Compound 6j (7-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)chroman-4-one) is a novel rasagiline-clorgyline hybrid designed and synthesized as a dual inhibitor targeting monoamine oxidase-B (MAO-B) and amyloid-β (Aβ) aggregation for the treatment of Alzheimer‘s disease. It features a chromanone moiety, a five-carbon flexible linker, and a propargylamine group. Among all synthesized hybrids, compound 6j demonstrated the most potent and selective inhibition of hMAO-B with an IC₅₀ of 4 nM and a selectivity index (SI) greater than 25,000, outperforming the reference drug rasagiline. It also exhibited moderate inhibition of Aβ₁₋₄₂ aggregation (40.78% at 25 μM). The compound was identified as a competitive and reversible MAO-B inhibitor with low cytotoxicity, good neuroprotective effects, and favorable pharmacokinetic properties, including good oral bioavailability (36.1%) and the ability to penetrate the blood-brain barrier. [1]

hMAO-B-IN-2 (compound 6j) exhibited strong inhibitory effects over a 15-minute period at IC50 values of 4. nM for hMAO-B and 6.04 nM for hMAO-A [1]. A reversible MAO-B inhibitor is called hMAO-B-IN-2 (30 min). Compound 6j restored the MAO-B enzyme activity to approximately 82% and 45%, respectively, after being diluted to 0.1 × IC50 and 1 × 50[1]. Significant inhibitory activity against MAO-B and good inhibitory performance against Aβ auto-induced aggregation (40.78 ± 6.27%) are demonstrated by hMAO-B-IN-2 (25 μM, 48 h)[1]. At a dose of 25 μM, hMAO-B-IN-2 (0-100 μM, 24 hours) does not appear to be hazardous [1]. In a dose-dependent manner, hMAO-B-IN-2 (0-25 μM, 24 hours) enhances cell survival and possesses neuroprotective properties against neurodegenerative disorders

---

Compound 6j exhibited potent hMAO-B inhibitory activity with an IC₅₀ of 4.0 ± 0.6 nM, significantly more potent than reference compounds rasagiline (IC₅₀ = 141.7 ± 6.3 nM) and iproniazid (IC₅₀ = 7,410 ± 340 nM). The compound showed no significant inhibition of hMAO-A (IC₅₀ > 100 μM), resulting in a selectivity index (SI) greater than 25,000. [1]
Compound 6j demonstrated moderate inhibition of Aβ₁₋₄₂ self-induced aggregation with an inhibition rate of 40.78 ± 6.27% at 25 μM, compared to the reference compound curcumin (46.1 ± 6.71%). Thioflavin-T fluorescence assay confirmed its anti-aggregation activity, and transmission electron microscopy (TEM) analysis showed that fewer Aβ₁₋₄₂ aggregates were observed when compound 6j was added to the samples. [1]
Compound 6j exhibited good blood-brain barrier permeability in the parallel artificial membrane permeability assay (PAMPA-BBB) with a Pe value of 10.25 ± 0.68 × 10⁻⁶ cm/s, which is higher than the threshold (3.78 × 10⁻⁶ cm/s) for high BBB permeation, predicting CNS+ activity. [1]
Kinetic studies using Lineweaver-Burk reciprocal plots revealed that compound 6j is a competitive inhibitor of hMAO-B, as all lines intersected on the Y-axis. Reversibility experiments demonstrated that after dilution of the enzyme-compound complex to 0.1× IC₅₀ and 1× IC₅₀, enzyme activity was restored to approximately 82% and 45%, respectively, confirming reversible inhibition. [1]
Molecular docking studies using the crystal structure of human MAO-B (PDB code 2V61) showed that the propargylamine moiety of compound 6j is oriented toward the FAD cofactor and establishes an ‘arene-H’ interaction with Tyr435, while the chromanone moiety occupies the entrance cavity and interacts with residues Leu171, Cys172, Ile198, Ile199, Ile316, Tyr326, Trp119, and Phe168 through van der Waals and hydrophobic interactions. [1]

The pharmacokinetic properties of hMAO-B-IN-2 (Compound 6j) (Sprague-Dawley rats; 3 mg/kg IV; 10 mg/kg PO; once) are deemed acceptable [1]. Male Sprague-Dawley rats' hMAO-B-IN-2 pharmacokinetic parameters [1]. AUC0-inf (μg/ L*h) 247.74 ± 11.48 268.49 ± 69.72 CL (L/h/kg) 3.33 ± 0.15 F (%) 36.10% Parameter IV (3 mg/kg) PO (10 mg/kg) T1/2 (h) 1.02 ± 0.17 1.33 ± 0.16 Tmax (h) 0.3 Cmax (μg/L) 639.29 ± 89.06 142.17 ± 72.21

---

Compound 6j demonstrated neuroprotective effects in SH-SY5Y cells against 6-hydroxydopamine (6-OHDA)-induced toxicity. When SH-SY5Y cells were treated with 6-OHDA (200 μM), cell viability significantly decreased. Co-incubation with increasing concentrations of compound 6j (1.563, 3.125, 6.25, 12.5, 25 μM) gradually increased cell survival rates in a concentration-dependent manner, comparable to the reference compound rasagiline. [1]
No direct in vivo efficacy studies (e.g., in animal models of Alzheimer‘s disease) are described for compound 6j in this paper; the study focuses on in vitro and ex vivo (cell-based) evaluations. [1]

hMAO inhibition assay (fluorescence-based Amplex Red method): The inhibitory activities of all synthesized compounds against hMAO-A and hMAO-B were tested using a fluorescence-based Amplex Red assay. Recombinant human MAO enzymes were used, with p-tyramine as the substrate. Each test compound was dissolved in DMSO and diluted with PBS buffer to different final concentrations (DMSO < 0.1%). The test compound was combined with hMAO-A (or hMAO-B) and substrate, then incubated for 15 minutes at 37°C in a 96-well black microtiter plate in the dark. Fluorescence was measured using a multidetection microplate fluorescence reader at excitation/emission wavelengths of 545/590 nm. Data were expressed as mean ± SD of three independent experiments. IC₅₀ values were calculated from concentration-response curves. For compound 6j, the IC₅₀ against hMAO-B was 4.0 ± 0.6 nM. [1]
Reversibility experiment: Compound 6j at concentrations of 10× IC₅₀ and 100× IC₅₀ was pre-incubated with hMAO-B enzyme (0.75 mg/mL) for 30 minutes at 37°C. A parallel control was run without compound. The enzyme-compound mixtures were then diluted 100-fold to final concentrations of 0.1× IC₅₀ and 1× IC₅₀. Residual enzyme activity was measured. Reversible inhibition was confirmed when enzyme activity was restored to approximately 50-90% after dilution. Pargyline was used as an irreversible reference inhibitor. [1]
Kinetic study: To determine the mechanism of inhibition, enzyme kinetic studies were performed. hMAO-B was incubated with various concentrations of compound 6j (2.5, 5, and 10 nM) and increasing concentrations of p-tyramine substrate (0.05-3.0 mM). Lineweaver-Burk reciprocal plots were constructed from the initial reaction rates. The intersection of all lines on the Y-axis indicated competitive inhibition. [1]

Cytotoxicity assay
Cell Types: SH-SY5Y neuroblastoma cells [1]
Tested Concentrations: 0, 6.25, 12.5, 25, 50, 100 μM
Incubation Duration: 24 h
Experimental Results: No toxicity was shown at 25 μM concentration.

---

SH-SY5Y neuroblastoma cell viability assay (MTT): Human neuroblastoma SH-SY5Y cells were cultured in DMEM supplemented with fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in 5% CO₂. Cells were seeded into 96-well plates at a density of 10,000 cells/well. Various concentrations of compound 6j (6.25, 12.5, 25, 50, 100 μM) or rasagiline were added and incubated for 24 hours. Then, 20 μL of MTT solution was added and incubated for 4 hours at 37°C. The medium was removed, and 200 μL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 630 nm. Compound 6j showed no significant cytotoxicity at concentrations up to 25 μM. [1]
Neuroprotection assay: SH-SY5Y cells were treated with 6-hydroxydopamine (6-OHDA, 200 μM) to induce neurotoxicity. Compound 6j at concentrations of 1.563, 3.125, 6.25, 12.5, and 25 μM was co-incubated with 6-OHDA for 24 hours. Cell viability was measured by MTT assay. Compound 6j gradually increased cell survival rates in a concentration-dependent manner, demonstrating neuroprotective effects comparable to rasagiline. [1]

Pharmacokinetic study in Sprague-Dawley rats: Male Sprague-Dawley rats (220 ± 20 g, n = 3 per route) received compound 6j by intravenous injection (3 mg/kg) or oral gavage (10 mg/kg). Blood samples were collected at appropriate intervals (0.083, 0.17, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, and 48 hours post-dose). Plasma was separated by centrifugation and stored at -20°C. Plasma proteins were precipitated using two volumes of 0.5% formic acid in acetonitrile. After centrifugation (14,000 rpm, 14 minutes), supernatants were analyzed by LC-MS/MS. Pharmacokinetic parameters were calculated using DAS 2.0 software. [1]

---

Animal/Disease Models: SD (SD (Sprague-Dawley)) rat (male, 220±20 g) [1]
Doses: 3 mg /kg (IV), 10 mg/kg (PO)
Route of Administration: IV, PO (pharmacokinetic/PK/PK analysis)
Experimental Results: Has acceptable pharmacokinetic/PK/PK properties and exhibits high maximum concentration, appropriate Half-life and good oral bioavailability.

Compound 6j exhibited the following pharmacokinetic parameters in male Sprague-Dawley rats: [1]
Intravenous administration (3 mg/kg): Half-life (t₁/₂) = 1.02 ± 0.17 h; Cmax = 639.29 ± 89.06 μg/L; AUC₀₋ᵢₙf = 247.74 ± 11.48 μg•h/L; Clearance (Cl) = 3.33 ± 0.15 L/h/kg. [1]
Oral administration (10 mg/kg): Half-life (t₁/₂) = 1.33 ± 0.16 h; Tmax = 0.3 h; Cmax = 142.17 ± 72.21 μg/L; AUC₀₋ᵢₙf = 268.49 ± 69.72 μg•h/L; Oral bioavailability (F%) = 36.10%. [1]
Blood-brain barrier permeability: In the PAMPA-BBB assay, compound 6j showed a Pe value of 10.25 ± 0.68 × 10⁻⁶ cm/s, which exceeds the threshold of 3.78 × 10⁻⁶ cm/s for high BBB permeation, indicating that the compound can penetrate the blood-brain barrier and is predicted to be CNS⁺. [1]

Compound 6j showed low cytotoxicity in SH-SY5Y neuroblastoma cells. At concentrations of 6.25, 12.5, 25, 50, and 100 μM, cell viability remained above 80% at concentrations up to 25 μM, with no significant toxicity observed. At the highest concentration tested (100 μM), cell viability was still approximately 75%, indicating a favorable safety profile. [1]
No acute toxicity studies (e.g., LD₅₀ determination), hepatotoxicity, nephrotoxicity, or long-term toxicity data are reported in this paper. The compound is intended for research use and further development as a potential Alzheimer‘s disease therapeutic. [1]

[1]. Design, synthesis and biological evaluation of rasagiline-clorgyline hybrids as novel dual inhibitors of monoamine oxidase-B and amyloid-β aggregation against Alzheimer's disease. Eur J Med Chem. 2020;202:112475.

Compound 6j (chemical name: 7-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)chroman-4-one) is a rasagiline-clorgyline hybrid synthesized via a multi-step procedure. The synthesis involves alkylation of 7-hydroxychroman-4-one with α,ω-dibromoalkanes (1,5-dibromopentane) followed by nucleophilic substitution with N-methylpropargylamine. The compound appears as a yellow oil with a yield of 89% and was characterized by ¹H NMR, ¹³C NMR, and HRMS. [1]
Molecular docking studies revealed that compound 6j binds to hMAO-B with the propargylamine moiety oriented toward the FAD cofactor, forming an ‘arene-H’ interaction with Tyr435. The chromanone moiety occupies the entrance cavity and interacts with hydrophobic residues (Leu171, Cys172, Ile198, Ile199, Ile316, Tyr326, Trp119, Phe168). Compared to rasagiline, compound 6j exhibits additional interactions in the entrance cavity, contributing to its higher binding affinity and selectivity. [1]
In the PAMPA-BBB assay using porcine brain lipid (PBL) as the membrane model, compound 6j showed high BBB permeability (Pe = 10.25 × 10⁻⁶ cm/s), classifying it as a CNS⁺ compound capable of crossing the blood-brain barrier. [1]
Compound 6j is a competitive and reversible MAO-B inhibitor, which is advantageous over irreversible inhibitors for long-term Alzheimer‘s disease treatment as it may reduce the risk of adverse effects associated with permanent enzyme inactivation. [1]

C18H23NO3

301.380125284195

301.167

2454459-87-9

162671381

Typically exists as solid at room temperature

2.6

0

4

8

22

400

0

C1OC2=CC(OCCCCCN(C)CC#C)=CC=C2C(=O)C1

CLKPCTKGZGRHKG-UHFFFAOYSA-N

InChI=1S/C18H23NO3/c1-3-10-19(2)11-5-4-6-12-21-15-7-8-16-17(20)9-13-22-18(16)14-15/h1,7-8,14H,4-6,9-13H2,2H3

7-[5-[methyl(prop-2-ynyl)amino]pentoxy]-2,3-dihydrochromen-4-one

hMAO-B-IN-2; hMAO-B-IN2; CHEMBL4790537; 2454459-87-9; orb1689350; BDBM50565155;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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