ML328 is a selective inhibitor of bacterial AddAB and RecBCD helicases with IC50s of 26 and 5.1 μM, respectively. ML328 is also an inhibitor (blocker/antagonist) of gyrase. ML328 has an inhibitory effect on the growth of E. coli in the presence of phage. ML328 may be utilized to study bacterial infections.

Physicochemical Properties

Molecular Formula	C22H21F3N6O3S
Molecular Weight	506.50
Exact Mass	506.134
CAS #	634175-34-1
PubChem CID	1517823
Appearance	Typically exists as solid at room temperature
Density	1.5±0.1 g/cm3
Boiling Point	643.0±65.0 °C at 760 mmHg
Flash Point	342.7±34.3 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.657
LogP	2.38
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	4
Heavy Atom Count	35
Complexity	864
Defined Atom Stereocenter Count	0
SMILES	CCN1C=C(C(=O)O)C(=O)C2=CN=C(N=C12)N1CCN(C(NC2=CC=CC(C(F)(F)F)=C2)=S)CC1
InChi Key	USYVBPXJSBDUTJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H21F3N6O3S/c1-2-29-12-16(19(33)34)17(32)15-11-26-20(28-18(15)29)30-6-8-31(9-7-30)21(35)27-14-5-3-4-13(10-14)22(23,24)25/h3-5,10-12H,2,6-9H2,1H3,(H,27,35)(H,33,34)
Chemical Name	8-ethyl-5-oxo-2-[4-[[3-(trifluoromethyl)phenyl]carbamothioyl]piperazin-1-yl]pyrido[2,3-d]pyrimidine-6-carboxylic acid
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

ML328 (0.1-1000 μM) exhibits nuclease inhibitory effects against AddAB and RecBCD, with IC50 values of 26 and 5.1 μM, respectively [1]. In the RecBCD, RecF, and RecE pathways, ML328 selectively inhibits high-frequency recombination [1]. ML328 inhibits RecBCD; its IC50 values are 3, 0.3, and 5 μM for RecBCD nuclease, enhancement of Hfr recombination, and phage lambda recombination, respectively [2]. The inhibition coefficients (IC50) of ML328 against E. coli RecBCD, E. coli AddAB, M. smeg AddAB, and M. smeg RecBCD are 4.6, 16, 2.4, and 5.5 μM, in that order [2]. The frequency of H2O2-induced mutations in E. coli is decreased by Ml328 (25 μM; 2 h) [2]. When applied to E. coli, Ml328 (25 μM; 1 h) decreases the frequency of H2O2-induced valine resistance (valineR) mutation [2]. In the presence of phage, ML328 (50 μM) strongly suppresses E. Coli growth, although it just slightly inhibits it otherwise [2].

References

[1]. Amundsen SK, et al. Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes. ACS Chem Biol. 2012 May 18;7(5):879-91. [2]. SMITH GERALD R, et al. ANTIBIOTIC COMPOUNDS AND COMPOSITIONS, AND METHODS FOR IDENTIFICATION THEREOF. WO/2013/142628. 2014.

Solubility Data

Solubility (In Vitro)

DMSO : ~25 mg/mL (~49.36 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	1.9743 mL	9.8717 mL	19.7433 mL
	5 mM	0.3949 mL	1.9743 mL	3.9487 mL
	10 mM	0.1974 mL	0.9872 mL	1.9743 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.