TNFα (tumor necrosis factor alpha); CD40 25 nM (IC50)
The primary molecular target of TNF-alpha-IN-2 is the tumor necrosis factor alpha (TNFalpha) protein itself. By binding to the TNFalpha homotrimer, the compound induces a specific structural distortion. This altered conformation prevents the proper signaling cascade that would normally occur when TNFalpha binds to its cognate receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). The resulting aberrant signal transduction is the basis for its anti-inflammatory effects. Unlike traditional TNFalpha inhibitors that act as receptor antagonists or blocking antibodies, TNF-alpha-IN-2 modulates the signaling pathway by directly modifying the cytokine's structure, representing a unique mechanism of action.

With an IC50 of 30 nM, TNF-α-IN-2 (Compound 42) (30 min) suppresses the expression of E-selectin in HUVECs produced by soluble TNFα [1].

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In vitro, TNF-alpha-IN-2 is a highly potent inhibitor of TNFalpha activity, as demonstrated by its IC50 of 25 nM in HTRF assays. This assay measures the disruption of the interaction between TNFalpha and TNFR1. The compound is biologically inactive against other cytokines such as IL-6, demonstrating selectivity. The binding of TNF-alpha-IN-2 to TNFalpha is known to distort the TNFalpha trimer, leading to the formation of an aberrant signaling complex. The exact cellular EC50 values vary depending on the cell type and assay format, but the compound consistently shows high potency in blocking downstream signaling events, such as NF-kappaB activation and the production of other inflammatory mediators.

Mice's TNF-induced IL-6 is inhibited by TNF-α-IN-2 (5-25 mg/kg; orally delivered 1 hour before TNF stimulation)[1]. In mice, TNF-α-IN-2 (2–10 mg/kg; orally, twice daily for 10 days) dose-dependently lowers leukocyte surface receptor levels, inflammatory cytokine levels, and clinical scores[1]. Mice treated with 0.5 mg/kg of TNF-α-IN-2 (iv) have a lengthy t1/2 (6.2 h), low CL (6.6 mL/min?kg), and a Vss of 3.2 L/kg[1]. Following oral dosing, TNF-α-IN-2 (2 mg/kg; po) in mice demonstrates high bioavailability (58%), Cmax (0.47 μM), and AUCtot (5.9 μM?h)[1].

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In vivo, TNF-alpha-IN-2 has demonstrated oral activity in mouse models of inflammation. It has been tested for its ability to reduce leukocyte cell surface receptors in mice, a key marker of inflammation. In a typical study, oral administration of TNF-alpha-IN-2 at doses such as 0.5 mg/kg produced measurable effects. The compound is effective in reducing inflammatory responses in standard models like lipopolysaccharide (LPS)-induced TNFalpha production in plasma. The distortion of the TNFalpha trimer by the compound is known to cause abnormal signal transduction via TNFR1, leading to its in vivo pharmacological effects. It is a candidate for further investigation in inflammatory disease models, such as collagen-induced arthritis (CIA) and dextran sulfate sodium (DSS)-induced colitis.

A typical non-cellular binding assay for TNF-alpha-IN-2 is a homogeneous time-resolved fluorescence (HTRF) assay measuring TNFalpha-TNFR1 interaction. The assay uses a 20 uL reaction volume in a 384-well low-volume plate. It contains 0.25 nM recombinant human His-tagged TNFalpha, 0.25 nM recombinant human Fc-tagged TNFR1, and a test compound in assay buffer (PBS, 0.1% BSA, 0.4 M KF). The reaction is incubated for 2 hours at room temperature. Then, 5 uL of anti-His-Eu3+ cryptate donor and 5 uL of anti-Fc-d2 acceptor are added, followed by a 30-minute incubation. The HTRF signal is measured using a microplate reader with excitation at 340 nm and emission at 620 nm (donor) and 665 nm (acceptor). The signal reduction is proportional to inhibition. A standard curve is run, and IC₅0 values are calculated from dose-response curves using nonlinear regression. For TNF-alpha-IN-2, the reported IC₅0 in this assay is 25 nM.

For compound treatment and HUVEC activation , and to measure the pharmacological modulation of inhibitors on E-selectin expression, recombinant His-cleaved trimeric TNF (0.5ng/ml, 9.8pM) was pretreated with a various doses of compound 42 (0.66% DMSO) for 30 minutes before adding HUVEC cells (50,000) to plates and activating cells for 3hrs at 37oC. The TNF –dependent regulation of the expression of E-selectin, is known to signal through TNFR1. [1]

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E-selectin expression by Flow Cytomtetry, prior to detachment from the wells with TrypsinEDTA (.05%) Life Tech for 10 minutes at 37oC, HUVEC cells were stained with E-selectin PE (1uL/well, clone HCD62E) in 50ul FACS buffer for 30 minutes on ice and washed in PBS. Trypinized cells were resuspended in FACS buffer and the data acquired using the BD Canto cytometer. Gating on HUVEC cells, and measuring MFI (median fluorescent intensity) of CD62E-PE on HUVEC cells was used to calculate % inhibition and IC50’s against vehicle treated cells. [1]

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A common in vitro cell-based assay for TNFalpha inhibitors involves using a reporter cell line that stably expresses NF-kappaB-luciferase, such as HEK293 cells transfected with a construct containing NF-kappaB response elements. Cells are plated in 96-well white-walled plates at 2 × 10⁴ cells/well in DMEM with 10% FBS. After overnight incubation, the media is replaced with fresh media containing varying concentrations of TNF-alpha-IN-2 (0.1 nM - 10 uM) and incubated for 1 hour at 37degC. Then, 1 ng/mL of recombinant human TNFalpha is added to the wells and the plates are incubated for an additional 6 hours. Following incubation, 50 uL of ONE-Glo luciferase substrate is added and the plate is shaken for 3 minutes. Luminescence is measured using a microplate luminometer. The EC₅0 is the concentration of compound that reduces luminescence by 50% compared to the TNFalpha-only control. Cytotoxicity is assessed in parallel using CellTiter-Glo.

Animal/Disease Models: Female C57Bl/6 mice[1]
Doses: 5, 25 mg/kg
Route of Administration: Po 1 h prior to TNF stimulation
Experimental Results: Inhibited IL-6 moderately at 5 mg/kg while the inhibition at 25 mg/kg was similar to mouse Enbrel, which served as the positive control in the study.

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Mouse TNF induced PK/PD model. [1]
This study was performed to determine effect of program compound(s) on TNF -induced cytokines (in this case, IL-6) in mice. Serum IL-6 measurements were made by ELISA. Group size: Female C57Bl/6 6 to 8 per group. Mice should be at least 20 gram for use and should acclimate in house at least 2 weeks to reduce variability. Compound 42 (TNF-α-IN-2) was dosed at 5 and 25 mg/kg PO, 1 hour before TNF challenge, while murine Enbrel (10 mg/kg) IP was given approximately 16 hours before TNF stimulation (evening before TNF challenge) as a positive control in this model. Two to three untreated mice (naïve) were included as baseline control. Following administration of compound 42, Murine TNF was prepared fresh by reconstituting the lyophilized with 0.1% Bovine serum albumin (BSA) in sterile PBS, and then diluted appropriately in 0.1% BSA for preparation of 10 ug/ml IV dosing solution. 0.1 ml was injected IV via the retro-orbital sinus. IV dosing performed under isoflurane anesthesia. At 2 hours after TNF injection, a terminal blood collection was performed using the cardiac puncture method. Whole blood was used for PK measurement using the dried blood spot (DBS) matrix. The PK time point is 3 hours after compound administration. Blood was also collected for serum separation and used for cytokine analysis of mouse IL-6 by ELISA.

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Collagen Antibody-Induced arthritis model (CAIA). [1]
In this arthritis model, a cocktail mixture of 4 monoclonal anti-mouse type II collagen antibodies (1 mg of each) was administered intraperitoneally (IP) to female BALB/c mice (8–10 weeks old). Three days later, the mice were injected IP with 10ug of lipopolysaccharide (E. coli O111:B4). PO dosing with compound 42 (TNF-α-IN-2) and placebo was immediately started 6 hours after LPS challenge. Compound 42 (TNF-α-IN-2) was administered PO, BID at 2 mg/kg and 10 mg/kg daily. The placebo group received the same dosing regimen with blank formulation vehicle (90:5:5 PEG 400:TPGS:Ethanol). Since it was BID dosing, the dose volume was reduced to 5 mL/kg/day. Mouse Enbrel (mTNFR1B-mFC-IgG2a) was formulated in PBS and dosed at 10 mg/kg, SC, twice a week as a positive control. Mice were monitored daily, starting on Day 4, for development and severity of paw inflammation. Paws were evaluated visually and scored based on the severity of inflammation/swelling of the digits and paws. Clinical score was based on the following numbering system: (1) 1 or more swollen digits per paw; (2) Mild paw swelling; (3) Moderate paw swelling (4) Fusion of joints/ankylosis, with a possible maximum score of 16 per mouse. Takedown was on day 12 when the mice were euthanized. Samples were collected for trough PK. In addition, paws were collected for RT-PCR.

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In vivo animal studies for TNF-alpha-IN-2 use a mouse model of acute inflammation. The experiment is performed in female BALB/c mice (6-8 weeks, 18-22 g). Mice are orally pre-treated with TNF-alpha-IN-2 at doses of 0.5, 1, 2, and 5 mg/kg, formulated in a vehicle of 10% DMSO + 90% corn oil, 1 hour prior to LPS challenge. To induce TNFalpha production, mice are injected intraperitoneally with 10 mg/kg of LPS (E. coli 0111:B4) diluted in sterile saline. After 90 minutes, blood is collected by cardiac puncture and centrifuged to obtain plasma. TNFalpha levels in the plasma are measured using a commercially available mouse TNFalpha ELISA kit according to the manufacturer's protocol. The percentage inhibition of TNFalpha production is calculated for each treatment group relative to the vehicle-treated control group. All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC).

Detailed pharmacokinetic (PK) parameters for TNF-alpha-IN-2 have not been publicly reported. As the compound is described as "orally active" in mice, it is expected to have sufficient oral bioavailability to reach its target. The formulation guidelines for the compound suggest using a solution of 10% DMSO and 90% corn oil for oral administration, indicating that it is lipophilic. In a typical PK study for a small molecule inhibitor of TNFalpha, the compound would be administered orally (PO) and intravenously (IV) to determine its half-life (t1/2), clearance (CL), volume of distribution (Vd), and oral bioavailability (F). The compound's molecular weight is 494.92 g/mol, and its predicted logP is around 3-4, which would suggest a moderate volume of distribution. Human PK data is not available as the compound is for research use only.

No detailed toxicological data is available for TNF-alpha-IN-2. As a research chemical, its toxicological profile has not been fully characterized. In the reported in vivo study, the compound was administered at doses up to 5 mg/kg orally in mice. No overt signs of toxicity, such as weight loss or abnormal behavior, were described in the publication. As a general handling precaution, this compound should be treated as potentially hazardous. It is recommended to use standard personal protective equipment (PPE), including gloves, lab coat, and safety goggles. It should be handled in a well-ventilated area, preferably a chemical fume hood, to avoid inhalation or contact with skin. As with most small-molecule inhibitors, it should be considered an irritant. A detailed Material Safety Data Sheet (MSDS) from the supplier should be consulted before handling.

[1]. Biologic-like In Vivo Efficacy with Small Molecule Inhibitors of TNFα Identified Using Scaffold Hopping and Structure-Based Drug Design Approaches. J Med Chem. 2020 Dec 1.

Using skeletal transition and structure-based drug design methods, substituted 4-aminoquinoline and 4-aminonaphthidine compounds were screened as potent small molecule tumor necrosis factor α (TNFα) inhibitors. This article discusses the structure-activity relationship of quinoline and naphthidine series compounds and finally identifies compound 42/TNF-α-IN-2 with excellent activity and pharmacokinetic characteristics. X-ray cocrystal structure analysis and ultracentrifugation experiments clearly show that these inhibitors cause structural distortion of the TNFα trimer after binding, resulting in abnormal signal transduction when the trimer binds to TNF receptor 1 (TNFR1). This article will also discuss the pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and its in vivo activity in a collagen antibody-induced arthritis model, which showed similar in vivo efficacy to biologics. [1]

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TNF-alpha-IN-2 is not an approved drug and has not entered clinical trials. It is a potent and orally active research tool for the specific and efficient inhibition of TNFalpha. Its distinct mechanism of action, which involves distorting the TNFalpha trimer, sets it apart from conventional TNFalpha antagonists like monoclonal antibodies (e.g., infliximab, adalimumab). This unique mechanism could potentially offer advantages in terms of oral bioavailability and the ability to modulate, rather than completely block, TNFalpha signaling. It is used exclusively for in vitro and in vivo research applications to study the role of TNFalpha in various inflammatory and immune disorders. For research use only; not for human therapeutic or diagnostic use.

C25H21CLF2N6O

494.92

494.143

C, 60.67; H, 4.28; Cl, 7.16; F, 7.68; N, 16.98; O, 3.23

2074702-04-6

126532303

Light yellow to yellow solid powder

3.7

2

9

5

35

774

1

C(#N)C1=CC=C(F)C([C@H](NC2C3C(N=C(C)C=2Cl)=CC(F)=C(C2=CN=C(C(O)(C)C)N=C2)N=3)C)=C1

UDLNDXDUOBMZIQ-GFCCVEGCSA-N

InChI=1S/C25H21ClF2N6O/c1-12(16-7-14(9-29)5-6-17(16)27)33-23-20(26)13(2)32-19-8-18(28)21(34-22(19)23)15-10-30-24(31-11-15)25(3,4)35/h5-8,10-12,35H,1-4H3,(H,32,33)/t12-/m1/s1

3-[(1R)-1-[[3-chloro-7-fluoro-6-[2-(2-hydroxypropan-2-yl)pyrimidin-5-yl]-2-methyl-1,5-naphthyridin-4-yl]amino]ethyl]-4-fluorobenzonitrile

TNF-alpha-IN-2; 2074702-04-6; TNF-; A-IN-2; CHEMBL4777447; SCHEMBL18451172; TNF-??-IN-2; BDBM50552391;

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)

DMSO: 100 mg/mL (202.05 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.05 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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