Ribocil is a novel, potenty and highly selective chemical modulator of bacterial riboflavin riboswitches. Ribocil strongly inhibits GFP expression, achieving a 50% effective concentration (EC50) of 0.3 μM. Ribocil is a highly specific bioactive synthetic mimic of FMN, which competes with the natural ligand to inhibit FMN riboswitch-mediated expression of ribB and inhibits bacterial growth. Ribocil-B demonstrates superior microbiological activity as compared to Ribocil-A (minimum inhibitory concentration (MIC) = 1 μg/ml versus MIC ≥ 64 μg/ml), inhibition of riboflavin synthesis (IC50 = 0.13 μM versus IC50 > 26 μM), and binding affinity to the E. coli FMN aptamer (Kd = 6.6 nM versus Kd ≥ 10,000 nM). Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors.

Physicochemical Properties

Molecular Formula	C19H22N6OS
Molecular Weight	382.482581615448
Exact Mass	382.157
Elemental Analysis	C, 59.66; H, 5.80; N, 21.97; O, 4.18; S, 8.38
CAS #	1381289-58-2
Related CAS #	Ribocil-C;1825355-56-3;Ribocil B;1825355-55-2;Ribocil-C Racemate;2309762-18-1
PubChem CID	136881500
Appearance	Solid powder
Density	1.3±0.1 g/cm3
Boiling Point	610.4±65.0 °C at 760 mmHg
Flash Point	323.0±34.3 °C
Vapour Pressure	0.0±1.8 mmHg at 25°C
Index of Refraction	1.673
LogP	0.99
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	5
Heavy Atom Count	27
Complexity	601
Defined Atom Stereocenter Count	0
SMILES	S1C=CC=C1C1=CC(NC(C2CN(CC3=CN=C(NC)N=C3)CCC2)=N1)=O
InChi Key	ZSXCVAIJFUEGJR-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H22N6OS/c1-20-19-21-9-13(10-22-19)11-25-6-2-4-14(12-25)18-23-15(8-17(26)24-18)16-5-3-7-27-16/h3,5,7-10,14H,2,4,6,11-12H2,1H3,(H,20,21,22)(H,23,24,26)
Chemical Name	2-(1-((2-(methylamino)pyrimidin-5-yl)methyl)piperidin-3-yl)-6-(thiophen-2-yl)pyrimidin-4(3H)-one
Synonyms	Ribocil;
HS Tariff Code	2934.99.03.00
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

Ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

References

[1]. Selective small-molecule inhibition of an RNA structural element. Nature. 2015 Oct 29;526(7575):672-7.

Solubility Data

Solubility (In Vitro)

DMSO : ~12.5 mg/mL (~32.68 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.27 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 12.5 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (3.27 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 12.5 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 3: ≥ 1.25 mg/mL (3.27 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 1.25 mg/mL (3.27 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6145 mL	13.0726 mL	26.1452 mL
	5 mM	0.5229 mL	2.6145 mL	5.2290 mL
	10 mM	0.2615 mL	1.3073 mL	2.6145 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.