SCH-546738 targets the chemokine receptor CXCR3, which is involved in immune cell trafficking, inflammation, and autoimmune responses. It is a potent antagonist with a Ki of 0.4 nM for the human CXCR3 receptor. It acts as a non-competitive antagonist. It specifically inhibits CXCR3-mediated chemotaxis of immune cells, particularly T cells.

SCH 546738's affinity for binding to the human CXCR3 receptor was assessed through a competitive binding assay, wherein the competitive tracer was 35S radiolabeled SCH 535390, a sulfonamide analog belonging to the CXCR3 compound series with a Kd of 0.6 nM. Furthermore, with an IC50 ranging from 0.8 to 2.2 nM, SCH 546738 non-competitively displaces radiolabeled CXCL10 and CXCL11 in human CXCR3. With an IC90 of roughly 10 nM, SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells [1].

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In vitro, SCH-546738 potently binds to the human CXCR3 receptor with a Ki of 0.4 nM. The affinity is determined by competition binding analysis using ³⁵S radiolabeled SCH 535390 as a competitive tracer. It specifically inhibits CXCR3-mediated signaling and chemotaxis.

With IC50s of 1.3, 6.4, 5.9, and 4.2 nM, respectively, SCH 546738 exhibits substantial cross-species action against the binding of [125I]hCXCL10 to CXCR3 in monkeys, dogs, mice, and rats. According to in vivo research, SCH 546738 is a strong and selective CXCR3 antagonist with good PK [1].

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In vivo, SCH-546738 attenuates the development of autoimmune diseases and delays graft rejection. By blocking CXCR3, it inhibits the recruitment of immune cells to sites of inflammation. It has been studied in animal models of autoimmune diseases and transplant rejection.

A scintillation proximity assay is used for radioligand competition binding assays with some modifications. For each assay point, 1 µg of membrane is preincubated for 1 hr with 300 µg wheat germ agglutinin (WGA) coated SPA beads in the binding buffer (50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 125 mM NaCl, 0.002% NaN3, 1.0% BSA) at room temperature. The beads are spun down, resuspended in the binding buffer and transferred to a 96-well Isoplate. The indicated concentrations of 125I-hCXCL10, 125I-hCXCL11 or 35S-SCH 535390 with a series of titrations of SCH-546738 (SCH546738) (1-10 μM) are added to start the reaction. After indicated reaction times at room temperature, the amount of radioactivity bound to the SPA beads is determined with a Wallac 1450 Microbeta counter[1].

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The in vitro receptor binding assay for SCH-546738 involves competition binding analysis using ³⁵S radiolabeled SCH 535390 as a competitive tracer. Membranes from cells expressing the human CXCR3 receptor are incubated with varying concentrations of SCH-546738 and a fixed concentration of radioligand. Ki values are calculated.

The preparation of human activated T cells is performed. Human peripheral blood lymphocytes are prepared by Ficoll-Hypaque centrifugation, depleted of monocytes, and stimulated for 2 days with 1 µg/mL PHA and 100 U/mL IL-2 in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 µg/mL Streptomycin, 100 U/mL Penecillin, 1% non-essential amino acids and 2 mM HEPES. Following stimulation, peripheral blood lymphocytes are cultured in above media containing 5% conditioned media for up to 15 days. uman activated T cell chemotaxis assays ae performed using 96-well ChemoTx microplates with a 3 µm filter. Activated T cells are washed with RPMI medium twice, and then resuspended in the medium containing 20% FBS. 1.25×105 cells/reaction are mixed with indicated concentrations of SCH 546738 (1, 10 or 100 nM) and placed on the filter. SCH 546738 and chemokines are mixed and placed in the bottom well of the ChemoTx system. After 2.5 hours incubation at 37°C/5% CO2, the cells are scraped off and the plate system is centrifuged for 5 minutes at 1000 RPM. The filter screen is then removed and the ChemoTx plate is inverted into a 96 well plate with a funnel plate. The plate system is centrifuged for 5 minutes at 1000 RPM. The volume in the wells is brought to 100 μL with assay buffer and the plates are rested for approximately 15 minutes at room temperature. The number of migrated cells is measured using the Cell Titer Glo Luminescent Assay. Chemotaxis is expressed as a chemotactic index, which is a ratio versus the one without chemokines[1]

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In vitro cell-based assays for SCH-546738 are performed using cells expressing CXCR3, such as T cells. Cells are treated with SCH-546738, and CXCR3-mediated chemotaxis is measured using a Boyden chamber assay. Calcium mobilization and other signaling readouts are also assessed to confirm antagonist activity.

Mice[1] Female C57BL/6 mice are used. For immunization, 150 μg MOG35-55 peptide prepared by Princeton Biomolecules and 300 μg killed Mycobacterium tuberculosis are mixed in CFA and injected s.c. in two 50-μL injections over the flanks on day 1. Also, 200 ng of pertussis toxin is injected i.v. on days 0 and 2. SCH 546738 is administered orally 30 mpk in C57BL/6 mice twice daily. Dosing with SCH 546738 started at day 0, 24 h prior to MOG35-55 immunization (day 1). Mice are monitored daily and assessed for clinical signs of disease in a blinded fashion according to the following criteria: 0, no signs of disease; 1, tail paralysis; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb plus forelimb paralysis; and 5, moribund or dead. Cumulative clinical scores are calculated by adding daily scores from the day of immunization until the end of the experiment. Mean clinical scores at separate days and mean maximal scores are calculated by adding the scores of individual mice and dividing with the number of mice in each group, including mice not developing signs of EAE. Rats[1] Male Lewis rats challenged by injection of 50 μL (30 mg) of a guinea pig spinal cord homogenate in complete Freunds adjuvant (CFA) into one footpad. SCH 546738 or 0.4% methylcellulose (vehicle) is orally administered at the indicated dose (0.2 mL) twice a day, starting on the day before transplantation until the day of graft rejection. SCH 546738 is administered orally at 10 mg/kg (mpk) in Lewis rats. To test whether SCH 546738 enhances the effect of conventional immunosuppressive reagent, the recipients are received treatment with subtherapeutic dose of CsA for one week combined with treatment with SCH 546738. Graft survival is analyzed using the log-rank test. The parametric data are analyzed by Student t test (2-tailed) using GraphPad InStat version.

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In vivo animal experiments for SCH-546738 have been conducted in models of autoimmune diseases and transplant rejection. Animals are administered the compound orally, and disease severity is assessed. Immune cell infiltration into target tissues is measured. Graft survival is monitored in transplantation models.

Pharmacokinetic properties of SCH-546738 have been characterized in preclinical species. It is orally active and achieves sufficient plasma concentrations to exert its pharmacological effects. Detailed PK parameters such as half-life, clearance, and oral bioavailability are determined from plasma concentration-time profiles.

Toxicological data for SCH-546738 are limited as it is a research compound. In standard preclinical safety assessments, it would be evaluated for potential off-target effects and general toxicity. The compound is intended for research use only and not for therapeutic use in humans.

[1]. A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection. BMC Immunol. 2012 Jan 10;13:2.

[2]. CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy. J Hepatol. 2016 Jan;64(1):160-70.

[3]. STAT3 in CD8+ T Cells Inhibits Their Tumor Accumulation by Downregulating CXCR3/CXCL10 Axis. Cancer Immunol Res. 2015 Aug;3(8):864-870.

SCH-546738 is a potent and selective CXCR3 antagonist that has been studied for the treatment of autoimmune diseases and transplant rejection. It plays a significant role in biochemical reactions by specifically inhibiting the CXCR3 receptor. It has not been approved for clinical use.

C23H31N7OCL2

492.44454

491.197

906805-42-3

11995774

White to light yellow solid powder

1.327 g/cm3

628.202ºC at 760 mmHg

333.725ºC

4.252

2

7

6

33

642

1

CC[C@H]1CN(CCN1C2CCN(CC2)CC3=CC=C(C=C3)Cl)C4=NC(=C(N=C4Cl)C(=O)N)N

UYDYJFWSPRQEAX-KRWDZBQOSA-N

InChI=1S/C23H31Cl2N7O/c1-2-17-14-31(23-20(25)28-19(22(27)33)21(26)29-23)11-12-32(17)18-7-9-30(10-8-18)13-15-3-5-16(24)6-4-15/h3-6,17-18H,2,7-14H2,1H3,(H2,26,29)(H2,27,33)/t17-/m0/s1

3-Amino-6-chloro-5-[(3S)-4-[1-[(4-chlorophenyl)methyl]piperidin-4-yl]-3-ethylpiperazin-1-yl]pyrazine-2-carboxamide

SCH-546738; SCH 546738; SCH546738

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 : ~4.5 mg/mL (~9.14 mM)
H2O : ~1 mg/mL (~2.03 mM)

Solubility in Formulation 1: 1 mg/mL (2.03 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.

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