ML-18 (the S-enantiomer, the enantiomer is EMY-98), a nonpeptide analogs of PD176252, is a BRS-3 (Bombesin receptor subtype 3) antagonist with an IC50 of 4.8 μM. ML-18 and EMY-98, with IC50 values of 4.8 and >100μM, respectively, inhibited specific (125)I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB(6-14) binding to NCI-H1299 lung cancer cells that were stably transfected with BRS-3. On the other hand, ML-18 exhibited a lower affinity for binding to the GRPR and NMBR, with IC50 values exceeding 100μM and 16μM, respectively. With lung cancer cells loaded with FURA2-AM, ML-18 (16μM) but not its enantiomer EMY-98 inhibited 10nM BA1's capacity to elevate cytosolic Ca(2+) in a reversible manner. Lung cancer cells' ability to become tyrosine phosphorylated on EGFR and ERK in response to 100nM BA1 was inhibited by ML-18 (16μM), but not by EMY-98. ML-18 but not EMY-98 inhibited the proliferation of lung cancer cells. The findings suggest that ML-18 is a nonpeptide antagonist of BRS-3 that can be used as a model to increase potency and selectivity. A G protein coupled receptor (GPCR) for the bombesin (BB)-family of peptides is known as the bombesin receptor subtype (BRS)-3. As an orphan GPCR, BRS-3's physiological function is poorly understood because there aren't any particular agonists or antagonists. While PD176252 is a nonpeptide antagonist for the gastrin releasing peptide (GRP) R and NMBR but not BRS-3, PD168368 is a nonpeptide antagonist for the neuromedin B (NMB) receptor (R).

Physicochemical Properties

Molecular Formula	C32H35N5O5
Molecular Weight	569.66
Exact Mass	569.263
Elemental Analysis	C, 67.47; H, 6.19; N, 12.29; O, 14.04
CAS #	1422269-30-4
Related CAS #	1422269-30-4
PubChem CID	91827363
Appearance	White to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	803.5±75.0 °C at 760 mmHg
Flash Point	439.7±37.1 °C
Vapour Pressure	0.0±3.0 mmHg at 25°C
Index of Refraction	1.652
LogP	8.03
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	9
Heavy Atom Count	42
Complexity	906
Defined Atom Stereocenter Count	1
SMILES	O=C([C@]([H])(C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-])=O)N([H])C([H])([H])C1(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]
InChi Key	JOKVJNCYOSFDGC-LJAQVGFWSA-N
InChi Code	InChI=1S/C32H35N5O5/c1-42-26-15-9-23(10-16-26)32(17-5-2-6-18-32)21-34-30(38)29(19-22-20-33-28-8-4-3-7-27(22)28)36-31(39)35-24-11-13-25(14-12-24)37(40)41/h3-4,7-16,20,29,33H,2,5-6,17-19,21H2,1H3,(H,34,38)(H2,35,36,39)/t29-/m0/s1
Chemical Name	(2S)-3-(1H-indol-3-yl)-N-[[1-(4-methoxyphenyl)cyclohexyl]methyl]-2-[(4-nitrophenyl)carbamoylamino]propanamide
Synonyms	ML 18; ML-18; ML18
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	BRS-3 ( IC50 = 4.8 μM )
ln Vitro	ML-18 blocks specific 125I-BA1 (DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2)BB6-14 binding to NCI-H1299 lung cancer cells that have been stably transfected with BRS-3 with IC50 values of 4.8 μM. ML-18 exhibits a reduced affinity for binding the GRPR and NMBR, with IC50 values exceeding 100 μM and 16 μM, respectively. ML-18 at 16 μM reversibly prevents 10 nM BA1 from increasing cytosolic Ca2+ in lung cancer cells that have been loaded with FURA2-AM. ML-18 at 16 μM prevents 100 nM BA1 from causing EGFR and ERK tyrosine phosphorylation in lung cancer cells. It prevents lung cancer cells from proliferating[1].
Enzyme Assay	The cells are cultured in SIT buffer supplemented with varying concentrations of unlabelled competitor (ML-18), 0.25% bovine serum albumin, 250 μg/mL bacitracin, and 1253I-BA1 (100,000 cpm). Following a 30-minute incubation period at 37°C, free 1253I-BA1 is eliminated by three rounds of buffer washing, and the cells containing bound 1253I-BA1 are dissolved in 0.2 N NaOH and counted using a gamma counter. For every competitor without a label, the IC50 is computed[1].
Cell Assay	The MTT assay is used to determine cell viability. After transfecting NCI-H727 or NCI-H1299 cells with BRS-3, ML-18 (0, 4.8, 16, 48 μM) or gefitinib is added. 15 μL of 0.1% MTT solution was added after two days. 150 μL of DMSO is added after 4 hours. The optical density at 570 nm is calculated after 16 hours[1].
References	[1]. ML-18 is a non-peptide bombesin receptor subtype-3 antagonist which inhibits lung cancer growth. Peptides. 2015 Feb;64:55-61.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 100 mg/mL (~175.6 mM) Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.39 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7554 mL	8.7772 mL	17.5543 mL
	5 mM	0.3511 mL	1.7554 mL	3.5109 mL
	10 mM	0.1755 mL	0.8777 mL	1.7554 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.