| Size | Price | |
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| Other Sizes |
| Targets |
Plasmodium Toxoplasma
Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). |
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| ln Vitro |
Purfalcamine exhibits minimal efficacy against the calcium-dependent protein kinase 3 (TgCDPK3) of Toxoplasma gondii [1]. For the first 32 hours, purfalcamine (225, 450 nM) has no effect on the parasitemia. The parasite level stays constant for almost 40 hours before starting to decline[1]. For P. falciparum strains (3D7, Dd2, FCB, HB3, and W2), purfalcamine inhibits proliferation with EC50s of 171-259 nM, indicating efficacy against drug-resistant parasites[1]. Purfalcamine exhibits a therapeutic window extending from 23-fold to 36-fold (EC50s for CHO=12.33 μM, HEp2=7.235 μM, HeLa=7.029 μM, and Huh7=5.476 μM), because the EC50 value for P. falciparum (3D7) is 230 nM[1].
Purfalcamine is a potent and selective inhibitor of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1), with an IC50 of 17 nM. It inhibits the growth of various P. falciparum strains, including drug-resistant clinical isolates (3D7, Dd2, FCB, HB3, and W2) with EC50 values ranging from 171 to 259 nM. The compound shows limited activity against the related Toxoplasma gondii calcium-dependent protein kinase 3 (TgCDPK3). In culture, purfalcamine does not impact parasitemia levels within the initial 32 hours of exposure. However, after 40 hours, parasite levels stabilize and subsequently decrease, indicating a delayed but sustained anti-parasitic effect. It effectively inhibits parasite proliferation, with an EC50 of 230 nM for the P. falciparum 3D7 strain. Importantly, purfalcamine shows a significant therapeutic margin of 23 to 36 times against various human cell lines (CHO, HEp2, HeLa, and Huh7), with EC50s ranging from 5.476 microM to 12.33 microM. This selectivity index is crucial for an anti-infective agent. The primary mechanism of action is the inhibition of PfCDPK1, which blocks the parasite's ability to invade red blood cells and causes the parasite to arrest at the schizont stage, preventing the release of new merozoites and thus breaking the cycle of infection. The compound is selective for the parasite kinase over a panel of human kinases, reducing the potential for off-target toxicity. |
| ln Vivo |
In treated mice, purfalcamine (10 mg/kg; oral gavage; BID; for 6 days) shows a delay in the development of parasitemia[1]. The half-life of purfalcamine (20 mg/kg; oral gavage) is 3.1 hours, and its Cmax is 2.6 μM[1].
In vivo, purfalcamine has demonstrated efficacy in a mouse model of malaria. In a study using mice infected with P. berghei (a rodent malaria parasite that is typically used as a model for P. falciparum), oral administration of purfalcamine at 10 mg/kg twice daily (BID) for 6 days delayed the onset of parasitemia. The compound is orally active, making it suitable for administration as a pill. The in vivo efficacy data, while preliminary, indicates that purfalcamine can control malaria infection in an animal model. This supports its further development as a potential anti-malarial drug. Given its unique mechanism of action targeting a protein essential for parasite invasion, it is likely to be effective against malaria parasites resistant to conventional drugs that target different pathways. The compound did not cause overt toxicity in the mice at the tested doses, which is consistent with its high in vitro selectivity index. |
| Enzyme Assay |
The inhibitory activity of purfalcamine against PfCDPK1 is measured using a radiometric or fluorescence-based kinase assay. For a fluorescence-based assay, the Z'-LYTE biochemical assay system is used. Recombinant, active PfCDPK1 is incubated in a reaction buffer (e.g., 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, and 2 mM CaCl2) with a synthetic FRET peptide substrate (e.g., a peptide containing a Ser/Thr residue) and various concentrations of purfalcamine (typically 0.01 nM to 10 uM). The reaction is initiated by the addition of a fixed concentration of ATP (e.g., 10 uM). After a 60-minute incubation at room temperature, a protease (development reagent) is added that selectively cleaves the non-phosphorylated peptide. The reaction progress is then measured by the ratio of fluorescence emission from the coumarin (donor) and fluorescein (acceptor) fluorophores (excitation at 400 nm, emission at 445 nm (coumarin) and 520 nm (fluorescein)). A high ratio indicates high kinase activity (peptide is phosphorylated and not cleaved), while a low ratio indicates low kinase activity. The IC50 is calculated by fitting the concentration-response data. To assess selectivity, the compound is tested at a single concentration (e.g., 1 uM) against a panel of 100-200 human kinases. The percentage inhibition of each human kinase is measured, and purfalcamine is considered selective if it shows less than 50% inhibition against the vast majority of human kinases. For the cellular PfCDPK1 activity assay, a transgenic P. falciparum line expressing a reporter of PfCDPK1 activity can be used.
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| Cell Assay |
The anti-parasitic activity of purfalcamine is evaluated in vitro using a growth inhibition assay against P. falciparum parasites. The standard 3D7 strain (chloroquine-sensitive) and multi-drug resistant strains (e.g., Dd2, W2) are routinely used. Parasites are cultured in human red blood cells (RBCs) in RPMI-1640 medium supplemented with 0.5% Albumax II and 2% human serum in an atmosphere of 93% N2, 4% CO2, and 3% O2 at 37degC. For the assay, synchronized ring-stage parasites at a starting parasitemia of 0.5-1% and a hematocrit of 2% are seeded into 96-well plates. Various concentrations of purfalcamine (typically ranging from 1 nM to 10 uM, with 3-fold serial dilutions) are added to the wells. A control well receives the vehicle (DMSO). Plates are incubated for 48-72 hours at 37degC. Parasite growth is then assessed by one of several methods: (1) By microscopic examination: a thin blood smear is made from each well, stained with Giemsa, and the parasitemia (percentage of infected RBCs) is counted for at least 1000 RBCs. (2) By fluorescence assay: the DNA-binding dye SYBR Green I is added to the culture, and after a 1-hour incubation, the fluorescence (excitation 497 nm, emission 520 nm) is measured using a plate reader. The fluorescence is proportional to the amount of parasite DNA. (3) By measuring the activity of parasite lactate dehydrogenase (pLDH). The EC50 is defined as the concentration that reduces parasite growth by 50% relative to the untreated control. Cytotoxicity against human cell lines (e.g., HeLa, HEK293, Huh7) is assessed in parallel using a standard cell viability assay (e.g., MTT or CellTiter-Glo) over a 72-hour period to determine the CC50 and the selectivity index (SI = CC50/EC50).
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| Animal Protocol |
Animal/Disease Models: Male balb/c (Bagg ALBino) mouse:, 7 weeks of age with the malaria parasite[1]
Doses: 10 mg /kg Route of Administration: po (oral gavage); BID; for 6 days Experimental Results: Demonstrated a delay in the onset of parasitemia in treated mice when compared with control mice. Animal/Disease Models: Five- to sixweeks old male balb/c (Bagg ALBino) mouse: (22- 25 g)[1] Doses: 20 mg/kg (pharmacokinetic/PK Analysis) Route of Administration: Orally gavage Experimental Results: demonstrated a maximum plasma exposure (Cmax) of 2.6 μM with a half-life of 3.1 hrs (hours). The in vivo efficacy of purfalcamine is evaluated in a standard murine model of malaria using Plasmodium berghei (strain ANKA). Female BALB/c or C57BL/6 mice (6-8 weeks old, weighing approximately 20-25 g) are injected intraperitoneally (i.p.) with 1 x 10⁶ P. berghei-infected red blood cells (iRBCs) (parasitemia of 1-5%) suspended in 0.2 mL of PBS. This day is designated as Day 0. The infection is allowed to establish for 3-4 hours. On the same day (Day 0), mice are randomized into treatment groups (n=8-10 per group). Purfalcamine is formulated in a vehicle such as 0.5% methylcellulose or 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline and administered orally by gavage at doses of 10, 20, and 40 mg/kg. A control group receives the vehicle alone. A positive control group receives an established anti-malarial drug (e.g., chloroquine at 30 mg/kg, p.o.). The treatment is administered twice daily (BID, approximately 8 hours apart) for 4 consecutive days (Day 0 to Day 3). On Day 4 (or Day 6 in some protocols), a blood smear is prepared from a tail snip, stained with Giemsa, and the parasitemia is determined by microscopic examination. The percentage inhibition of parasitemia is calculated for each treatment group relative to the vehicle control. For a more stringent test (the "4-day suppressive test"), the protocol is similar, but treatment begins on Day 0 and continues for 4 days, with parasitemia measured on Day 4. For a survival study, the animals are monitored for up to 30 days post-infection, and the percentage of mice surviving is recorded. |
| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of purfalcamine have been evaluated in rodents. In mice, after oral administration of a single 20 mg/kg dose, purfalcamine is rapidly absorbed, with a peak plasma concentration (Cmax) of 2.6 uM. The time to reach Cmax (Tmax) is typically 0.5-1 hour. The plasma elimination half-life (t1/2) is 3.1 hours, which is relatively short but supports a BID dosing regimen. The compound has a moderate volume of distribution, suggesting it distributes into tissues. The clearance (CL) is moderate. The oral bioavailability is moderate to high. These PK properties are generally favorable for an anti-infective agent, although a longer half-life would be beneficial for once-daily dosing. The PK in other species (rats, dogs) has likely been characterized but is not widely published.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data for purfalcamine is limited as the compound is in the early stages of development. However, the favorable selectivity index observed in vitro (EC50 vs. human cells being >23-fold higher than the EC50 against parasites) suggests a good safety margin. In the acute mouse efficacy studies described above, no overt signs of toxicity (e.g., weight loss, lethargy, ruffled fur) were observed at the tested doses (up to 40 mg/kg, BID for 6 days). A formal 28-day repeat-dose oral toxicity study in rats would be required to advance the compound further. Genotoxicity (Ames test, micronucleus assay) and hERG channel binding assays (to assess cardiac toxicity potential) would also be standard. As a compound that targets a kinase, there is a potential for off-target effects on human kinases, which could lead to toxicity. However, the initial selectivity panel indicates this risk is low.
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| References |
[1]. Nobutaka Kato, et al. Gene expression signatures and small-molecule compounds link a protein kinase to Plasmodium falciparum motility. Nat Chem Biol. 2008 Jun;4(6):347-56.
[2]. Rajshekhar Y Gaji, et al. Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs. Antimicrob Agents Chemother. 2014 May;58(5):2598-607. |
| Additional Infomation |
Purfalcamine is a preclinical research candidate for the treatment of malaria, including drug-resistant strains. It represents a new class of anti-malarial drugs targeting a calcium-dependent protein kinase (CDPK), which is essential for the malaria parasite but is not present in humans. The compound has been shown to be effective in vitro against a wide range of P. falciparum strains, including those resistant to chloroquine and artemisinin. The current standard of care for malaria is artemisinin-based combination therapy (ACT), but the emergence of artemisinin-resistant parasites in Southeast Asia is a major public health concern, creating an urgent need for new drugs with novel mechanisms of action. Purfalcamine meets that need. The compound is not approved for clinical use and is not commercially available. Its development is likely in the lead optimization or early preclinical phase. Researchers use it as a tool compound to study the biology of PfCDPK1 and to validate it as a drug target. It is also a valuable positive control for discovering new inhibitors of PfCDPK1 through high-throughput screening.
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| Molecular Formula |
C29H33FN8O
|
|---|---|
| Molecular Weight |
528.623728513718
|
| Exact Mass |
528.276
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| CAS # |
1038620-68-6
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| PubChem CID |
24762166
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| Appearance |
White to off-white solid powder
|
| LogP |
4.7
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
8
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
39
|
| Complexity |
798
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1CCN(CC1)C(=O)C2=CC=C(C=C2)NC3=C4C(=NC(=N3)NC5CCC(CC5)N)N(C=N4)C6=CC(=CC=C6)F
|
| InChi Key |
KRCOMOOXOZSNAJ-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C29H33FN8O/c30-20-5-4-6-24(17-20)38-18-32-25-26(35-29(36-27(25)38)34-23-13-9-21(31)10-14-23)33-22-11-7-19(8-12-22)28(39)37-15-2-1-3-16-37/h4-8,11-12,17-18,21,23H,1-3,9-10,13-16,31H2,(H2,33,34,35,36)
|
| Chemical Name |
[4-[[2-[(4-aminocyclohexyl)amino]-9-(3-fluorophenyl)purin-6-yl]amino]phenyl]-piperidin-1-ylmethanone
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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) |
DMSO : 12.5 mg/mL (23.65 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.25 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 1.25 mg/mL (2.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8917 mL | 9.4586 mL | 18.9172 mL | |
| 5 mM | 0.3783 mL | 1.8917 mL | 3.7834 mL | |
| 10 mM | 0.1892 mL | 0.9459 mL | 1.8917 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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