TNFα (tumor necrosis factor alpha); CD40 25 nM (IC50)

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].

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].

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]

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.

[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.

Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure-activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42/TNF-α-IN-2 with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed. [1]

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.)