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| Targets |
DHODH-IN-4 targets human dihydroorotate dehydrogenase (HsDHODH) and Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). DHODH is a flavin-dependent mitochondrial enzyme that catalyzes the fourth and rate-limiting step of de novo pyrimidine biosynthesis, converting dihydroorotate to orotate. This pathway is essential for the synthesis of pyrimidine nucleotides required for DNA and RNA production. The malaria parasite relies exclusively on de novo pyrimidine synthesis for its rapid replication within the host, as it lacks pyrimidine salvage pathways. DHODH-IN-4 shows differential potency between the parasite and human enzymes, with IC₅₀ values of 4 μM for PfDHODH and 0.18 μM for HsDHODH. This indicates that the compound is more potent against the human enzyme than the parasite enzyme.
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| ln Vitro |
In vitro, DHODH-IN-4 demonstrates potent inhibition of human DHODH with an IC₅₀ of 0.18 μM and inhibition of P. falciparum DHODH with an IC₅₀ of 4 μM. The compound exhibits antimalarial activity. The differential potency (0.18 μM vs 4 μM) suggests that the compound is more potent against the human enzyme than the parasite enzyme, which may limit its selectivity for antimalarial applications. The compound's activity is concentration-dependent, with efficacy observed in the low micromolar range. These in vitro data indicate that DHODH-IN-4 is a dual inhibitor of both human and parasite DHODH, making it a useful tool for studying DHODH biology and comparing enzyme inhibition across species.
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| ln Vivo |
In vivo data for DHODH-IN-4 are not extensively reported in the available literature. As an inhibitor of DHODH with antimalarial activity, the compound would be expected to demonstrate efficacy in animal models of malaria infection. However, its higher potency against human DHODH (0.18 μM) compared to parasite DHODH (4 μM) may limit its therapeutic window due to potential host toxicity. DHODH inhibitors have been studied in various preclinical models for their immunomodulatory and antiproliferative properties. Further studies are needed to establish the compound's in vivo efficacy, safety, and pharmacokinetic profile. The compound is supplied as a solid with purity ≥98% and can be formulated for in vivo administration using appropriate vehicles.
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| Enzyme Assay |
In vitro enzyme/receptor binding (non-cellular) assays for DHODH-IN-4 involve measuring inhibition of recombinant human and P. falciparum DHODH enzymatic activity. The assay typically uses a colorimetric method detecting the conversion of dihydroorotate to orotate coupled with the reduction of the electron acceptor (e.g., 2,6-dichloroindophenol, DCIP). The enzyme is incubated with varying concentrations of the compound, substrate (dihydroorotate), and coenzyme (ubiquinone or decylubiquinone) in assay buffer (typically 50 mM Tris-HCl, pH 8.0, with 0.1% Triton X-100). The reaction is monitored spectrophotometrically by following the decrease in absorbance at 600 nm (DCIP reduction). IC₅₀ values are calculated from concentration-response curves. The IC₅₀ values of 4 μM for PfDHODH and 0.18 μM for HsDHODH are determined using this assay format.
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| Cell Assay |
In vitro cellular experiments with DHODH-IN-4 are performed using P. falciparum cultures to assess antimalarial activity. Parasites are cultured in human erythrocytes in RPMI 1640 medium supplemented with Albumax or human serum. Cultures are maintained at 37°C in a gas mixture of 5% O₂, 5% CO₂, and 90% N₂. For drug susceptibility testing, synchronized ring-stage parasites are treated with varying concentrations of the compound for 48-72 hours. Parasite growth inhibition is assessed by measuring [³H]hypoxanthine incorporation, by fluorescence-based methods using SYBR Green I, or by microscopic examination of Giemsa-stained blood smears. IC₅₀ values are calculated from concentration-response curves. Cytotoxicity against mammalian cells (e.g., HepG2 or Vero cells) is assessed in parallel to determine the selectivity index.
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| Animal Protocol |
In vivo animal studies with DHODH-IN-4 have not been extensively reported. For antimalarial compounds, standard in vivo efficacy studies are performed in mouse models of P. berghei or P. falciparum (using SCID mice with human erythrocytes). Mice are infected with the parasite and treated with the compound via oral, intraperitoneal, or intravenous administration at various doses and schedules. Efficacy is assessed by measuring parasitemia in blood smears, survival rates, and recrudescence following treatment. Pharmacokinetic studies in rodents assess absorption, distribution, metabolism, and excretion. The compound's higher potency against human DHODH (0.18 μM) compared to parasite DHODH (4 μM) may be a concern for host toxicity, and selectivity optimization would be needed for therapeutic development.
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| ADME/Pharmacokinetics |
Comprehensive pharmacokinetic data for DHODH-IN-4 are not extensively reported. The compound has molecular formula C₁₇H₁₂Cl₂N₂O₂ and molecular weight 347.20. It is typically supplied as a solid and may dissolve in DMSO. Storage recommendations: powder at -20°C for 3 years or 4°C for 2 years; in solvent at -80°C for 6 months or -20°C for 1 month. For in vivo administration, the compound can be formulated using vehicles such as DMSO/Tween 80/saline (10:5:85) or DMSO/PEG300/Tween 80/saline (10:40:5:45). Detailed pharmacokinetic parameters including half-life, clearance, bioavailability, and tissue distribution require further investigation from primary research publications.
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| Toxicity/Toxicokinetics |
Toxicological information for DHODH-IN-4 is not extensively detailed in the available literature. As a research compound with enzyme inhibitory activity, it should be handled with appropriate safety precautions. Standard safety guidelines for handling potent pharmaceutical compounds apply, including use of personal protective equipment (gloves, safety goggles, lab coat), working in a well-ventilated area, and proper chemical waste disposal. The compound is intended for research use only and is not approved for human therapeutic use. The compound's higher potency against human DHODH (0.18 μM) compared to parasite DHODH (4 μM) suggests potential for host toxicity, and caution should be exercised in handling and experimental use.
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| References | |
| Additional Infomation |
DHODH-IN-4 (CAS 1148125-93-2) is an inhibitor of both human and Plasmodium falciparum dihydroorotate dehydrogenase (DHODH). The compound has molecular formula C₁₇H₁₂Cl₂N₂O₂ and molecular weight 347.20. It exhibits IC₅₀ values of 4 μM for PfDHODH and 0.18 μM for HsDHODH. DHODH-IN-4 demonstrates antimalarial activity. The compound is also known as compound 17 and is supplied as a solid with purity ≥98%. Storage: powder at -20°C for 3 years or 4°C for 2 years; in solvent at -80°C for 6 months or -20°C for 1 month. The compound is for research use only and is not approved for clinical use.
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| Molecular Formula |
C17H12CL2N2O2
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| Molecular Weight |
347.20
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| Exact Mass |
346.027
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| CAS # |
1148125-93-2
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| PubChem CID |
54727973
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
5.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
526
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C/C(=C(\C#N)/C(=O)NC1=C(C=C(C=C1)C2=CC=CC=C2Cl)Cl)/O
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| InChi Key |
KEZXIYKJTYTGOO-RAXLEYEMSA-N
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| InChi Code |
InChI=1S/C17H12Cl2N2O2/c1-10(22)13(9-20)17(23)21-16-7-6-11(8-15(16)19)12-4-2-3-5-14(12)18/h2-8,22H,1H3,(H,21,23)/b13-10-
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| Chemical Name |
(Z)-N-[2-chloro-4-(2-chlorophenyl)phenyl]-2-cyano-3-hydroxybut-2-enamide
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| Synonyms |
DHODHIN4; DHODH IN 4
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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 | 2.8802 mL | 14.4009 mL | 28.8018 mL | |
| 5 mM | 0.5760 mL | 2.8802 mL | 5.7604 mL | |
| 10 mM | 0.2880 mL | 1.4401 mL | 2.8802 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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