Mitochondrial electron transport
NSC-102533 (2-hydroxy-3-phenylnaphthalene-1,4-dione) is a selective inhibitor of mitochondrial electron transport in Plasmodium falciparum, which is essential for the parasite's energy production [1]
- The compound specifically disrupts the mitochondrial membrane potential (ΔΨmit) of malaria parasites, with an IC50ΔΨmit of 16 μM after 1 hour of incubation [1]
- No other specific molecular targets beyond mitochondrial electron transport chain components are identified in the literature [1]

Antimalarial 14 (Compound N3) (zero-400 μM, 48 hours) has a minimal cytotoxic effect on human cells and inhibits Plasmodium falciparum [1]. Plasmodium falciparum's mitochondrial membrane potential is inhibited by antimalarial drug 14 with an IC50ΔΨmit of 16 μM [1].Anti-malarial activity was assessed in vitro for a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluating the parasitaemia after 48 hours of incubation. Potential cytotoxicity in HEK293T cells was assessed using the MTT assay. Changes in mitochondrial membrane potential of Plasmodium were measured using the fluorescent dye Mitrotracker Red CMXROS [1].

---

NSC-102533 exhibits potent anti-malarial activity against Plasmodium falciparum with an IC50 of 443 nM in 48-hour incubation assays [1]
- Among a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluated, NSC-102533 (compound N3) was the most active, with the other compounds showing mid-micromolar range IC50 values (1-10 μM) [1]
- The compound demonstrates minimal cytotoxicity against human HEK293T cells, with a CC50 of 54.6 ± 0.23 μM, indicating high selectivity for the parasite over host cells [1]
- At concentrations up to 16 μM, NSC-102533 showed no significant cytotoxicity to human cells, while effectively inhibiting parasite growth [1]
- The anti-malarial activity of NSC-102533 is comparable to atovaquone, another hydroxynaphthoquinone anti-malarial drug, despite structural differences in their chemical backbones [1]

Changes in mitochondrial membrane potential (ΨΔmit)[1]
Loss of parasite mitochondrial membrane potential (ΨΔmit) was determined using 5 μM Mitrotracker Red CMXROS as described previously [18]. Cultures were incubated for 30 min at 37°C with the dye and then for 1 h with 10-fold serial dilutions (0.001-100 μM) of N3 and atovaquone. As a control, 5 μM cyanide m-chlorophenylhydrazone (CCCP), a protonophore that dissipate the membrane potential, was used. Results were analysed by flow cytometry as described above.

---

Mitochondrial membrane potential (ΔΨmit) disruption assay [1]: - Plasmodium falciparum 3D7 strain parasites were loaded with 5 μM Mitotracker Red CMXROS (a fluorescent dye sensitive to mitochondrial membrane potential) - After 30-minute incubation at 37°C, serial dilutions of NSC-102533 (0.001-100 μM) were added and incubated for 1 hour - 5 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP, a protonophore that dissipates membrane potential) was used as a positive control - Changes in fluorescence were analyzed by flow cytometry to determine the concentration causing 50% disruption of ΔΨmit (IC50ΔΨmit)

Cytotoxicity assay[1]
Cell Types: Plasmodium falciparum and HEK293T
Tested Concentrations: 0.128, 0.64, 3.2, 16, 80 and 400 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: demonstrated inhibitory effect against Plasmodium falciparum with IC50 of 443 nM and Exhibits a cytotoxic CC50 of 54.6 ± 0.23 μM.

---

Anti-malarial activity assay [1]: - Plasmodium falciparum cultures were incubated with NSC-102533 at concentrations ranging from 0.128 to 400 μM for 48 hours - Parasite growth inhibition was assessed by measuring parasitemia (percentage of infected erythrocytes) - The concentration causing 50% inhibition (IC50) was calculated using dose-response curves - The assay was performed in A+ human erythrocytes (1-3% parasitemia and 2% hematocrit) in RPMI-1640 medium supplemented with 0.2% sodium bicarbonate, 50 mg/L hypoxanthine, and 10% type A+ human serum - Cultures were maintained in a gas mixture of 92% N2, 5% CO2, and 3% O2 at 37°C - Cytotoxicity assay [1]: - Human HEK293T cells were treated with NSC-102533 at concentrations from 0.128 to 400 μM for 48 hours - Cell viability was assessed using the MTT assay, which measures mitochondrial dehydrogenase activity as an indicator of viable cells - The concentration causing 50% cytotoxicity (CC50) was determined - Results showed NSC-102533 had minimal cytotoxic effect on human cells compared to its anti-malarial activity

3D7 strain parasites were cultured and synchronized as described previously. Briefly, parasites were routinely maintained in A+ human erythrocytes (1-3% parasitaemia and 2% haematocrit) in RPMI-1640 media supplemented with 0.2% sodium bicarbonate, 50 mg/L hypoxanthine and 10% type A+ human serum in 92% N2, 5% CO2 and 3% O2.

---

No animal protocols for NSC-102533 are described in the literature [1]
- The study focuses on in vitro evaluation and does not include animal experiments [1]
- While the article mentions that the compound may serve as a starting point for design of new hydroxynaphthoquinone anti-malarials, it does not report on in vivo testing [1]

No specific ADME/pharmacokinetic data for NSC-102533 have been reported in the literature [1] - This article does not include studies on absorption, distribution, metabolism, or excretion [1] - However, as a hydroxynaphthoquinone derivative similar to atovaquinone, NSC-102533 may share some common pharmacokinetic characteristics with such compounds, such as: - relatively low bioavailability - mainly excreted in feces - high protein binding - long elimination half-life (these characteristics are based on the pharmacokinetics of atovaquinone, rather than direct measurements of NSC-102533) [105]

No specific toxicity or toxicokinetic data for NSC-102533 have been reported in the literature [1] - This article only assessed its in vitro cytotoxicity in HEK293T cells (CC50 = 54.6 ± 0.23 μM) [1] - No in vivo toxicity studies, LD50 values, or safety profiles have been described [1] - Although other hydroxynaphthoquinone compounds have been shown to cause hemolysis and kidney injury in animal models, there have been no reports of such effects of NSC-102533 [68]

[1]. Biological evaluation of hydroxynaphthoquinones as anti-malarials. Malar J. 2013 Jul 10;12:234.

Background: Hydroxynaphthoquinones have been extensively studied over the past 50 years due to their antimalarial activity. Atorvaquinone, one of these compounds, is used in combination with guanidine as an important drug for the treatment and prevention of malaria, Malalong®. Methods: This study evaluated the antimalarial activity of a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) in vitro, detecting parasitemia after 48 hours of incubation. The potential cytotoxicity of these compounds to HEK293T cells was assessed using the MTT assay. Changes in the mitochondrial membrane potential of Plasmodium parasites were detected using the fluorescent dye Mitrotracker Red CMXROS. Results: The IC50 values of the four compounds were all in the medium micromolar range, with the most active compound, N3, having an IC50 of 443 nM. N3 disrupted the mitochondrial membrane potential, with an IC50 ΔΨmit of 16 μM after 1 hour. In in vitro cytotoxicity assays of HEK 293T cells, N3 did not exhibit cytotoxicity at concentrations up to 16 μM. Conclusion: N3 is a potent inhibitor of mitochondrial electron transport with nanomolar activity against cultured Plasmodium falciparum and extremely low cytotoxicity. N3 can serve as a starting point for designing novel hydroxynaphthoquinone antimalarial drugs. [1] NSC-102533 (compound N3) is a 3-alkyl-2-hydroxy-1,4-naphthoquinone with a phenyl substituent at the 3-position. [1] - The chemical structure of this compound is 2-hydroxy-3-phenylnaphtho-1,4-dione (molecular formula: C16H10O3, molecular weight: 250.25) [1] - It also has other names: 2-hydroxy-3-phenyl-1,4-naphthoquinone, 4-hydroxy-3-phenylnaphtho-1,2-dione and antimalarial drug 14. [1] - The compound is a yellow to brown solid with a melting point of 192-195°C (estimated based on structural similarity). (Compared with related compounds) [33] - The solubility of NSC-102533 in DMSO is approximately 125 mg/mL (499.5 mM), which is beneficial for in vitro studies [1] - The mechanism of action of this compound involves disrupting the mitochondrial membrane potential, similar to atovaquinone, but with a different chemical skeleton [1] - The cyclohexane ring in atovaquinone is not essential for antimalarial activity, as replacing the ring with a CH2 group in NSC-102533 only slightly reduces the activity [1] - NSC-102533 can serve as a starting point for designing novel hydroxynaphthoquinone antimalarial drugs with potentially improved performance [1]

C16H10O3

250.249

250.063

1150-59-0

265893

Yellow to brown solid

1.392g/cm3

445.3ºC at 760 mmHg

237.2ºC

1.04E-08mmHg at 25°C

1.693

3.034

1

3

1

19

429

0

C1=CC=C(C2C(=O)C(=O)C3=CC=CC=C3C=2O)C=C1

HCKUADBGJIXWTR-UHFFFAOYSA-N

InChI=1S/C16H10O3/c17-14-11-8-4-5-9-12(11)15(18)16(19)13(14)10-6-2-1-3-7-10/h1-9,17H

2-hydroxy-3-phenylnaphthalene-1,4-dione

NSC102533; NSC 102533; 2-Hydroxy-3-phenyl-1,4-naphthoquinone; 4-hydroxy-3-phenylnaphthalene-1,2-dione; 2-hydroxy-3-phenylnaphthalene-1,4-dione; NSC 102533; 1,4-Naphthalenedione, 2-hydroxy-3-phenyl-; Antimalarial agent 14; 4-hydroxy-3-phenyl-naphthalene-1,2-dione; NSC-102533

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~125 mg/mL (499.5 mM)

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 Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

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
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)