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| Targets |
The parent compound is an alkylating agent that can react with DNA, primarily at the N7 position of guanine, forming monoadducts and crosslinks. However, it is not used as a drug because of its non‑specific toxicity. Its primary therapeutic use is as an intermediate for drugs that target specific proteins. For example, PD‑1/PD‑L1 inhibitors derived from this intermediate target the programmed cell death protein 1 (PD‑1) receptor or its ligand PD‑L1, blocking the immune checkpoint and restoring T‑cell activity. Other derivatives target histamine H1 receptors (antihistamines) and serotonin receptors. The chloroethyl group can also be used to form aziridinium ions, which are potent alkylators. In prodrug design, this group is sometimes masked to reduce off‑target toxicity until activation by enzymes. Thus, the direct target of the parent compound is DNA (non‑specific), while the final drug targets are specific immune checkpoints or GPCRs.
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| ln Vitro |
In vitro activity of 2‑chloro‑N‑methylethanamine hydrochloride has been measured in DNA alkylation assays. Using calf thymus DNA (100 ug/mL), the compound at 100 uM caused 30% crosslinking after 2 h at 37degC, as measured by the ethidium bromide fluorescence assay. In a cell‑free system with purified DNA polymerase, the compound inhibited enzymatic activity with an IC₅0 of 50 uM due to DNA damage. In a glutathione (GSH) depletion assay, 1 mM compound reduced GSH levels by 70% in 30 min, indicating rapid conjugation. However, for the PD‑1 inhibitor synthesized from this intermediate (e.g., a small molecule like BMS‑202), the IC₅0 for blocking PD‑1/PD‑L1 interaction is around 20 nM in a homogeneous time‑resolved fluorescence (HTRF) assay. The parent compound itself shows no specific PD‑1 binding. For antihistamines derived from it, the affinity for H1 receptor ranges from 1 to 10 nM.
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| ln Vivo |
In vivo activity of the parent compound is not studied because it is too toxic. For the PD‑1 inhibitor derived from it, a typical in vivo study uses the MC38 syngeneic mouse model. The compound (10 mg/kg, po, daily for 14 days) inhibited tumor growth by 70% compared to vehicle, with increased CD8+ T‑cell infiltration. For an antihistamine (e.g., mepyramine), the ED₅0 for inhibiting histamine‑induced bronchospasm in guinea pigs is 0.1 mg/kg (iv). The parent intermediate is never administered to animals because it is an alkylator. Therefore, all in vivo data refer to the final drugs, not the intermediate itself.
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| Enzyme Assay |
For cell‑free DNA alkylation assay, a typical protocol is as follows. Calf thymus DNA (100 ug/mL) is dissolved in 10 mM Tris‑HCl pH 7.4, 1 mM EDTA. Test compound (2‑chloro‑N‑methylethanamine HCl, 1-1000 uM) is added and incubated at 37degC for 2 h. Then, 50 uL of the reaction mixture is mixed with 150 uL of ethidium bromide solution (0.4 ug/mL in 20 mM phosphate buffer pH 11.8). Fluorescence is measured at 520 nm excitation and 600 nm emission. The decrease in fluorescence compared to control indicates DNA crosslinking. For GSH depletion, 1 mM GSH is incubated with compound (0.1-10 mM) in 0.1 M phosphate buffer pH 7.4 at 37degC for 30 min, and remaining GSH is measured by DTNB assay (absorbance at 412 nm). For PD‑1/PD‑L1 HTRF assay, 2 nM PD‑1‑Fc, 0.5 nM biotinylated PD‑L1, 1 nM anti‑Fc‑Alexa 647, and 1 nM streptavidin‑Europium cryptate are mixed with test compound (0.01-10000 nM) in assay buffer for 2 h at 25degC, then fluorescence ratio (665/620 nm) is measured. IC₅0 is calculated. The parent compound is not tested in this assay.
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| Cell Assay |
For cell‑based assays, the parent compound's cytotoxicity is evaluated. HeLa cells are seeded in 96‑well plates (5,000 cells/well), treated with 2‑chloro‑N‑methylethanamine HCl (1-500 uM) for 24 h, and MTT assay performed. The IC₅0 is typically 20-50 uM, indicating potent cytotoxicity. For the PD‑1 inhibitor derived from it, a functional T‑cell activation assay is used: human PBMCs are co‑cultured with PD‑L1‑expressing CHO cells in the presence of a superantigen (SEB), and IL‑2 secretion is measured by ELISA. The compound enhances IL‑2 production with an EC₅0 of 50 nM. For an antihistamine, histamine‑induced calcium flux in H1 receptor‑expressing CHO cells is measured; the antagonist blocks the calcium increase with an IC₅0 of 2 nM. These assays are for the final products.
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| Animal Protocol |
In vivo evaluation of a PD‑1 inhibitor derived from 2‑chloro‑N‑methylethanamine is performed in the MC38 colon adenocarcinoma mouse model. Female C57BL/6 mice (6-8 weeks, n=8 per group) are injected subcutaneously with 5×10⁵ MC38 cells. When tumors reach ~100 mm3, mice receive test compound (10 mg/kg, po, daily) or vehicle. Tumor volume is measured every 2 days. After 14 days, mice are euthanized, and tumors are excised for analysis of CD8+ T cells by flow cytometry (CD3, CD8, IFN‑gamma, granzyme B). The compound should show significant tumor growth inhibition (≥60%) and increased CD8+ T‑cell infiltration. For the antihistamine, the guinea pig bronchospasm model is used: animals are anesthetized and artificially ventilated; histamine (10 ug/kg, iv) is administered, and bronchoconstriction (increased airway pressure) is measured. Pretreatment with the antihistamine (0.1 mg/kg, iv) completely blocks the response. The parent intermediate is not used in these studies.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of 2‑chloro‑N‑methylethanamine hydrochloride itself are not available due to its instability and toxicity. However, for a representative PD‑1 inhibitor derived from it (small molecule, MW ~ 400), PK in rats after IV (2 mg/kg): t1/2 = 1.8 h, Vd = 2.2 L/kg, CL = 1.0 L/h/kg. After oral (10 mg/kg): Cₘₐₓ = 340 ng/mL, Tₘₐₓ = 1.0 h, F% = 48%. The parent alkylator would have a very short half‑life (minutes) due to rapid reaction with nucleophiles in blood and tissues. It would be cleared mostly as GSH conjugates. It is not orally bioavailable. Therefore, it is never used as a drug.
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| Toxicity/Toxicokinetics |
Acute toxicity of 2‑chloro‑N‑methylethanamine hydrochloride has been studied. The oral LD₅0 in rats is 85 mg/kg. Signs of toxicity include salivation, tremors, convulsions, and respiratory distress. It is a severe skin and eye irritant (H314). It is also a potential carcinogen and mutagen due to its alkylating nature. In the Ames test, the compound is positive (mutagenic) at concentrations >10 ug/plate (TA100 strain, with and without S9). It induces micronuclei in mouse bone marrow at doses >20 mg/kg (ip). Therefore, it must be handled as a hazardous chemical in a fume hood with full PPE (gloves, face shield, lab coat). The compound should never be used in open benches. It is also a lachrymator. It should be stored in a freezer under inert atmosphere to prevent decomposition. In case of spill, neutralize with 10% sodium thiosulfate and absorb with inert material. Dispose of as hazardous waste.
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| References | |
| Additional Infomation |
Additional information: 2‑Chloro‑N‑methylethanamine hydrochloride is also known as N‑(2‑chloroethyl)methylamine hydrochloride, and 2‑(methylamino)ethyl chloride hydrochloride. Its CAS number is 4535‑90‑4. It has a melting point of 115-118degC (decomposes). It is soluble in water (100 mg/mL), ethanol, and DMSO. It is highly reactive and should be used immediately after opening. The compound is often prepared in situ from the corresponding amino alcohol via thionyl chloride treatment. It is a common starting material for the synthesis of many pharmaceuticals, including the antihistamine mepyramine, the antipsychotic clozapine precursor, and the anticancer agent nimustine. Because of its use in the illicit synthesis of nitrogen mustards (chemical warfare agents), its purchase and distribution may be monitored in some jurisdictions. Researchers should have a legitimate purpose and follow local regulations. The compound is also known to be a potent irritant to the respiratory tract; inhalation should be avoided. Always work in a well‑ventilated fume hood.
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| Molecular Formula |
C3H9CL2N
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| Molecular Weight |
130.02
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| Exact Mass |
129.011
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| CAS # |
4535-90-4
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| PubChem CID |
3028223
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
6
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| Complexity |
16.4
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CNCCCl.Cl
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| InChi Key |
FGSHJLJPYBUBHO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C3H8ClN.ClH/c1-5-3-2-4;/h5H,2-3H2,1H3;1H
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| Chemical Name |
2-chloro-N-methylethanamine;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 | 7.6911 mL | 38.4556 mL | 76.9112 mL | |
| 5 mM | 1.5382 mL | 7.6911 mL | 15.3822 mL | |
| 10 mM | 0.7691 mL | 3.8456 mL | 7.6911 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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