IC50: 35.0?nM (RIPK1); EC50: 10.0 nM (HT29 cells), 4.2 nM (L929 cells), 11.4 nM (J774A.1 cells), 17.8?nM (U937 cells)[1].

RI-962 exhibits strong RIPK1 inhibitory activity, as evidenced by its IC50 value of 35.0 nM[1]. With EC50 values of 10.0 nM, 4.2 nM, 11.4 nM, and 17.8 nM for HT29, L929, J774A.1, and U937 cells, respectively, RI-962 has a protective effect against necroptotic death[1]. RI-962 (0-100 μM; 24 h) prevents cells from necroptosis caused by TSZ by blocking RIPK1's kinase activity[1].

RI-962 (ip; 40 mg/kg; once daily for 10 days) decreases inflammation in acute DSS-induced colitis and improves TNFα-induced SIRS[1]. Rats' pharmacokinetic parameters (iv, ip, po; 5, 20 mg/kg) for RI-962 [1]. AUC0–t(ng*h/mL) 4526.1 ± 546.0 1594.9 ± 891.8 6459.7 ± 1131.6 AUC0–∞ (ng* h/mL) 4538.1 ± 546.3 1604.5 ± 896.1 6609.3 ± 1121.4 Vss (L/kg) 0.4 ± 0.1 - - MRT0–∞ (h) 0.4 ± 0.0 1.8 ± 0.2 2.8 ± 0.1 CL (mL/min/kg) 18.5 ± 2.1 - - F (%) - 8.8 ± 5.0 35.7 ± 6.3

In vitro kinase activity assays[1]
In vitro kinase activity assays were conducted through the Kinase Profiling Services provided by Eurofins. The protocol for the RIPK1 assay is briefly described as follows (Protocols for other kinases are very similar and can be found in http://www.eurofins.com/pharmadiscovery). RIPK1 kinase was incubated with the test compound in assay buffer containing 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 250 μM KKKSPGEYVNIEFG, 10 mM magnesium acetate, and 10 μM [γ-33P]-ATP for 15 min at room temperature. The reaction was initiated by the addition of the Mg/ATP mixture. After incubation for 40 min at room temperature, and the reaction was stopped by the addition of 3% phosphoric acid. A 10 μL portion of the reaction mixture was then spotted onto a P30 filter mat and washed four times for 4 min in 0.425% phosphoric acid and once in methanol prior to drying and scintillation counting.

Cell Viability Assay[1]
Cell Types: HT29, L929, J774A.1, and U937 cells
Tested Concentrations: 0-100 μM
Incubation Duration: 24 h
Experimental Results: Exerted a dose-dependent protective effect against necroptotic death.

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Western Blot Analysis[1]
Cell Types: HT29 cells
Tested Concentrations: 0-400 nM
Incubation Duration:
Experimental Results: Markedly inhibited the phosphorylation of RIPK1 and its downstream signaling proteins RIPK3 and MLKL in a dose- dependent manner.

Animal/Disease Models: C57BL/6 female mice[1]
Doses: 40 mg/kg
Route of Administration: intraperitoneal (ip)for 15 min; one time/day for 10 day
Experimental Results: Ameliorated TNFα-induced SIRS by inhibiting RIPK1 activity. Suppressed the RIPK1 signaling in the mouse model of DSS-induced colitis.

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Animal/Disease Models: SD (Sprague-Dawley) rats[1]
Doses: 5, 20 mg/kg
Route of Administration: intravenous (iv)(iv) (5 mg/kg), intraperitoneal (ip)(20 mg/kg) and oral (po) (20 mg/kg)
Experimental Results: Had good metabolic stability in rats.

[1]. Generative deep learning enables the discovery of a potent and selective RIPK1 inhibitor. Nat Commun. 2022 Nov 12;13(1):6891.

DSS-induced IBD experiment [1]
DSS (3% w/v) was added to drinking water and allowed free access for 7 days (from day 0 to day 7). The DSS solution was changed 3 times on day 2, day 4 and day 6. Female C57BL/6 mice were intraperitoneally injected with excipients, RI-962 (40 mg/kg) or GSK3145095 (40 mg/kg) for 10 days (from day 0 to day 9). Three mice from each group were randomly sacrificed and distal colon tissue was collected for analysis on day 7. Mouse weight and survival rate were recorded daily.

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Pharmacokinetic (PK) characterization assessment [1]
The PK characterization of compounds was assessed in male Sprague-Dawley rats (n = 3 per group, weight: 180–220 g). The compounds were dissolved in physiological saline containing 5% (v/v) DMSO and 40% (v/v) PEG400. Animals were administered single doses of 5 mg/kg (intravenous injection (iv)) and 20 mg/kg (intraperitoneal injection (ip) or oral gavage (po)). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 hours, and plasma was separated by centrifugation. The concentrations of compounds in plasma were then determined using LC-MS/MS-13, and pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0. Screening for lead compounds with novel skeletons is an important but highly challenging task in the early stages of drug development. Various generative models have been proposed to construct drug-like molecules. However, the ability of these generative models to design target-specific molecules with novel skeletons and validated in wet labs has not been fully validated. This paper proposes a generative deep learning (GDL) model, namely a distributed learning conditional recurrent neural network (cRNN), to generate a customized virtual compound library for a given biological target. We then apply the GDL model to the RIPK1 target. By virtually screening and subsequently evaluating the bioactivity of the generated custom compound library, we discovered a highly selective RIPK1 inhibitor, RI-962, with a previously unreported scaffold. This compound exhibited potent protective activity against necrotizing apoptosis in vitro and demonstrated good in vivo efficacy in two inflammation models. In summary, these findings demonstrate that our GDL model can generate lead compounds with unreported scaffolds, highlighting the great potential of deep learning in the field of drug discovery. [1]

C28H28N6O2

480.56

480.23

C, 69.98; H, 5.87; N, 17.49; O, 6.66

2763831-53-2

165437244

Off-white to light yellow solid powder

4.2

2

4

6

36

786

0

CC(C)C(=O)NC1=NN2C=CC(=CC2=N1)C3=CC4=C(C=C3)N(C=C4C(=O)NC(C)C5=CC=CC=C5)C

XYAYQAFPZOGMAA-UHFFFAOYSA-N

InChI=1S/C28H28N6O2/c1-17(2)26(35)31-28-30-25-15-21(12-13-34(25)32-28)20-10-11-24-22(14-20)23(16-33(24)4)27(36)29-18(3)19-8-6-5-7-9-19/h5-18H,1-4H3,(H,29,36)(H,31,32,35)

1-methyl-5-[2-(2-methylpropanoylamino)-[1,2,4]triazolo[1,5-a]pyridin-7-yl]-N-(1-phenylethyl)indole-3-carboxamide

RI962; 2763831-53-2; SCHEMBL25830764; BDBM636322; US20230365546

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.)