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]

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]

[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.

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.)