TTT-28 is a thiazole-valine peptide mimetic, a potent ABCB1 (P-gp/MDR1) selective inhibitor (antagonist) with low toxicity. TTT-28 reverses ABCB1-mediated multidrug resistance (MDR) by selectively blocking the efflux function of ATP-binding cassette subfamily B member 1 (ABCB1).

Physicochemical Properties

Molecular Formula	C31H31N3O6S
Molecular Weight	573.659346818924
Exact Mass	573.193
CAS #	1609138-51-3
PubChem CID	86765226
Appearance	White to off-white solid powder
LogP	6.1
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	11
Heavy Atom Count	41
Complexity	870
Defined Atom Stereocenter Count	1
SMILES	S1C=C(C(NC2=CC=CC=C2C(C2C=CC=CC=2)=O)=O)N=C1[C@H](C(C)C)NC(C1C=C(C(=C(C=1)OC)OC)OC)=O
InChi Key	VUSXKXGCZNKQFS-SANMLTNESA-N
InChi Code	InChI=1S/C31H31N3O6S/c1-18(2)26(34-29(36)20-15-24(38-3)28(40-5)25(16-20)39-4)31-33-23(17-41-31)30(37)32-22-14-10-9-13-21(22)27(35)19-11-7-6-8-12-19/h6-18,26H,1-5H3,(H,32,37)(H,34,36)/t26-/m0/s1
Chemical Name	N-(2-benzoylphenyl)-2-[(1S)-2-methyl-1-[(3,4,5-trimethoxybenzoyl)amino]propyl]-1,3-thiazole-4-carboxamide
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	TTT-28 (0-100 μM; 72 hours) reverses ABCB1-mediated MDR in drug-selected SW620/Ad300 cells and transfected HEK293/ABCB1 cells; TTT-28 in CCD-18Co, SW620, and SW620/Ad300 cells The IC50 values in are 213.4±11.0 μM, 89.4±3.9 μM and 92.0±5.0 μM respectively[1]. TTT-28 (10 μM; 2 hours) promotes ABCB1-mediated MDR and increases [3H]-paclitaxel accumulation in ABCB1-overexpressing cells [1]. TTT-28 (10 μM; 0-72 hours) does not interfere with the expression level and localization of ABCB1 and is created by blocking the efflux function of ABCB1 [1]. TTT-28 (0-40 μM; 2 hours) binds at the drug-substrate binding site and increases the ATPase activity of ABCB1 in a concentration-dependent manner [1].
ln Vivo	Because TTT-28 (oral; 30 mg/kg; every 3 days; 18 days) inhibits the efflux function of ABCB1, it increases the anticancer activity of paclitaxel by inhibiting the growth of SW620/Ad300 tumors and promoting apoptosis[1].
Cell Assay	Cell Viability Assay[1] Cell Types: SW620 cells, SW620/Ad300 cells Tested Concentrations: 0.1 μM, 1 μM, 10 μM, 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: ABCB1 substrate (paclitaxel, doxorubicin, vincristine) The fold resistance of SW620/Ad300 cells was diminished compared with SW620 cells.
Animal Protocol	Animal/Disease Models: 5-10 weeks old male athymic NCR (nu/nu) nude mice ABCB1 overexpression SW620/Ad300 cell tumor xenograft model Doses: 30mg/kg Route of Administration: oral; Route of Administration: oral. Once every three days; 18-day Experimental Results: Causes increased intratumoral accumulation of paclitaxel in the tumor.
References	[1]. Thiazole-valine peptidomimetic (TTT-28) antagonizes multidrug resistance in vitro and in vivo by selectively inhibiting the efflux activity of ABCB1. Sci Rep. 2017 Feb 9;7:42106.

Solubility Data

Solubility (In Vitro)

DMSO : ~250 mg/mL (~435.80 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	1.7432 mL	8.7160 mL	17.4319 mL
	5 mM	0.3486 mL	1.7432 mL	3.4864 mL
	10 mM	0.1743 mL	0.8716 mL	1.7432 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.