Interleukin-1 receptor-associated kinase 4 (IRAK4). ND-2158 is a potent and selective small molecule inhibitor of IRAK4 kinase activity, acting as a competitive inhibitor that binds in the ATP pocket. The in vitro inhibitory constant (Ki) against IRAK4 is 1.3 nM. [1]

ND-2158 potently blocks the production of the proinflammatory cytokine TNF by human white blood cells (WBCs) stimulated with the TLR4 agonist LPS or the TLR9 agonist CpG. The structurally related compound ND-1659, which does not inhibit IRAK4 kinase activity, is inactive. [1]
In a panel of diffuse large B-cell lymphoma (DLBCL) lines, ND-2158 reduces the viability of ABC DLBCL lines harboring the MYD88 L265P mutation but does not affect GCB DLBCL lines with wild-type MYD88 or ABC DLBCL lines with other MYD88 mutations (S222R and S219C). [1]
In ABC DLBCL lines (OCI-Ly10, TMD8, HBL1, OCI-Ly3), ND-2158 treatment leads to down-modulation of survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK-STAT3 pathway, as shown by gene expression profiling. [1]
ND-2158 decreases BCL2L1 and BCL2A1 mRNA levels but increases BCL2 expression in ABC DLBCL lines. [1]
In combination studies, ND-2158 synergistically enhances the ability of the BTK inhibitor ibrutinib, the Syk inhibitor PRT062607, and the Bcl-2 inhibitor ABT-199 to decrease viability and promote apoptosis (increased cleaved PARP and activated caspase-3) in ABC DLBCL lines. Synergy was observed even in the CARD11 mutant ABC line OCI-Ly3 that is insensitive to ibrutinib. [1]

In a rat model of LPS-induced TNF production, ND-2158 significantly reduces serum TNF levels in a dose-dependent manner (1, 3, and 30 mg/kg, i.p.). [1]
In a mouse collagen-induced arthritis model, ND-2158 (30 mg/kg i.p., twice daily) is effective within 2 days of initiating treatment, reducing clinical scores and improving histological parameters of inflammation (inflammatory cell influx, cartilage damage, bone resorption, and periosteal bone formation). [1]
In a mouse air pouch model of gout, ND-2158 (3, 10, and 30 mg/kg i.p., once daily for 6 days) blocks leukocyte infiltration in a dose-dependent manner, with higher doses achieving more than a two-fold reduction in leukocyte influx compared to vehicle control. [1]
In a mouse xenograft model of ABC DLBCL (OCI-Ly10, MYD88 L265P+), ND-2158 (100 mg/kg/day, i.p., twice daily) retards tumor growth and is well tolerated, without significant body weight loss. ND-2158 reduces IRAK4 phosphorylation (Thr-345/Ser-346) in the tumors. [1]
In a mouse xenograft model of ABC DLBCL (OCI-Ly3, CARD11 mutant), ND-2158 (100 mg/kg/day, i.p., twice daily) combined with ABT-199 (50 mg/kg/day, oral) produces near-complete inhibition of tumor growth, whereas each agent alone only moderately suppresses growth. [1]

Kinase Assay: The inhibitory activity of ND-2158 against IRAK4 and IRAK1 was evaluated using radioisotope-based enzymatic assays. IC50 and Ki values were obtained from dose-inhibition assays. Broad kinase selectivity was assessed against 334 kinases at a single concentration (10 μM) of ND-2158. For kinases showing ≥75% inhibition at 10 μM, IC50 and Ki were subsequently determined. Dose-response curves were generated using four-parameter fit. [1]

MTS Viability Assay: Approximately 10⁴ cells per well of each DLBCL line were cultured in triplicate in 96-well plates with vehicle (DMSO) or indicated drugs. On day 2, wells were replenished with freshly prepared drugs. On day 4, cell viability was assayed by adding MTS tetrazolium reagent and an electron coupling reagent, incubating for 2 hours at 37°C, and measuring absorbance at 490 nm. For single-agent experiments, data were normalized to DMSO-treated cells. For combination experiments, data were first normalized to DMSO-treated cells and then to cells treated with ND-2158 alone. [1]
Apoptosis Measurement (Flow Cytometry): DLBCL lines were cultured for 72 hours with indicated drugs. Apoptosis was measured by intracellular flow cytometry for activated caspase-3 and cleaved PARP. Apoptotic cells were defined as the percentage of cells double-positive for both markers. [1]
NF-κB and IκBα Reporter Assays: Stable NF-κB firefly luciferase reporter ABC DLBCL lines were generated. Cells were seeded at 2.5 × 10⁵ cells/mL in 96-well plates and treated with drugs for indicated times. Firefly luciferase and Renilla activity were measured using the Dual-Glo Luciferase Reporter Assay System. Relative NF-κB activity was determined by normalizing firefly to Renilla luciferase titers. [1]
IL-6 and IL-10 ELISA: ABC DLBCL lines were cultured for 48 hours with ND-2158 or vehicle control. Concentrations of IL-6 and IL-10 in cell culture supernatants were measured using ELISA kits according to the manufacturer's protocol. Each experiment was performed in triplicate. [1]
LPS/CpG-Induced Cytokine Release: Human white blood cells were isolated from buffy coat by red blood cell lysis. Cells were preincubated for 1 hour with ND-2158 (0.3% DMSO final) before stimulation with 1 μg/mL LPS or 0.5 μM CpG for 20 hours. TNF production in the supernatant was detected using an ELISA kit. [1]
Gene Expression Profiling: ABC DLBCL lines were treated with either 10 μM ND-2158 or 10 μM negative control compound ND-1659 for 6, 12, 24, or 36 hours. RNA was extracted using TRIzol and cleaned up with an RNeasy kit. Gene expression profiling was performed using two-color human gene expression arrays, comparing signal from ND-1659-treated control cells (Cy3) with ND-2158-treated cells (Cy5). [1]

LPS-Induced TNF Production Model (Rat): Female Lewis rats (n=10 per group) were dosed intraperitoneally (i.p.) with ND-2158 (1, 3, and 30 mg/kg) or vehicle (10% hydroxypropyl-β-cyclodextrin in saline). Dexamethasone was administered orally as a positive control. Thirty minutes after dosing, rats were intravenously administered 0.1 mg/kg LPS in PBS. One hour later, rats were bled and serum analyzed for TNF production by ELISA. [1]
Collagen-Induced Arthritis Model (Mouse): Male DBA mice were immunized with type II collagen emulsified in complete Freund's adjuvant on days 0 and 21. Mice were randomly enrolled at disease onset (n=8 per group, except n=4 naive controls). ND-2158 or vehicle (10% hydroxypropyl-β-cyclodextrin in ddH₂O) was administered i.p. Dexamethasone (0.1 mg/kg i.p., twice daily) served as a positive control. ND-2158 was dosed at 30 mg/kg i.p. twice daily. Mice were evaluated daily for clinical scores until day 11. Histological analysis was conducted on harvested tissue sections. [1]
MSU Air Pouch Gout Model (Mouse): BALB/c mice were distributed randomly into groups of 10. ND-2158 or vehicle (10% hydroxypropyl-β-cyclodextrin in ddH₂O) was administered i.p. (3, 10, and 30 mg/kg). After the first dose, an air pouch was generated by injecting 6 mL of air into the nape of the neck. Mice were subsequently dosed once daily with ND-2158 or vehicle for 6 days. Thirty minutes after the final dose, 3 mL of monosodium urate (MSU) suspension was injected into the air pouch. Four hours after MSU injection, exudate was removed and used for white blood cell count and differential analysis. [1]
Tumor Xenograft Models (Mouse): Female NOD/SCID mice (6-8 weeks old) were inoculated subcutaneously with a tumor cell suspension containing Matrigel/PBS (1:1). For OCI-Ly10, 10 × 10⁶ cells were inoculated; for OCI-Ly3, 10⁶ cells were inoculated. When tumors reached a mean volume of 150 mm³, treatment was initiated. ND-2158 (100 mg/kg/day) or vehicle was administered i.p. twice daily. For combination experiments, ibrutinib (9 mg/kg/day, i.p., once daily) or ABT-199 (50 mg/kg/day, oral gavage, once daily) was administered 4 hours after ND-2158. Tumor growth was monitored daily by digital caliper measurement. [1]
Mouse Pharmacokinetic Analysis: For ND-2158, male C57BL/6 mice were dosed either i.v. or i.p. at 3 mg/kg in 10% hydroxypropyl-β-cyclodextrin. Blood was collected before dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after dosing. Plasma was prepared and ND-2158 levels were quantified by LC/MS/MS. Pharmacokinetic parameters were calculated using Phoenix WinNonlin. [1]

In mice, ND-2158 exhibits attractive drug-like properties including good solubility, cell permeability, and a pharmacokinetic profile suitable for in vivo studies. [1]
For ND-2158, a mouse pharmacokinetic study was performed with i.v. or i.p. dosing at 3 mg/kg in 10% hydroxypropyl-β-cyclodextrin. Blood samples were collected over 24 hours, and plasma drug levels were quantified by LC/MS/MS. [1]

In the mouse OCI-Ly10 xenograft model, ND-2158 (100 mg/kg/day, i.p., twice daily for 14 days) was well tolerated, with no significant body weight loss observed in treated mice compared to vehicle controls. [1]
In the mouse collagen-induced arthritis model, ND-2158 (30 mg/kg i.p., twice daily) was administered without reported overt toxicity. [1]

[1]. Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy. J Exp Med. 2015;212(13):2189-2201.

In the mouse OCI-Ly10 xenograft model, ND-2158 (100 mg/kg/day, i.p., twice daily for 14 days) was well tolerated, with no significant body weight loss observed in treated mice compared to vehicle controls. [1]
In the mouse collagen-induced arthritis model, ND-2158 (30 mg/kg i.p., twice daily) was administered without reported overt toxicity. [1]

C22H30N4O4S

446.563004016876

446.198

C, 59.17; H, 6.77; N, 12.55; O, 14.33; S, 7.18

1388896-07-8

1388896-07-8;

86343751

Typically exists as solid at room temperature

2

2

8

6

31

629

2

C1CC2=C([C@H]1C[C@@H](C(=O)N)O)C3=C(N=CN=C3S2)OC4CCC(CC4)N5CCOCC5

ZSMULWAVKYFPSS-FPCVCCKLSA-N

InChI=1S/C22H30N4O4S/c23-20(28)16(27)11-13-1-6-17-18(13)19-21(24-12-25-22(19)31-17)30-15-4-2-14(3-5-15)26-7-9-29-10-8-26/h12-16,27H,1-11H2,(H2,23,28)/t13-,14-,15-,16+/m1/s1

(S)-2-hydroxy-3-((R)-4-(((1r,4R)-4-morpholinocyclohexyl)oxy)-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-5-yl)propanamide

ND-2158; ND 2158; 1388896-07-8; CHEMBL3361255; (S)-2-Hydroxy-3-((R)-4-((trans-4-morpholinocyclohexyl)oxy)-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-5-yl)propanamide; (2S)-2-hydroxy-3-[(3R)-12-(4-morpholin-4-ylcyclohexyl)oxy-7-thia-9,11-diazatricyclo[6.4.0.02,6]dodeca-1(12),2(6),8,10-tetraen-3-yl]propanamide; ND2158.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)