MAO
(S)-Rasagiline has minimal affinity for monoamine oxidase B (MAO-B), the primary target of the R-enantiomer rasagiline. Rasagiline (R-enantiomer) is a highly potent, selective, and irreversible inhibitor of mitochondrial MAO-B with an IC50 of 4.43 nM for rat brain MAO B activity. In contrast, (S)-Rasagiline (TVP1022) is approximately 1000-fold less active as an MAO-B inhibitor, making it a valuable negative control for distinguishing MAO-B-dependent and MAO-B-independent effects. Despite this, (S)-Rasagiline appears to share the same neuroprotective activity as the R-isomer, suggesting that the neuroprotective effects of rasagiline may be independent of MAO-B inhibition. The compound may interact with other cellular targets involved in neuroprotection, including anti-apoptotic and anti-inflammatory pathways.

Determination of MAO-A and -B inhibition in vitro [1]
MAO-A and -B activities were determined in rat brain homogenate in vitro following incubation with varying concentrations of rasagiline, TVP1022, and selegiline (Figure 2 and Table 1). The IC50 values calculated for each inhibitor are presented in Table 1. The optical enantiomers of N-propargyl-1-aminoindan can be seen to possess a striking degree of stereoselectivity, since the (−)-isomer (TVP 1022) is 1/3,800 as active as the (+)-isomer (rasagiline) for inhibition of MAO-B. Whereas rasagiline is 93 times more potent for inhibition of MAO-B than MAO-A, TVP 1022 shows little or no selectivity for MAO-A or -B. Rasagiline and selegiline had similar IC50 values for inhibition of MAO-B (P>0.05), but selegiline was significantly less potent than rasagiline for inhibition of MAO-A (P<0.01). In human tissue, there was no significant difference between the IC50 values for rasagiline and selegiline for inhibition of MAO-A or MAO-B (Table 1), and TVP 1022 was again seen to be largely devoid of inhibitory activity. The racemic form of N-propargyl-1-aminoindan (AGN 1135) had approximately half the potency of rasagiline, indicating that no interaction occurred between the optical isomers at the MAO active site (similar data were obtained in preliminary studies using rat tissue). [1]
In vitro studies demonstrate that (S)-Rasagiline (TVP1022) exhibits cytoprotective effects without significant MAO-B inhibition, distinguishing it from rasagiline. The compound shows neuroprotective activity similar to that of the R-isomer in various neuronal cell models, protecting against oxidative stress, glutamate toxicity, and apoptosis. Preclinical studies have shown potential benefits in myocardial infarction, heart failure, and neuronal injury models. In cellular assays, (S)-Rasagiline has been shown to reduce cell death and promote cell survival through mechanisms that are independent of MAO-B inhibition, likely involving modulation of mitochondrial function, reduction of oxidative stress, and activation of survival signaling pathways. The compound's cytoprotective effects are observed at concentrations that do not inhibit MAO-B activity, confirming its MAO-B-independent mechanism of action.

In vivo studies have demonstrated the neuroprotective and cardioprotective effects of (S)-Rasagiline in animal models. In models of neuronal injury and neurodegenerative disease, (S)-Rasagiline exhibits neuroprotective activity comparable to that of rasagiline, despite its lack of MAO-B inhibitory activity. This suggests that the neuroprotective effects are mediated through MAO-B-independent mechanisms. In cardiovascular research, (S)-Rasagiline has shown potential benefits in models of myocardial infarction and heart failure. In vivo protocols typically involve oral or intraperitoneal administration of (S)-Rasagiline at doses comparable to those used for rasagiline (e.g., 0.1-10 mg/kg). Endpoints include assessment of neuronal survival, behavioral outcomes in neurological models, cardiac function, and histopathological examination. The compound serves as an essential control for differentiating MAO-B-dependent from MAO-B-independent effects in vivo.

Determination of MAO inhibitory activity in vitro [1]
The activities of MAO-A and -B were determined by the adapted method of Tipton & Youdim (1983). Rat or human cerebral cortical tissue was homogenized in 0.3 M sucrose (one part tissue to 20 parts sucrose) using a glass-teflon motor-driven homogenizer (brain and liver), or Ultraturrax (gut). The inhibitor under test was added to a suitable dilution of the enzyme preparation in 0.05 M phosphate buffer (pH 7.4) and incubated together with selegiline 0.1 μM (for determination of MAO-A) or clorgyline 0.1 μM (for determination of MAO-B). Incubation was carried on for 60 min at 37°C before addition of labelled substrates (14C-5-hydroxytryptamine creatinine disulphate 100 μM for determination of MAO-A, or 14C-phenylethylamine 10 μM for determination of MAO-B) and incubation continued for 30 or 20 min respectively. The reaction was then stopped by addition of citric acid (2 M). Radioactive metabolites were extracted into toluene/ethyl acetate (1 : 1 v v−1), a solution of 2,5-diphenyloxazole was added to a final concentration of 0.4% (w v−1), and metabolite content estimated by liquid scintillation counting. Activity in presence of drug was expressed as a percentage of that in control samples. [1]
The preincubation was carried out in the presence of clorgyline or selegiline because phenylethylamine is also metabolized quite effectively by MAO-A (O'Carroll et al., 1983), leading to inhibition curves for MAO-B, which showed a plateau at about 80% inhibition with selegiline or rasagiline if MAO-A was not inactivated. For comparison between two inhibitors with potentially different inhibitory effects on MAO-A and MAO-B, therefore, it was thought necessary to employ the system in which opposite enzyme forms are inactivated before assay.
For MAO-B activity assays, (S)-Rasagiline is used as a negative control to compare with the R-enantiomer. MAO activity is measured using purified MAO-B enzyme or mitochondrial preparations from rat brain. Enzyme preparations are incubated with varying concentrations of (S)-Rasagiline (0.001-100 µM) or R-rasagiline (positive control) in assay buffer (50 mM potassium phosphate buffer, pH 7.4) at 37°C for 20 minutes. Substrate (e.g., kynuramine or [¹⁴C]-phenylethylamine) is added, and the reaction is allowed to proceed for 20-30 minutes. Product formation is measured fluorometrically (for kynuramine, 4-hydroxyquinoline is detected at excitation 318 nm/emission 380 nm) or by radiometric detection. IC50 values are calculated. For MAO-B selectivity studies, MAO-A activity is also measured using serotonin as substrate. (S)-Rasagiline shows minimal inhibition of both MAO-A and MAO-B compared to the R-enantiomer.

For neuroprotection studies in vitro, primary neuronal cultures or neuronal cell lines (e.g., SH-SY5Y, PC12) are cultured in appropriate medium (DMEM/F12 or Neurobasal with supplements) at 37°C in 5% CO₂. Cells are seeded in 96-well or 24-well plates and allowed to differentiate (if applicable). (S)-Rasagiline is dissolved in DMSO or PBS and diluted in culture medium to final concentrations (typically 0.1-100 µM). Cells are pre-treated with (S)-Rasagiline for 1-2 hours, then exposed to neurotoxic insults such as glutamate (1-10 mM), 6-hydroxydopamine (50-100 µM), or hydrogen peroxide (100-500 µM) for 24-48 hours. Cell viability is assessed by MTT, LDH release, or calcein-AM staining. Apoptosis is measured by caspase-3/7 activity and Annexin V/PI staining. Mitochondrial membrane potential is assessed using JC-1 dye. Oxidative stress markers (ROS, MDA, GSH) are measured using fluorescent probes or colorimetric assays. For anti-inflammatory studies, microglial cells (BV-2) are stimulated with LPS and cytokine production is measured by ELISA.

Determination of inhibition of MAO activity in vivo [1]
In in vivo studies, drugs were administered orally by gavage (p.o.). The animals weighed 250 – 300 g at the time of killing. For estimation of in vivo inhibitory effect, varying doses of the inhibitors were administered to groups of five or six rats for the stated times, the animals were killed by decapitation, tissues removed and frozen at −20°C, and enzyme activity determined subsequently as above. Enzyme activity in drug-treated tissues were expressed as a percentage of that in control tissues.
For in vivo neuroprotection studies, adult mice or rats (6-8 weeks old) are used. (S)-Rasagiline is dissolved in vehicle (saline or 0.5% methylcellulose) and administered via oral gavage or intraperitoneal injection at doses of 0.1-10 mg/kg. For the MPTP model of Parkinson's disease, mice are treated with MPTP (20-30 mg/kg, i.p.) for 5 days, with (S)-Rasagiline or vehicle given daily starting before MPTP treatment. After 7-14 days, striatal dopamine levels, tyrosine hydroxylase staining, and behavioral tests (rotarod, pole test) are assessed. For the 6-OHDA model, rats receive stereotaxic injection of 6-OHDA into the striatum or substantia nigra, and (S)-Rasagiline is administered daily for 2-4 weeks. Apomorphine-induced rotations are measured as an index of lesion severity. For the MCAO model of stroke, rats undergo middle cerebral artery occlusion for 60-90 minutes, followed by reperfusion. (S)-Rasagiline is administered before or after ischemia, and infarct volume, neurological scores, and neuronal survival are assessed.

Pharmacokinetic data for (S)-Rasagiline are similar to those of rasagiline, as they are enantiomers with comparable physicochemical properties. Rasagiline is rapidly absorbed after oral administration, with Tmax of 0.5-1 hour, and has a plasma half-life of 1-2 hours in humans. The compound is extensively metabolized by CYP450 enzymes (primarily CYP1A2) via N-dealkylation to aminoindan, which is then glucuronidated or sulfated. (S)-Rasagiline is expected to have similar absorption, distribution, metabolism, and excretion properties, including high oral bioavailability (>80%), extensive plasma protein binding (~80%), and hepatic clearance. The compound's alkyne group does not significantly affect its pharmacokinetic properties. As a research tool, (S)-Rasagiline is used at doses that match those of rasagiline for comparative studies.

Toxicological data for (S)-Rasagiline are limited as the compound is primarily used as a research reagent and negative control. Rasagiline (R-enantiomer) has been extensively studied in toxicology and has a well-established safety profile, with hepatotoxicity being the most significant adverse effect. (S)-Rasagiline is expected to have a better safety profile because it lacks MAO-B inhibitory activity, which is the primary source of rasagiline's pharmacological effects and potential adverse events. In preclinical studies, (S)-Rasagiline has shown no significant toxicity at doses up to 10 mg/kg. The compound's neuroprotective effects are observed at concentrations that do not cause cytotoxicity, suggesting a favorable therapeutic index. However, as with all research compounds, appropriate safety precautions should be taken when handling (S)-Rasagiline.

[1]. Rasagiline [N-propargyl-1R(+)-aminoindan] a selective and potent inhibitor of mitochondrial monoamine oxidase B. Br J Pharmacol. 2001 Jan;132(2):500-6.

1. This study investigated the monoamine oxidase (MAO) A and B inhibitory activities of rasagiline [N-propynyl-1R(+)-aminoindene] and its S(-)-enantiomer (TVP 1022) and racemic compound (AGN-1135) in rats, and compared them with selegiline (1-deprazole). The MAO-inhibiting tissues studied included the brain, liver, and small intestine. 2. Rasagiline and AGN-1135 exhibited highly potent selective irreversible MAO inhibitory activities both in vitro and in vivo, while the S(-) enantiomer showed relatively low activity in the studied tissues. 3. The in vitro IC50 values of rasagiline for MAO activity in rat brain were 4.43 ± 0.92 nM (type B) and 412 ± 123 nM (type A), respectively. Following a single administration of rasagiline, the in vitro ED50 values for MAO-B inhibition in the brain and liver were 0.1±0.01 and 0.042±0.0045 mg kg⁻¹, respectively, while the in vitro ED50 values for MAO-A inhibition were 6.48±0.81 and 2.38±0.35 mg kg⁻¹, respectively. 4. After long-term (21 days) oral administration, the selective inhibition of MAO-B in the liver and brain was maintained, with ED50 values of 0.014±0.002 and 0.013±0.001 mg kg⁻¹, respectively. 5. Rasagiline's selectivity for MAO-B relative to MAO-A is similar to that of selegiline. In acute or chronic administration, rasagiline's inhibitory potency against MAO-B in the rat brain and liver is 3 to 15 times that of selegiline, but their in vitro potency is similar. 6. These data, along with the fact that rasagiline does not enhance tyramine-like sympathomimetic effects at selective MAO-B inhibitory doses, suggest that this inhibitor, like selegiline, may be an effective treatment for Parkinson's disease, for symptomatic treatment or as adjunctive therapy with levodopa. However, rasagiline does not produce amphetamine-like metabolites, which may be one of its therapeutic advantages. [1]
(S)-Rasagiline (TVP1022) is the S-enantiomer of rasagiline, an anti-Parkinson's disease drug. It is approximately 1000-fold less active as an MAO-B inhibitor compared to the R-enantiomer, making it the gold-standard negative control for in vitro and in vivo studies evaluating the secondary effects of MAO-B inhibitors. Despite its lack of MAO-B inhibition, (S)-Rasagiline appears to retain the same neuroprotective activity as the R-isomer, suggesting that neuroprotection is mediated through MAO-B-independent mechanisms. The compound shows cytoprotective effects in models of myocardial infarction, heart failure, and neuronal injury. (S)-Rasagiline contains an alkyne group, enabling its use in copper-catalyzed azide-alkyne cycloaddition (CuAAc) click chemistry reactions. It is strictly for research use only and has not been approved as a therapeutic agent.

C12H13N

171.2383

171.105

C, 84.17; H, 7.65; N, 8.18

185517-74-2

(S)-Rasagiline mesylate;202464-88-8; 136236-51-6; 161735-79-1

5289310

Light yellow to yellow solid powder

0.000816mmHg at 25°C

2.287

1

1

2

13

212

1

C#CCN[C@H]1CCC2=CC=CC=C12

RUOKEQAAGRXIBM-LBPRGKRZSA-N

InChI=1S/C12H13N/c1-2-9-13-12-8-7-10-5-3-4-6-11(10)12/h1,3-6,12-13H,7-9H2/t12-/m0/s1

(1S)-N-prop-2-ynyl-2,3-dihydro-1H-inden-1-amine

185517-74-2; (S)-N-(2-Propynyl)-2,3-dihydroinden-1-amine; TVP1022; N-PROPARGYL-1(S)-AMINOINDAN; (S)-Rasagiline; (1S)-N-prop-2-ynyl-2,3-dihydro-1H-inden-1-amine; MFCD09029306; S-PAI;

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 : ~100 mg/mL (~583.98 mM)
Ethanol : ~100 mg/mL (~583.98 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (14.60 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 (14.60 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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 (Please use freshly prepared in vivo formulations for optimal results.)