(+)-SJ733 is a potent inhibitor of Plasmodium falciparum ATPase 4 (PfATP4), a putative Na+-efflux ATPase. Whole-genome sequencing of resistant mutants identified point mutations in the pfatp4 gene as the sole determinant of resistance, and theoretical docking studies suggest a binding site within the transmembrane channel of PfATP4. The EC₅₀ for disrupting parasite Na+ homeostasis ([Na+]ᵢ) is ~200 nM. [1]

In P. falciparum-infected erythrocytes, (+)-SJ733 binds to a single receptor site with an affinity equivalent to its growth inhibitory potency (kd=50 nM). In extensive in vitro assays, as well as in single or repeated dose studies at any tested dose in any preclinical species safety or tolerability (no adverse effect levels observed and maximum tolerated dose >240 mg/kg in a 7-day repeated-dose study in rats), (+)-SJ733 did not demonstrate significant safety at any dose. Consequently, it is anticipated that (+)-SJ733 will have a safety margin of at least 43 times [1].

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(+)-SJ733 exhibited high potency against all tested strains of P. falciparum, with EC₅₀ values ranging from 10 to 60 nM, including strains resistant to other antimalarials. The (+)-enantiomer was significantly more potent than the (-)-enantiomer. [1]
Treatment of saponin-isolated P. falciparum trophozoites with (+)-SJ733 caused a rapid, dose-dependent increase in cytosolic Na+ concentration ([Na+]ᵢ) from a resting level of ~6 mM to a maximum of ~110 mM within 1.5 hours (EC₅₀ ~200 nM). This effect was greatly diminished (~50-fold decrease in sensitivity) in a resistant strain (ATP4^L350H). [1]
In a proliferation assay using luciferase-labeled P. falciparum (3D7 strain), (+)-SJ733 rapidly arrested parasite growth, reaching maximal effect within 24 hours. [1]
In clonal dilution cidality assays, continuous exposure to (+)-SJ733 at concentrations above the EC₉₉ for 96 hours was required for maximal killing of parasites in vitro. [1]
In fluorescence-activated cell sorting (FACS) analysis, treatment of P. falciparum-infected erythrocytes with (+)-SJ733 (or another PfATP4 inhibitor, NITD246) induced a cryptosis/senescence-like phenotype. This was characterized by cell shrinkage, increased phosphatidylserine (PS) externalization (EC₅₀ ~30 nM), and a shift to a more spherical morphology. These effects were specific to infected erythrocytes and not observed with other antimalarial classes (e.g., artesunate) or in uninfected erythrocytes. [1]
Treatment of infected erythrocytes with (+)-SJ733 significantly increased membrane rigidity, peaking around 7 hours post-treatment. No effect was observed on uninfected erythrocytes. [1]
Time-lapse microscopy showed that (+)-SJ733 treatment led to immediate arrest of parasite motility and development within the erythrocyte, followed in some cases by parasite swelling, lysis, and subsequent erythrocyte lysis. [1]
(+)-SJ733 was approximately 10-fold less potent against ex vivo blood stages of rodent malaria species (P. berghei, P. vinckei, P. chabaudi) compared to P. falciparum. [1]

After administering (+)-SJ733 to P. falciparum-infected NOD-scid IL2R·null mice, the parasites were rapidly cleared, becoming undetectable within 48 hours and 80% eliminated in the first 24. In a NOD-scid IL2Rγnull mouse model, (+)-SJ733 was highly potent and effective against Plasmodium falciparum 3D70087/N9 in vivo, with an effective dose of 90% (ED90 1.9 mg) when given orally four times a day. When compared to artesunate (11.1 mg/kg; AUCED90 undetermined), chloroquine (4.3 mg/kg; AUCED90 3.1 μM·h), and pyrimethamine (0.9 mg/kg; AUCED90 5. μM⋅h) in the same model, (+)-SJ733 was superior. Blood concentrations of (+)-SJ733 treated with ED90 dosages stayed above the mean in vitro EC90 for 6 to 10 hours following each dose [1].

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In the NOD-scid IL2Rγ^null (NSG) mouse model infected with P. falciparum (3D70087/N9 strain), oral administration of (+)-SJ733 (four sequential daily doses) was highly efficacious, with an ED₉₀ of 1.9 mg/kg and an associated AUC_ED90 of 1.5 µM·h. Its potency was superior to artesunate (ED₉₀ 11.1 mg/kg) and chloroquine (ED₉₀ 4.3 mg/kg) in the same model. Parasitemia was reduced by 80% within 24 hours and cleared by 48 hours, a rate comparable to artesunate. [1]
Against P. berghei in mice (four sequential daily oral doses of the racemate), the ED₉₀ was 40 mg/kg (AUC_ED90 80 µM·h), consistent with its lower ex vivo potency against rodent malarias. [1]
The rapid clearance rate in vivo was independent of host spleen presence or Kupffer cell depletion, suggesting it was not solely dependent on these immune clearance mechanisms. [1]
(+)-SJ733 potently blocked transmission of P. berghei from infected mice to mosquitoes when mice were treated 1 hour before mosquito feeding, with an ED₅₀ of 5 mg/kg. [1]

For Na+ homeostasis assays, saponin-isolated P. falciparum trophozoites were loaded with the sodium-sensitive dye Sodium-Binding Benzofuran Isophthalate (SBFI). Parasites were treated with (+)-SJ733 or other compounds, and fluorescence was measured to determine cytosolic Na+ concentration ([Na+]ᵢ) over time, typically >60 minutes after compound addition. [1]
For proliferation/growth inhibition assays (EC₅₀ determination), P. falciparum strains were co-cultured with human erythrocytes in vitro and treated with serial dilutions of (+)-SJ733. Parasite growth was assessed, for example, by measuring luciferase activity from transgenic parasites or by SYBR Green staining. [1]
For cryptosis/senescence phenotyping, P. falciparum-infected erythrocytes were treated with (+)-SJ733 and analyzed by multimodal FACS. Cells were stained with SYBR Green (to identify infected cells) and Annexin V (to detect phosphatidylserine exposure). Forward scatter (FSC) and side scatter (SSC) were measured to assess cell size and shape/complexity. Data were analyzed to characterize shifts in cell populations indicative of cryptosis. [1]
For erythrocyte rigidity measurement, a microfluidic biomechanical assay was used. Erythrocytes (infected or uninfected, treated or untreated) were passed through a constriction under fluid pressure, and their deformability was analyzed to determine rigidity. [1]
For time-lapse microscopy, ring-stage P. falciparum-infected erythrocytes were treated with (+)-SJ733 and imaged by phase-contrast microscopy at regular intervals over 18 hours to observe parasite motility, development, and morphological changes. [1]

P. falciparum efficacy in NSG mice: NOD-scid IL2Rγ^null (NSG) mice were engrafted with human erythrocytes and infected with P. falciparum (3D70087/N9 strain). Mice were treated orally with (+)-SJ733 (as the (+)-enantiomer) administered as four sequential daily doses. Parasitemia was monitored over time to determine efficacy (ED₉₀) and clearance kinetics. Some experiments used splenectomized mice or mice treated with liposomal clodronate to deplete Kupffer cells. [1]
P. berghei efficacy in mice: Normal mice were infected with P. berghei. Mice were treated orally with racemic SJ733 (or the (+)-enantiomer) as four sequential daily doses to determine ED₉₀ and clearance kinetics. [1]
Transmission blocking assay: Mice infected with P. berghei were treated with a single oral dose of (+)-SJ733 (1 hour before mosquito feeding). Mosquitoes were allowed to feed on the mice, and later dissected to count oocysts in the midgut, measuring the compound's ability to block transmission. [1]
In vivo resistance selection: A P. berghei strain with weak resistance to (+)-SJ733 was generated by serially passaging parasites in mice with multiple rounds of intermittent high-dose treatment with a close analog, SJ311. Resistance emerged slowly (after 4 weeks). [1]

(+)-SJ733 exhibited favorable pharmacokinetic characteristics in preclinical animal models (mice, rats, and dogs). After oral administration (at doses of 20-30 mg/kg), peak plasma concentrations (C_max) in rodents were 3-10 µM, and peak plasma concentrations in dogs were >20 µM, with AUC values of 5-40 µM·h. [1] Oral bioavailability in rats and dogs was >65%. [1] In the mouse model of Plasmodium falciparum NSG, after administration of ED₉₀ dose (1.9 mg/kg), plasma concentrations of (+)-SJ733 were higher than the in vitro EC₉₀ average within 6-10 hours after each administration. [1] The compound showed relatively high clearance in human microsomal models. Based on allometric growth-derived predictions, the effective human dose range was 3-6 mg/kg. [1]

In extensive in vitro analyses, (+)-SJ733 did not exhibit significant safety issues. [1]
In preclinical animal studies (e.g., rats) with single and repeated doses, no significant safety or tolerability issues were observed at any of the tested doses. In a 7-day repeated-dose study in rats, no adverse event response level (NOAEL) and no maximum tolerated dose (MTD) greater than 240 mg/kg were observed. [1]
By comparing the AUC at the maximum non-toxic dose in rat safety studies with the AUC at the dose that produced the maximum parasitological response in mice, its safety margin (treatment ratio) was estimated to be at least 43 times. [1]

[1]. (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium. Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):E5455-62.

SJ-733 has been used in clinical trials for malaria research.

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(+)-SJ733 is the (+)-enantiomer of SJ733, a dihydroisoquinolone (DHIQ) compound identified through high-throughput phenotypic screening. It was selected as a clinical candidate for a rapid clearance component (target candidate component 1) of a potential single-exposure eradication and prophylaxis (SERCaP) malaria drug. [1]
Its mechanism of action is thought to be the inhibition of parasite PfATP4, leading to rapid disruption of Na+ homeostasis. This triggers physical changes (causing occultation/senescence) in infected host erythrocytes, including phosphatidylserine exposure, increased rigidity, and spherical morphology. These changes facilitate rapid host-mediated clearance in vivo (e.g., through phagocytosis or mechanical filtration in the spleen/liver), explaining why it acts faster in vivo than in vitro. [1]
Resistant mutations in pfatp4 (e.g., L350H) are associated with higher adaptation costs in competitive trials and higher quiescent [Na+]ᵢ in the parasite, which may slow the emergence of resistance in vivo. [1]
(+)-SJ733 exhibits complete cross-resistance with other chemical families believed to target PfATP4 (e.g., spironindolones). [1]
It can be easily synthesized from a commercially available precursor via a five-step reaction. [1]
(+)-SJ733 was selected for clinical development by Medicines for Malaria Venture in March 2013. [1]

C24H16F4N4O2

468.403059005737

468.12

1424799-20-1

89508529

White to off-white solid powder

1.4±0.1 g/cm3

599.0±50.0 °C at 760 mmHg

316.0±30.1 °C

0.0±1.7 mmHg at 25°C

1.591

3.8

1

8

4

34

813

2

C1=CC=C2C(=C1)[C@@H]([C@H](N(C2=O)CC(F)(F)F)C3=CN=CC=C3)C(=O)NC4=CC(=C(C=C4)F)C#N

VKCPFWKTFZAOTO-LEWJYISDSA-N

InChI=1S/C24H16F4N4O2/c25-19-8-7-16(10-15(19)11-29)31-22(33)20-17-5-1-2-6-18(17)23(34)32(13-24(26,27)28)21(20)14-4-3-9-30-12-14/h1-10,12,20-21H,13H2,(H,31,33)/t20-,21+/m0/s1

(3S,4S)-N-(3-cyano-4-fluorophenyl)-1-oxo-3-pyridin-3-yl-2-(2,2,2-trifluoroethyl)-3,4-dihydroisoquinoline-4-carboxamide

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 : ~50 mg/mL (~106.75 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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