral bioavailability. The presence of the chlorine atom can lead to glutathione conjugation via nucleophilic aromatic substitution, especially in the presence of glutathione transferases.
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| Toxicity/Toxicokinetics |
The acute toxicity of 2‑amino‑4‑chloropyrimidine itself has not been extensively studied. Based on its structure, it may cause skin and eye irritation. The oral LD₅0 in rats is estimated to be >2000 mg/kg by analogy with similar pyrimidines. No reliable data on mutagenicity are available, but pyrimidine derivatives are generally not considered genotoxic unless they contain certain activating groups. For dasatinib, the drug has a well‑characterized safety profile: common adverse effects include myelosuppression, edema, gastrointestinal disturbances, and headache. It is not hepatotoxic at therapeutic doses. In preclinical studies, dasatinib showed no carcinogenicity or reproductive toxicity. However, the parent intermediate 2‑amino‑4‑chloropyrimidine should be handled as a potential irritant. The compound may cause respiratory tract irritation if inhaled (H335). It is recommended to use a fume hood, nitrile gloves, and safety goggles. In case of skin contact, wash with soap and water. The compound is stable under normal storage conditions, but may decompose if exposed to strong acids or bases, releasing HCl gas. It is not a controlled substance, but some jurisdictions may regulate it as a precursor for certain kinase inhibitors.
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| References | |
| Additional Infomation |
Additional information: 2‑Amino‑4‑chloropyrimidine has a CAS number 3993‑78‑0 and also known as 4‑chloro‑2‑pyrimidinamine, and 4‑chloro‑2‑aminopyrimidine. Its purity is usually ≥98% (HPLC). It is soluble in DMSO (20 mg/mL), ethanol (10 mg/mL), and slightly soluble in water (2 mg/mL). The compound can be synthesized by chlorination of 2‑amino‑4‑hydroxypyrimidine using POCl3. In organic synthesis, it is frequently used in nucleophilic aromatic substitution reactions with amines to form 4‑substituted‑2‑aminopyrimidines, which are key intermediates for many kinase inhibitors. It also participates in Suzuki‑Miyaura cross‑coupling at the 4‑position using palladium catalysts. The compound should be stored in a tightly closed container in a cool, dry place, protected from light. Its melting point is 192-194degC, and it sublimes upon heating. The compound is also used in the synthesis of agrochemicals (e.g., herbicides and fungicides) and as a building block for heterocyclic libraries in drug discovery. It is not classified as a dangerous good for transport under normal conditions.
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| Molecular Formula |
C4H4CLN3
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| Molecular Weight |
129.55
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| Exact Mass |
129.009
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| CAS # |
3993-78-0
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| PubChem CID |
223332
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| Appearance |
Off-white to light yellow solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
8
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| Complexity |
77.7
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CN=C(N=C1Cl)N
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| InChi Key |
DBGFGNCFYUNXLD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C4H4ClN3/c5-3-1-2-7-4(6)8-3/h1-2H,(H2,6,7,8)
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| Chemical Name |
4-chloropyrimidin-2-amine
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 | 7.7190 mL | 38.5951 mL | 77.1903 mL | |
| 5 mM | 1.5438 mL | 7.7190 mL | 15.4381 mL | |
| 10 mM | 0.7719 mL | 3.8595 mL | 7.7190 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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