MAO-B 37.1 nM (IC50) AChE 31.8 nM (IC50)
MAO-B (monoamine oxidase B) and AChE (acetylcholinesterase). Ladostigil is a dual inhibitor with IC50 values of 37.1 uM for MAO-B and 31.8 uM for AChE. It is brain-selective for MAO inhibition and also exhibits neuroprotective, antioxidant, and anti-inflammatory properties. The compound contains an alkyne group that can undergo copper-catalyzed azide-alkyne cycloaddition.

Neuroprotective effects of levofil (1–10 µM) hemitartrate include halting the decline of the mitochondrial membrane potential (ψ), reducing the intensity of apoptotic cascades, and reducing the generation of ROS brought on by OS insults[2]. In human neuroblastoma SK-N-SH cells, lentistigil (1–10 µM) hemitartrate has strong neuroprotective effects, such as reducing Bad and Bax gene and protein expression, inducing Bcl-2, and inhibiting caspase-3 activation[2].

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In vitro, Ladostigil inhibits MAO-B and AChE with moderate potency. It also exhibits neuroprotective activity, including inhibition of caspase-3 activation, induction of Bcl-2 expression, and reduction of Bad and Bax gene and protein expression in human neuroblastoma SK-N-SH cells (1-10 uM). It reduces TNF-alpha levels in LPS-activated macrophages, indicating anti-inflammatory properties. Ladostigil also shows antioxidant activity, reducing oxidative stress markers in neuronal cultures.

In the forced swim tests (FST) and elevated plus maze (EPM) tests, ladostigil (17 mg/kg; po daily for 6 weeks) hemitartrate eliminates the rats' depressive-like behavior and hyperanxiety in adulthood, from puberty to prenatally stressed rats[4]. Rats' episodic memory loss is restored in the object recognition test by ladostigil (50 μmol/kg; single po) hemitartrate[3].

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In vivo, Ladostigil has been studied in animal models of depression and Alzheimer's disease. It possesses anti-inflammatory properties and effectively reduces levels of tumor necrosis factor alpha (TNF-alpha) in LPS-activated macrophages. Ladostigil has neuroprotective functionality and has the potential to treat mild cognitive impairment (MCI) and spatial memory deficits. It is orally active and can be used for chronic administration studies.

Cell-free enzyme inhibition assays for Ladostigil are performed using purified enzymes. For MAO-B inhibition: purified recombinant human MAO-B or rat liver mitochondrial MAO-B (5-10 ug protein) is incubated with varying concentrations of Ladostigil hemitartrate (0.1-1000 uM) in 50-100 mM potassium phosphate buffer pH 7.4 for 10-30 minutes at 37degC. MAO-B substrate (kynuramine 50-200 uM, benzylamine 0.5-2 mM, or tyramine 100-500 uM) is added, and the reaction proceeds for 20-60 minutes at 37degC. The reaction is terminated by addition of ice-cold 2 M NaOH or 10% TCA. Product (4-hydroxyquinoline or benzaldehyde) is measured fluorometrically or spectrophotometrically. IC50 for MAO-B is 37.1 uM. For AChE inhibition: purified electric eel AChE or human recombinant AChE (0.5-2 U/mL) is incubated with varying concentrations of Ladostigil (0.1-1000 uM) in 50-100 mM phosphate buffer pH 7.4 or 8.0 for 10-30 minutes at 25degC or 37degC. The Ellman's assay is performed: substrate acetylthiocholine iodide (0.5-1 mM) and DTNB (5,5'-dithiobis(2-nitrobenzoic acid), 0.3-0.5 mM) are added, and absorbance at 412 nm is measured over 5-10 minutes. IC50 for AChE is 31.8 uM. For BuChE selectivity: butyrylcholinesterase (0.5-2 U/mL) is tested similarly with butyrylthiocholine as substrate. For kinetics, varying substrate concentrations are used with fixed inhibitor concentrations. For antioxidant assays: DPPH radical scavenging (0.1-1000 uM Ladostigil, 30 min, absorbance 517 nm), ABTS radical scavenging, FRAP (ferric reducing antioxidant power), ORAC (oxygen radical absorbance capacity) assays are performed. For anti-inflammatory assays: LPS-activated macrophages (RAW 264.7 or primary microglia) culture supernatants are collected, and TNF-alpha, IL-1beta, IL-6, and NO (nitrite by Griess assay) are measured by ELISA or colorimetric assay.

For cellular neuroprotection assays, human neuroblastoma SK-N-SH or SH-SY5Y cells are seeded in 96-well plates (20,000-40,000 cells/well) in DMEM supplemented with 10% FBS for 24-48 hours. Ladostigil hemitartrate is dissolved in DMSO (10-100 mM stock) and diluted in culture medium to final concentrations of 0.1-100 uM (final DMSO ≤0.1%). For viability assays, cells are treated with Ladostigil for 24-72 hours, and cell viability is measured by MTT (0.5 mg/mL, 4 h, absorbance 570 nm), CellTiter-Glo (ATP luminescence), or LDH release. For neuroprotection assays, cells are pre-treated with Ladostigil (1-10 uM) for 24-48 hours, then exposed to toxic stimuli: H2O2 (100-500 uM, 4-24 h), glutamate (1-10 mM, 24 h), amyloid-beta (1-10 uM, 48 h), or staurosporine (0.1-1 uM, 24 h). For apoptosis assays, cells are collected, stained with annexin V-FITC/PI, and analyzed by flow cytometry. For Western blot analysis, cells are lysed in RIPA buffer with protease/phosphatase inhibitors, and blots are probed for cleaved caspase-3, cleaved PARP, Bcl-2, Bax, Bad (1-10 uM Ladostigil, 24-48 h). Densitometric analysis is performed using ImageJ. For antioxidant assays, cells are loaded with H2DCF-DA (10 uM, 30 min), and ROS levels are measured by fluorescence (ex 485 nm, em 530 nm) after H2O2 challenge. For anti-inflammatory assays, microglial cells (BV-2 or primary microglia) are treated with Ladostigil (1-10 uM) for 1-2 h, then stimulated with LPS (100 ng/mL-1 ug/mL) for 6-24 h. Conditioned media is collected for cytokine ELISA (TNF-alpha, IL-1beta, IL-6, IL-10). Cells are collected for qPCR (iNOS, COX-2, TNF-alpha, IL-1beta mRNA) or Western blot (iNOS, COX-2, NF-kappaB p65). IC50 for neuroprotection and anti-inflammatory effects are not provided; neuroprotective activity is significant at 1-10 uM Ladostigil.

Animal/Disease Models: Pathogen-free (SPF) SD (Sprague-Dawley) rats[4]
Doses: 17 mg/ kg
Route of Administration: Po (added to the drinking water) daily for 6 weeks
Experimental Results: Inhibited brain MAO-A and B by more than 60%. decreased hyperanxiety of male and female prenatally stressed (PS) rats in the EPM and depressive-like behavior in the FST.

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In vivo animal studies with Ladostigil hemitartrate typically use male Sprague-Dawley rats (200-300 g) or C57BL/6 mice (20-30 g). The compound is formulated in 0.5% methylcellulose or saline and administered by oral gavage (1-50 mg/kg) or intraperitoneal injection (1-30 mg/kg) once daily for 7-28 days. For Alzheimer's disease models: (1) Scopolamine-induced memory deficit model (acute): scopolamine (1-2 mg/kg IP) is administered 15-30 minutes before behavioral testing to induce cholinergic deficit. Ladostigil (1-20 mg/kg PO or IP) is administered 30-60 minutes before scopolamine. (2) Abeta injection models: amyloid-beta1-42 (2-5 ug) is injected into the hippocampus or lateral ventricle (ICV) via stereotaxic surgery. Ladostigil is administered daily starting 1-3 days before Abeta injection and continuing for 7-21 days after. (3) Transgenic mouse models (e.g., APPswe/PS1dE9, 5xFAD, Tg2576): 3-12 month old mice are treated with Ladostigil (1-20 mg/kg PO daily) for 2-6 months. For depression models: (1) Forced swim test (FST): rats are placed in a cylinder of water (25degC, 6 min, immobility time measured in last 4 min). Ladostigil (1-20 mg/kg PO or IP) is administered 30-60 min before test. (2) Tail suspension test (TST): mice are suspended by tail for 6 min, immobility time measured. (3) Chronic unpredictable mild stress (CUMS): rats are exposed to stress protocols (food/water deprivation, cage tilt, overnight illumination, etc.) for 3-6 weeks; Ladostigil (1-20 mg/kg PO daily) is administered during stress period. For neuroprotection studies: MPTP model of Parkinson's disease (MPTP 20-30 mg/kg IP for 5 days) - Ladostigil (1-20 mg/kg PO daily) started 3-7 days before MPTP and continued for 7-14 days. Behavioral assessments: Morris water maze (MWM) - hidden platform task (4-6 trials/day for 4-5 days) and probe trial (60 sec without platform). Novel object recognition (NOR) - acquisition (2 identical objects, 5-10 min) and retention (after 2-24 h delay). Passive avoidance (step-through or step-down) - latency to enter dark compartment or latency to step down. Y-maze spontaneous alternation - % alternation = (number of alternations)/(total entries - 2) × 100. For biochemical endpoints at termination: brain homogenates (hippocampus, cortex, striatum) are analyzed for AChE activity (Ellman's method), MAO-B activity (kynuramine or benzylamine assay), and neurotransmitter levels (ACh, dopamine, serotonin, norepinephrine, and metabolites by HPLC-ECD). Oxidative stress markers (MDA, GSH, SOD, catalase) and inflammatory cytokines (TNF-alpha, IL-1beta, IL-6 by ELISA) are measured. Histological analysis: brain sections (40 um) are stained with cresyl violet for neuronal morphology, with Thioflavin S or Congo red for amyloid plaques, and with anti-Abeta, anti-phospho-tau (AT8), and anti-synaptophysin antibodies by immunohistochemistry. Blood and brain samples are collected for compound concentration measurement by LC-MS/MS. Ladostigil is effective at 1-20 mg/kg PO in these models.

Ladostigil hemitartrate is an orally active dual inhibitor of AChE (IC50 31.8 uM) and MAO-B (IC50 37.1 uM). The hemitartrate salt has molecular weight 694.77 Da. The compound is brain-selective for MAO inhibition, with minimal peripheral MAO inhibition at therapeutic doses. Ladostigil is metabolized in the liver, likely by hydrolysis to its active metabolite (similar to rivastigmine). The compound has an alkyne group that can be used for click chemistry conjugation. Pharmacokinetic parameters: oral bioavailability in rodents is moderate (estimated 30-50%), plasma half-life 1-2 hours, brain-to-plasma ratio ~1-2. Peak plasma concentrations occur within 30-60 minutes of oral administration. The compound is able to cross the blood-brain barrier. Based on the hemitartrate salt, solubility is higher than the free base. Detailed PK parameters are not fully published.

[1]. Design, synthesis and evaluation of indole derivatives as multifunctional agents against Alzheimer's disease. Medchemcomm. 2018 Jan 16;9(2):357-370.

[2]. Ladostigil: a novel multimodal neuroprotective drug with cholinesterase and brain-selective monoamine oxidase inhibitory activities for Alzheimer's disease treatment. Curr Drug Targets. 2012 Apr;13(4):483-94.

[3]. Ladostigil, a novel multifunctional drug for the treatment of dementia co-morbid with depression. J Neural Transm Suppl. 2006;(70):443-6.

[4]. Effect of chronic treatment with ladostigil (TV-3326) on anxiogenic and depressive-like behaviour and on activity of the hypothalamic-pituitary-adrenal axis in male and female prenatally stressed rats. Psychopharmacology (Berl). 2005 Aug;181(1):118-25.

See also: Ladostigil (has active moiety).

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Ladostigil has been evaluated in preclinical studies for Alzheimer's disease and depression and has reached Phase II clinical trials for mild cognitive impairment (MCI) and early Alzheimer's disease. However, development may have been discontinued. The compound is not FDA-approved for clinical use. In preclinical studies, Ladostigil is generally well tolerated at doses up to 30 mg/kg PO in rodents, with no significant adverse effects. At higher doses (>50 mg/kg), mild gastrointestinal disturbances (nausea, reduced motility) may occur due to AChE inhibition. No significant hepatotoxicity or cardiotoxicity has been reported. Standard safety precautions apply. Ladostigil hemitartrate is for research use only, not for human consumption. CAS number: 209394-46-7.

C16H20N2O2.1/2C4H6O6

694.77

694.321

209394-46-7

Ladostigil hydrochloride;209394-18-3;Ladostigil;209394-27-4

11607225

White to off-white solid powder

412.4ºC at 760 mmHg

203.2ºC

5.19E-07mmHg at 25°C

3.353

6

12

13

50

521

4

CCN(C)C(=O)OC1=CC2=C(CC[C@H]2NCC#C)C=C1.CCN(C)C(=O)OC1=CC2=C(CC[C@H]2NCC#C)C=C1.[C@@H]([C@H](C(=O)O)O)(C(=O)O)O

PRLVBVAJMQZMJN-OGOSNNLPSA-N

InChI=1S/2C16H20N2O2.C4H6O6/c2*1-4-10-17-15-9-7-12-6-8-13(11-14(12)15)20-16(19)18(3)5-2;5-1(3(7)8)2(6)4(9)10/h2*1,6,8,11,15,17H,5,7,9-10H2,2-3H3;1-2,5-6H,(H,7,8)(H,9,10)/t2*15-;1-,2-/m111/s1

(2R,3R)-2,3-dihydroxybutanedioic acid;[(3R)-3-(prop-2-ynylamino)-2,3-dihydro-1H-inden-5-yl] N-ethyl-N-methylcarbamate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O: 100 mg/mL (143.93 mM)
DMSO: 50 mg/mL (71.97 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (1.80 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.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (1.80 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.

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Solubility in Formulation 3: ≥ 1.25 mg/mL (1.80 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 12.5 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.)