ML-184 (CID244033) is a selective GPR55 agonist with EC50 of 250 nM and is more than 100 times more selective for GPR55 than GPR35, CB1 and CB2. ML-184 induces ERK1/2 phosphorylation and PKCβII translocation to the plasma membrane through activation of GPR55. ML-184 (CID2440433) increases neural stem cell growth/proliferation and promotes neuronal differentiation in vitro.

Physicochemical Properties

Molecular Formula	C25H34N4O3S
Molecular Weight	470.63
Exact Mass	470.235
Elemental Analysis	C, 63.80; H, 7.28; N, 11.90; O, 10.20; S, 6.81
CAS #	794572-10-4
PubChem CID	2440433
Appearance	White to off-white solid powder
Density	1.2±0.1 g/cm3
Boiling Point	681.4±65.0 °C at 760 mmHg
Flash Point	365.9±34.3 °C
Vapour Pressure	0.0±2.1 mmHg at 25°C
Index of Refraction	1.607
LogP	2.29
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	33
Complexity	769
Defined Atom Stereocenter Count	0
SMILES	O=C(N1CCN(C2C(C)=C(C)C=CC=2)CC1)C1C(N2CCCC2)=CC=C(S(N(C)C)(=O)=O)C=1
InChi Key	VRSJAHQGJHDACS-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H34N4O3S/c1-19-8-7-9-23(20(19)2)28-14-16-29(17-15-28)25(30)22-18-21(33(31,32)26(3)4)10-11-24(22)27-12-5-6-13-27/h7-11,18H,5-6,12-17H2,1-4H3
Chemical Name	3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide
Synonyms	ML184; CID 2440433; ML-184; CID-2440433; CID2440433; ML184; 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-(pyrrolidin-1-yl)benzene-1-sulfonamide; 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide; ML-184; MLS001002737; SMR000369190; ML184; CID2440433
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

Targets	GPR55 (EC50 = 250 nM)
ln Vitro	Activation of GPR55 significantly increased proliferation rates of hNSCs in vitro. These effects were attenuated by ML193, a selective GPR55 antagonist. ML184 significantly promoted neuronal differentiation in vitro while ML193 reduced differentiation rates as compared to vehicle treatment. Continuous administration of O-1602 into the hippocampus via a cannula connected to an osmotic pump resulted in increased Ki67+ cells within the dentate gyrus. O-1602 increased immature neuron generation, as assessed by DCX+ and BrdU+ cells, as compared to vehicle-treated animals. GPR55-/- animals displayed reduced rates of proliferation and neurogenesis within the hippocampus while O-1602 had no effect as compared to vehicle controls.[2]
Enzyme Assay	Chemical Library Screening[1] A β-arrestin (see methods below), high-throughput, high-content screen (HCS) of 300,000 compounds was used here to identify potent GPR55 selective antagonists. This work was performed in collaboration with the Molecular Libraries Probe Production Centers Network program. For more details about this library of compounds, see http://mli.nih.gov/mli/compound-repository/mlsmr-compounds/. Compounds were screened for antagonism (PubChem AID 2026) at GPR55 (using LPI as the agonist), as well as for both agonism and antagonism at GPR35 (PubChem AIDs 2809, 2815), CB1 (PubChem AIDs 2814, 2835) and CB2 (PubChem AIDs 2822, 2836). A cell line permanently expressing a β-arrestin GFP biosensor and an enhanced receptor of interest (i.e., GPR55, GPR35, CB1 or CB2) were employed in the high content imaging assay. Assay protocol descriptions (according to AID number) are accessible at the PubChem website (http://pubchem.ncbi.nlm.nih.gov/). Potent GPR55 antagonist compounds that lacked agonism or antagonism at GPR35, CB1 or CB2, were further evaluated for inhibition of pERK activation and PKCβII translocation produced by the GPR55 agonists, LPI or ML186 (see methods below). A set of novel GPR55 antagonist molecular scaffolds were selected from the screen ML191 (CID23612552), ML192 (CID1434953) and M193(CID1261822), and the binding of each compound was explored using a computer model of the GPR55 inactive state.
Cell Assay	To investigate the effects of GPR55 activation on hNSC proliferation, cells were plated on laminin‐coated 6‐well plates. Cells were allowed to adhere overnight and then treated with LPI (1 μM), the endogenous ligand for GPR55, or synthetic agonists, O‐1602 (1 μM) or ML184 (1 μM), in a reduced growth factor media (5% growth factor). Reduced growth factor medium was utilized to better mimic a less proliferative phenotype while still maintaining a ‘stemness’ state. Analysis by flow cytometry showed no significant reduction of nestin+ or Sox2+ populations after 48 h (data not shown). Cells treated with the selective GPR55 antagonist ML193 (5 μM) were pretreated for 30 min prior to addition of agonist. Vehicle‐treated cells received 0.1% DMSO in 5% growth factor media. For differentiation studies, cells were treated with either vehicle, ML184 (1 μM), ML193 (5 μM), or a combination of ML184 (1 μM) and ML193 (5 μM) in ReNcell medium that did not contain growth factors.[2]
References	[1]. Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands. Biochemistry. 2011 Jun 28;50(25):5633-47. [2]. Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis. Br J Pharmacol. 2018 Aug;175(16):3407-3421.
Additional Infomation	3-[[4-(2,3-dimethylphenyl)-1-piperazinyl]-oxomethyl]-N,N-dimethyl-4-(1-pyrrolidinyl)benzenesulfonamide is a member of piperazines.

Solubility Data

Solubility (In Vitro)

DMSO: ~12.5 mg/mL (~26.6 mM)

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	2.1248 mL	10.6241 mL	21.2481 mL
	5 mM	0.4250 mL	2.1248 mL	4.2496 mL
	10 mM	0.2125 mL	1.0624 mL	2.1248 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.