DRI-C21045 (compound 10) is a potent and specific inhibitor of the CD40-CD40L costimulatory protein-protein interaction (PPI) with IC50 of 0.17 μM. DRI-C21045 showed concentration-dependent inhibition of CD40L-induced NF-κB activation and B cell growth/proliferation with IC50 of 17.1 μM and 4.5 μM, respectively.

Physicochemical Properties

Molecular Formula	C32H24N2O7S
Molecular Weight	580.607167243958
Exact Mass	580.13
CAS #	2101765-81-3
PubChem CID	129275747
Appearance	Off-white to gray solid powder
LogP	5.1
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	8
Heavy Atom Count	42
Complexity	1050
Defined Atom Stereocenter Count	0
SMILES	S(C1C=CC=C2C=CC=C(C2=1)NC(C1C=CC(=CC=1)C1C=CC(=CC=1)NC(C1C=CC(C(=O)OC)=CC=1)=O)=O)(=O)(=O)O
InChi Key	BZAWLSZPOBGURT-UHFFFAOYSA-N
InChi Code	InChI=1S/C32H24N2O7S/c1-41-32(37)25-14-12-23(13-15-25)30(35)33-26-18-16-21(17-19-26)20-8-10-24(11-9-20)31(36)34-27-6-2-4-22-5-3-7-28(29(22)27)42(38,39)40/h2-19H,1H3,(H,33,35)(H,34,36)(H,38,39,40)
Chemical Name	8-[[4-[4-[(4-methoxycarbonylbenzoyl)amino]phenyl]benzoyl]amino]naphthalene-1-sulfonic 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

Targets	DRI-C21045 targets CD40-CD40L costimulatory protein-protein interaction (PPI) (IC₅₀ = 0.17 μM) [1, 2, 4, 6] DRI-C21045 inhibits CD40L-induced NF-κB activation (IC₅₀ = 17.1 μM) and CD40L-induced B cell proliferation (IC₅₀ = 4.5 μM) [2, 4, 6]
ln Vitro	CD40L-induced NF-κB activation in CD40 sensor cells is concentration-dependently inhibited by DRI-C21045 (3.2-100 μM; 18 h) [1]. Primary B cells' CD40L-induced functional activation is blocked by DRI-C21045 (0.6–50 μM; 48 hours) [1]. In THP-1 cells, CD40L-induced MHC-II upregulation is inhibited by DRI-C21045 (0.4-50 μM; 48 hours) [1]. The 48-hour DRI-C21045 (2-100 μM) inhibits the proliferation of B cells induced by CD40L [1]. At concentrations of up to 500 µM, DRI-C21045 exhibits no potential genotoxicity and no indications of cytotoxicity [1]. - In cell-free ELISA-type assay, DRI-C21045 concentration-dependently inhibited human CD40-CD40L interaction, with no significant inhibition of human TNF-R1-TNF-α interaction at effective concentrations [1] - In CD40 sensor cells, DRI-C21045 (3.2–100 μM, 18 h) concentration-dependently inhibited CD40L (20 ng/mL)-induced NF-κB activation, with an anti-CD40L antibody as positive control [1, 2] - In primary human B cells stimulated with IL-4 (0.2 μg/mL) and CD40L (0.2 μg/mL), DRI-C21045 (0.6–50 μM, 48 h) blocked CD40L-induced functional activation (AID activation quantified via fluorescent marker by flow cytometry) and (2–100 μM, 48 h) inhibited CD40L-induced B cell proliferation [1, 2] - In THP-1 myeloid cells, DRI-C21045 (0.4–50 μM, 48 h) inhibited CD40L-induced MHC-II upregulation [2] - At concentrations up to 500 μM, DRI-C21045 showed no potential genotoxicity or cytotoxicity [2]
ln Vivo	In a mouse skin allograft model, DRI-C21045 (30 mg/kg; daily, subcutaneous; in 20% HPβCD) prolongs graft life [1]. In draining lymph nodes (DLN), DRI-C21045 (20–60 mg/kg; subcutaneous injection twice daily; dissolved in 20% HPβCD) suppresses T cell growth induced by alloantigen [1]. - In a mouse skin allograft model (full-thickness ear skin transplantation from Balb/c to dorsal thorax of C57BL/6 mice), DRI-C21045 (30 mg/kg, daily subcutaneous injection in 20% HPβCD) combined with CTLA4-Ig (250 μg on days 0, 2, 4, 6) significantly prolonged skin allograft survival compared to CTLA4-Ig alone [1, 2] - In Balb/c mice receiving footpad injection of DBA-2 mouse splenocytes, DRI-C21045 (20–60 mg/kg, twice daily subcutaneous injection in 20% HPβCD from day -1 to 3) dose-dependently suppressed alloantigen-induced T cell expansion in draining popliteal lymph nodes [1, 2]
Enzyme Assay	- Cell-free CD40-CD40L interaction assay: The interaction between human CD40 and CD40L was quantified using an ELISA-type assay. DRI-C21045 at different concentrations was added, and the inhibitory effect was evaluated by measuring normalized percent binding. Data were presented as average ± SD from 3 independent experiments with triplicates per condition, and standard binding curves were fitted [1] - CD40L-induced NF-κB activation assay in CD40 sensor cells: CD40 sensor cells were treated with DRI-C21045 at various concentrations (3.2–100 μM) for 18 h, then stimulated with CD40L (20 ng/mL). NF-κB activation level was detected, and data were collected from 4 independent experiments with duplicates per condition, normalized to CD40L-activated cells alone [1, 2]
Cell Assay	- Primary human B cell functional activation assay: Human B cells were transfected with a lentiviral construct containing the aicda promoter/enhancer fused to Ds-Red reporter. Transfected cells were treated with DRI-C21045 (0.6–50 μM) and stimulated with IL-4 (0.2 μg/mL) + CD40L (0.2 μg/mL) for 48 h. AID activation was evaluated by Ds-Red level via flow cytometry, with an anti-CD40L antibody (33.3 nM) as positive control [1, 2] - Primary human B cell proliferation assay: Primary human B cells were treated with DRI-C21045 (2–100 μM) and stimulated with CD40L for 48 h. Cell proliferation was measured to assess inhibitory effect [2] - THP-1 cell MHC-II upregulation assay: THP-1 cells were treated with DRI-C21045 (0.4–50 μM) and stimulated with CD40L for 48 h. MHC-II expression level was detected to evaluate inhibitory activity [2]
Animal Protocol	Animal/Disease Models: Full-thickness ear skin transplantation from BALB/c to dorsal thorax of C57BL/6 [1] Doses: 30 mg/kg Route of Administration: daily subcutaneous injection; in 20% w/v hydroxypropyl-β-cyclo Administration of dextrin (HPβCD) in solution resulted in prolonged skin allograft survival. - Mouse skin allograft experiment: C57BL/6 mice were transplanted with full-thickness ear skin from Balb/c mice. Mice were treated with DRI-C21045 (30 mg/kg, daily subcutaneous injection) dissolved in 20% w/v hydroxypropyl-β-cyclodextrin (HPβCD), combined with CTLA4-Ig (250 μg administered on days 0, 2, 4, 6). Graft survival time was monitored, with 5–8 mice per group [1, 2] - Mouse draining lymph node (DLN) T cell expansion experiment: Balb/c mice received footpad injection of DBA-2 mouse splenocytes. Mice were treated with DRI-C21045 (20–60 mg/kg, twice daily subcutaneous injection) dissolved in 20% HPβCD from day -1 to 3. On day 3 post-alloantigen challenge, popliteal DLNs were collected and DLN cell count was performed to assess T cell expansion, with 3–4 mice per group [1, 2] - Mouse pharmacokinetic experiment: Mice received a single subcutaneous dose of DRI-C21045 (1.6 mg/mouse). Plasma concentrations of DRI-C21045 were measured at different time points, and data were fitted with a one-compartment pharmacokinetic model with first-order absorption and elimination (n = 4, average ± SD) [1]
ADME/Pharmacokinetics	- In mice, following a single subcutaneous dose of DRI-C21045 (1.6 mg/mouse), plasma concentration-time profile was fitted to a one-compartment model with first-order absorption and first-order elimination [1]
Toxicity/Toxicokinetics	- In vitro, DRI-C21045 exhibited no cytotoxicity or genotoxicity at concentrations up to 500 μM [2] - In vivo, no overt toxicity (e.g., weight loss, clinical signs) was observed in mice treated with DRI-C21045 at doses up to 60 mg/kg (twice daily subcutaneous injection) [1, 2]
References	[1]. Small-Molecule Inhibitors of the CD40-CD40L Costimulatory Protein-Protein Interaction. J Med Chem. 2017 Nov 9;60(21):8906-8922. [2]. Toward Small-Molecule Inhibition of Protein-Protein Interactions: General Aspects and Recent Progress in Targeting Costimulatory and Coinhibitory (Immune Checkpoint) Interactions. Curr Top Med Chem. 2018;18(8):674-699.
Additional Infomation	- Chemical properties of DRI-C21045: CAS No. 2101765-81-3; molecular weight 580.61; molecular formula C₃₂H₂₄N₂O₇S [4, 6] - Solubility of DRI-C21045: 2 mg/mL (3.44 mM) in DMSO (requires ultrasonic treatment) [6] - Storage conditions of DRI-C21045: Powder stored at -20°C for 3 years; solution stored at -80°C for 2 years [6] - DRI-C21045 is a potent and selective small-molecule inhibitor of CD40-CD40L PPI, developed for immunomodulation in transplantation and autoimmune diseases [1, 2]

Solubility Data

Solubility (In Vitro)

DMSO : ~2 mg/mL (~3.44 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 0.5 mg/mL (0.86 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 5.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.7223 mL	8.6116 mL	17.2233 mL
	5 mM	0.3445 mL	1.7223 mL	3.4447 mL
	10 mM	0.1722 mL	0.8612 mL	1.7223 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.