ML375 (VU0483253) is a potent, selective, BBB (blood-brain barrier) permeable/penetrable and orally bioactive M5 mAChR negative allosteric modulator (NAM) with IC50 of 300 nM and 790 nM for human and rat M5, respectively. . ML375 is inactive against human and rat M1-M4.

Physicochemical Properties

Molecular Formula	C23H15CLF2N2O2
Molecular Weight	424.83
Exact Mass	424.079
CAS #	1488362-55-5
Related CAS #	1488362-55-5 (S-isomer);1488362-56-6 (R-isomer);
PubChem CID	71598521
Appearance	White to off-white solid powder
Density	1.5±0.1 g/cm3
Boiling Point	611.6±55.0 °C at 760 mmHg
Flash Point	323.7±31.5 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.699
LogP	2.65
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	30
Complexity	701
Defined Atom Stereocenter Count	1
SMILES	C1CN([C@]2(N1C(=O)C3=CC=CC=C32)C4=CC=C(C=C4)Cl)C(=O)C5=CC(=C(C=C5)F)F
InChi Key	GXBAKXRLQAPKEE-QHCPKHFHSA-N
InChi Code	InChI=1S/C23H15ClF2N2O2/c24-16-8-6-15(7-9-16)23-18-4-2-1-3-17(18)22(30)28(23)12-11-27(23)21(29)14-5-10-19(25)20(26)13-14/h1-10,13H,11-12H2/t23-/m0/s1
Chemical Name	(9bS)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydroimidazo[2,1-a]isoindol-5-one
Synonyms	ML-375 VU-0483253 ML 375 VU0483253 ML375VU 0483253
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	In multiple species, ML375 has been found to exhibit low predicted hepatic clearance (CLhep; human, 2.3 mL/min/kg, cynomolgus monkey, 14 mL/min/kg, rat, 18 mL/min/kg) and high metabolic stability. Additionally, ML375 has been found to have low hepatic microsomal intrinsic clearance (CLint; human 2.6 mL/min/kg, cyno 20 mL/min/kg, rat 24 mL/min/kg) [1].
ln Vivo	Cocaine's relative intensity and reinforcing effects are lessened by ML375 (10–30 mg/kg; i.p.; once) [2]. ML375 demonstrates a low clearance (CLp, 2.5 mL/min/kg) and an extended elimination half-life in non-human primates (IV) T1/2, 80 hours) and male Sprague-Dawley rats (1 mg/kg IV). Male, cynomolgus monkey, T1/2 = 10 hours, CLp = 3.0 mL/min/kg, 1 mg/kg) [1]. Following the administration of a suspension dosage to male SD rats, ML375 exhibited high oral bioavailability (%F, 80), with a maximum plasma concentration (Cmax) of 1.4 μM and a corresponding time to Cmax (Tmax) of 7 hours [1].
Animal Protocol	Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats (70 days old; 260-300 g) injected with cocaine [2] Doses: 10 mg/kg, 30 mg/kg Route of Administration: intraperitoneal (ip) injection; primary outcome: cocaine self-administration Produces dose-related reductions.
References	[1]. Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one (ML375). J Med Chem. 2013 Nov 27;56(22):9351-5. [2]. Selective inhibition of M 5 muscarinic acetylcholine receptors attenuates cocaine self-administration in rats. Addict Biol. 2018 Sep;23(5):1106-1116.

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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 Formulations (e.g. IP/IV/IM/SC) 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). 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 Formulations 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). 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3539 mL	11.7694 mL	23.5388 mL
	5 mM	0.4708 mL	2.3539 mL	4.7078 mL
	10 mM	0.2354 mL	1.1769 mL	2.3539 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.