IC50: 1.6 μM (TRPML1), 2.3 μM (TRPML2), 12.5 (TRPML3) for the (-)-isome of (1S,2S)-ML-SI3; 5.9 μM (TRPML1) for the (+)-enantiomer[1]
(1S,2S)-ML-SI3 targets all three isoforms of the TRPML channel family: TRPML1, TRPML2, and TRPML3. The compound exhibits differential activity depending on the enantiomer. The (-)-enantiomer of (1S,2S)-ML-SI3 is a potent inhibitor of TRPML1 and TRPML2 with IC50 values of 1.6 μM and 2.3 μM, respectively, and shows weaker inhibition of TRPML3 with an IC50 of 12.5 μM. In contrast, the (+)-enantiomer (which is (1S,2S)-ML-SI3 itself) inhibits TRPML1 with an IC50 of 5.9 μM and serves as an activator of TRPML2 and TRPML3 with EC50 values of 2.7 μM and 10.8 μM, respectively.

The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been published, albeit without clear information about stereochemical details. In this investigation we developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer. Separation of the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 values 1.6 and 2.3 μM) and a weak inhibitor (IC50 12.5 μM) of TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC50 5.9 μM), but an activator on TRPML 2 and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first insights into structure-activity relationships in this chemotype, and showed that a broad variety of modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1 and strong activator of TRPML2).[1]

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In vitro studies have established that (1S,2S)-ML-SI3 is a trans-isomer of ML-SI3 that targets TRPML channels with stereospecific activity. The (-)-enantiomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 = 1.6 μM and 2.3 μM) and a weak inhibitor of TRPML3 (IC50 = 12.5 μM), while the (+)-enantiomer inhibits TRPML1 (IC50 = 5.9 μM) but activates TRPML2 and TRPML3. TRPML channels are involved in lysosomal and endosomal function regulation. The compound represents a valuable membrane-permeable chemical tool for investigating TRPML functions, as only two TRPML inhibitors (ML-SI1 and ML-SI3) have been published to date.

In vivo studies of (1S,2S)-ML-SI3 remain limited in publicly available literature, as the compound is primarily used as a research tool for in vitro characterization of TRPML channel function. The compound has been developed through stereoselective synthesis to confirm that the trans-isomer is significantly more active than the cis-isomer. Given its role as a TRPML modulator and the association of TRPML1 mutations with mucolipidosis type IV, the compound may have potential for in vivo investigations of lysosomal storage disorders, but specific animal model studies have not been reported. Further in vivo research is needed to establish its pharmacokinetic and pharmacodynamic profiles.

For TRPML channel binding assays, cell membranes expressing recombinant TRPML channels are prepared and incubated with radiolabeled ligands or fluorescent probes in the presence of varying concentrations of (1S,2S)-ML-SI3. Non-specific binding is determined using excess unlabeled reference compound. Following incubation at appropriate temperature (typically 4-25°C for 30-60 minutes), bound and free ligands are separated by rapid filtration through glass fiber filters or by centrifugation. Filters are washed, dried, and radioactivity counted by liquid scintillation or fluorescence measured by plate reader. IC50 and EC50 values are calculated from dose-response curves using nonlinear regression analysis. Assays are performed in triplicate with appropriate vehicle controls.

For in vitro cellular assays, cells expressing TRPML channels (e.g., HEK293 cells transfected with TRPML1, TRPML2, or TRPML3) are cultured in appropriate media under standard conditions (37°C, 5% CO2). Cells are seeded in multi-well plates and allowed to adhere overnight. (1S,2S)-ML-SI3 is dissolved in DMSO and diluted in culture medium to desired concentrations (typically ranging from nM to μM). Cells are treated with compound for specified durations. TRPML channel activity is assessed by measuring intracellular calcium flux using fluorescent calcium indicators (e.g., Fura-2 or Fluo-4) or by electrophysiological patch-clamp recordings. Each concentration is tested in replicate wells, and appropriate vehicle controls and positive controls (e.g., known TRPML agonists/antagonists) are included.

For in vivo animal studies of (1S,2S)-ML-SI3, no specific protocols have been published in the available literature. The compound is primarily utilized as a research tool for in vitro characterization of TRPML channel function. For general in vivo administration of similar TRPML modulators, compounds are typically formulated in suitable vehicles (e.g., DMSO/PEG300/saline mixtures) and administered via intraperitoneal (i.p.) or intravenous (i.v.) injection. Dosing regimens vary by study objective. Blood samples are collected at predetermined time points for pharmacokinetic analysis by LC-MS/MS. Tissue distribution and target engagement can be assessed post-mortem. All procedures must follow institutional animal care and use committee guidelines.

Detailed pharmacokinetic data for (1S,2S)-ML-SI3 are not extensively reported in publicly available sources. The compound has a molecular weight of 429.58, a formula of C23H31N3O3S, and a CAS number of 2563870-87-9. Storage recommendations: powder at -20°C for up to 3 years; in solvent at -80°C for up to 1 year. Solubility information suggests the compound may be soluble in DMSO and other organic solvents. As a small-molecule TRPML modulator with membrane permeability, it is expected to have reasonable bioavailability, but specific parameters such as half-life, clearance, volume of distribution, and oral bioavailability have not been reported in the available literature.

According to available safety information, (1S,2S)-ML-SI3 is intended for research use only and not for human therapeutic applications. Standard laboratory safety precautions should be followed when handling this compound, including the use of appropriate personal protective equipment (gloves, lab coat, safety goggles). The compound should be handled in a well-ventilated area, preferably under a fume hood. Avoid dust formation and inhalation. In case of skin contact, wash with plenty of soap and water. In case of eye contact, rinse cautiously with water for several minutes. The compound is not for human use and no clinical toxicity data are available. Specific GHS hazard classifications for this compound have not been detailed in publicly available sources.

[1]. Chemical and pharmacological characterization of the TRPML calcium channel blockers ML-SI1 and ML-SI3. Eur J Med Chem. 2021 Jan 15;210:112966.

Mounting evidence suggests that impaired autophagy plays a crucial role in myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms underlying cardiomyocyte autophagy dysfunction following I/R injury remain unclear. Therefore, there are currently no effective treatments targeting autophagy to prevent myocardial I/R injury. This study used in vitro and in vivo I/R models, exposing neonatal rat ventricular myocytes to hypoxia/reoxygenation and subjecting mice to I/R treatment, respectively, to monitor autophagy flux in cardiomyocytes. We observed that I/R injury impaired cardiomyocyte autophagy flux in both in vitro and in vivo models. Downregulation of the lysosomal cation channel TRPML1 significantly restored ischemia/reperfusion (I/R)-induced myocardial autophagy flux blockade, indicating that TRPML1 directly participates in the blockade of cardiomyocyte autophagy flux in I/R-injured cardiomyocytes. Mechanistically, elevated reactive oxygen species (ROS) levels after ischemia/reperfusion subsequently activate TRPML1, inducing lysosomal zinc release into the cytoplasm, ultimately blocking cardiomyocyte autophagy flux. This may be achieved by disrupting the fusion of autophagosomes and lysosomes. Therefore, TRPML1-induced inhibition of cardiomyocyte autophagy flux disrupts mitochondrial turnover, leading to the accumulation of damaged mitochondria and further ROS release, ultimately resulting in cardiomyocyte death. More importantly, inhibiting the TRPML1 channel through pharmacological and genetic means can significantly reduce the infarct area of ischemia/reperfusion (I/R) injury in mice and salvage cardiac function by restoring damaged cardiomyocyte autophagy. In summary, our study shows that elevated ROS leads to TRPML1 activation, which in turn inhibits cardiomyocyte autophagy in ischemia/reperfusion injury and directly causes cardiomyocyte death by disrupting mitochondrial turnover. Therefore, targeting TRPML1 represents a novel therapeutic strategy for preventing myocardial ischemia/reperfusion injury. Basic Res Cardiol. 2022 Apr 7;117(1):20.

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(1S,2S)-ML-SI3 ((+)-trans-ML-SI3) is a trans-isomer of ML-SI3 and a TRPML inhibitor. The compound was developed through stereoselective synthesis to investigate the stereochemical requirements for TRPML modulation. The (-)-enantiomer is a potent inhibitor of TRPML1 and TRPML2 (IC50 = 1.6 μM and 2.3 μM) and a weak inhibitor of TRPML3 (IC50 = 12.5 μM), while the (+)-enantiomer inhibits TRPML1 (IC50 = 5.9 μM) but activates TRPML2 and TRPML3. TRPML channels are involved in lysosomal and endosomal function regulation, and TRPML1 mutations are linked to mucolipidosis type IV. The compound is for research use only with no clinical development or regulatory approvals reported.

C23H31N3O3S

429.58

429.21

C, 64.31; H, 7.27; N, 9.78; O, 11.17; S, 7.46

2563870-87-9

(1R,2R)-ML-SI3;2418594-00-8;(rel)-ML-SI3;2108567-79-7;ML-SI3;891016-02-7

94784693

White to off-white solid powder

1.3±0.1 g/cm3

589.3±60.0 °C at 760 mmHg

310.2±32.9 °C

0.0±1.7 mmHg at 25°C

1.629

3.8

1

6

6

30

624

2

S(C1C=CC=CC=1)(N[C@H]1CCCC[C@@H]1N1CCN(C2C=CC=CC=2OC)CC1)(=O)=O

OVTXOMMQHRIKGL-SFTDATJTSA-N

InChI=1S/C23H31N3O3S/c1-29-23-14-8-7-13-22(23)26-17-15-25(16-18-26)21-12-6-5-11-20(21)24-30(27,28)19-9-3-2-4-10-19/h2-4,7-10,13-14,20-21,24H,5-6,11-12,15-18H2,1H3/t20-,21-/m0/s1

N-[(1S,2S)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide

(1S,2S)-ML-SI3; CHEMBL4856175; 2563870-87-9; N-{(1S,2S)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl}benzenesulfonamide; N-[(1S,2S)-2-[4-(2-methoxyphenyl)piperazin-1-yl]cyclohexyl]benzenesulfonamide; DTXSID801336630; BDBM50569843; DA-48624;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: 100 mg/mL (232.79 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.82 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 (5.82 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 (5.82 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.)