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Purity: ≥98%
RP-54745 (RP 54745) is a novel amino-dithiole-one compound that is active at micromolar concentration on the metabolism of stimulated macrophages, for example, the hexose monophosphate pathway (HMP) and the exocytosis of lysosomal enzymes. LPS-induced interleukin-1 (IL-1) production by murine peritoneal macrophages was also diminished by this compound in vitro as well as in vivo. This effect was confirmed at the mRNA level; at the concentration of 3 x 10(-6) M, the IL-1 alpha and beta mRNA signals were inhibited, whereas the TNF alpha mRNA signal was only slightly lessened. These observations were confirmed in vivo, with a dose of RP 54745 of 25 mg kg-1. These results led us to consider that RP 54745 might influence certain cells and cytokines implicated in the regulation of the immune system, the disfunctioning of which can lead to inflammatory disorders or autoimmune pathologies. In all, as an inhibitor of macrophage stimulation and interleukin-1 production, RP-54745 has a potential to be used as an antirheumatic compound.
| Targets |
The specific molecular target of RP-54745 is not described in this study. However, it inhibits the metabolic and secretory functions of stimulated macrophages, particularly the hexose monophosphate pathway, lysosomal enzyme exocytosis, and interleukin-1 (IL-1) production. It has a more selective inhibitory effect on IL-1 production compared to TNFα. [1]
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| ln Vitro |
LPS-induced interleukin 1 (IL-1) production by mouse peritoneal macrophages is inhibited by RP-54745 [1].
RP-54745 inhibited the hexose monophosphate pathway (HMP) activity in zymosan-stimulated murine peritoneal macrophages in a concentration-dependent manner, with an IC₅₀ of 3 µM. This effect was reversible upon drug washout. At a concentration of 2.5 µM, it inhibited the release of the lysosomal enzyme β-glucuronidase from zymosan-stimulated macrophages by 70%. At a concentration of 10⁻⁶ M, RP-54745 added 15 minutes before LPS stimulation potently inhibited cell-associated IL-1 production by murine peritoneal macrophages: by 80% at 2 or 4 hours post-stimulation, and by 55% at 24 hours. Extracellular IL-1 production was also inhibited by 90% at 4 hours and 70% at 24 hours. At the mRNA level, RP-54745 (3 × 10⁻⁶ M) significantly reduced the IL-1α and IL-1β mRNA signals in LPS-stimulated macrophages, while the TNFα mRNA signal was only slightly affected. This is in contrast to dexamethasone (10⁻⁶ M), which inhibited signals for all three mRNAs. RP-54745 (10⁻⁵ M) did not inhibit cyclooxygenase (CO) activity. At 5 × 10⁻⁶ M, it slightly reduced 5-lipoxygenase (LO) activity. RP-54745 (10⁻⁵ M) did not affect the binding of IL-1β to EL4-6.1 murine thymoma cells or Raji human B lymphoma cells. [1] |
| ln Vivo |
In various mouse models of induced arthritis, as well as in MRL/lpr mice, which are genetically predisposed to develop autoimmune pathologies including arthritis, RP-54745 (5 mg/kg) is effective at oral dosage modulation. Following three months of treatment with RP-54745, the clinical condition of MRL mice as well as some of its disturbed biochemical and immunological parameters were improved [2].
In thioglycollate-stimulated female OF₁ mice, oral administration of RP-54745 at 25 mg kg⁻¹ day⁻¹ for 4 days significantly reduced the ex vivo LPS-induced production of cell-associated IL-1 by peritoneal macrophages in a dose-dependent manner. Extracellular IL-1 production was inhibited only at the 25 mg kg⁻¹ day⁻¹ dose. In a combined in vivo stimulation model, mice were orally treated with RP-54745 (5 or 25 mg kg⁻¹ day⁻¹ for 4 days) and then challenged intraperitoneally with LPS (0.5 µg per mouse). RP-54745 significantly inhibited the LPS-stimulated production of both cell-associated and extracellular IL-1. At the mRNA level, the oral dose of 25 mg kg⁻¹ day⁻¹ for 5 days significantly reduced IL-1β mRNA expression by 71% and slightly reduced TNFα mRNA expression by 22%. [1] |
| Cell Assay |
Hexose Monophosphate Pathway (HMP) Assay: Murine peritoneal macrophages were harvested 4 days after thioglycollate injection and cultured. After adherence and rest, cells were pre-incubated with or without RP-54745 for 17 hours. Following washing (for reversibility studies, some cells were cultured overnight without drug), macrophages were stimulated with zymosan (0.04%) for phagocytosis for 30 minutes in the presence of ¹⁴C₁-glucose. The liberated ¹⁴CO₂ was collected over 15 minutes, and radioactivity was measured. Results were corrected for cell viability and protein content.
β-Glucuronidase Release Assay: Adherent peritoneal macrophages, stimulated by zymosan phagocytosis for 17 hours (in the presence or absence of drug), released the lysosomal enzyme. The enzyme activity in supernatants was measured by incubating with phenolphthalein mono-β-glucuronic acid substrate (20 mM in acetate buffer, pH 4.5) for 17 hours at 37°C. The reaction was stopped and color developed by adding glycine buffer (pH 10.45), and absorbance was read at 540 nm. IL-1 Production and Bioassay: Peritoneal macrophages were cultured, pre-treated with RP-54745 15 minutes before LPS stimulation (100 ng mL⁻¹), and incubated for 2-24 hours. Supernatants (extracellular) and cell lysates (cell-associated) were collected. IL-1 activity was measured using the lymphocyte-activating factor (LAF) test: thymocytes from C₃H/Ouj mice were cultured with PHA and co-incubated with test samples or IL-1 standard for 72 hours. Thymocyte proliferation was assessed by MTT reduction, with absorbance read at 560/690 nm. IL-1 and TNFα mRNA Expression: Total cellular RNA was extracted from LPS-stimulated macrophages (with or without drug) after 6 hours. RNA was denatured, separated by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with specific oligonucleotide probes for IL-1α, IL-1β, and TNFα. Signals were detected by Northern blot analysis and quantified by densitometry. [1] |
| Animal Protocol |
In vivo IL-1 production studies: Female OF₁ mice were stimulated with an intraperitoneal injection of thioglycollate broth. Four days later, they were orally administered RP-54745 (dissolved in water with 0.5% Tween 20) at doses of 5 or 25 mg kg⁻¹ day⁻¹ for 4 consecutive days. Control groups received vehicle (0.5% Tween 20). Peritoneal macrophages were then harvested for ex vivo LPS stimulation and IL-1 measurement.
In vivo LPS challenge studies: Thioglycollate-stimulated mice were orally treated with RP-54745 (5 or 25 mg kg⁻¹ day⁻¹ for 4 days). Four hours before macrophage harvesting, mice received an intraperitoneal injection of LPS (0.5 µg per mouse). Peritoneal macrophages were then collected for IL-1 measurement and mRNA analysis. [1] |
| References |
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| Additional Infomation |
RP-54745 is an aminodithioketone compound selected for its ability to inhibit macrophage activation. It is a potential antirheumatic drug that works by inhibiting key inflammatory functions of macrophages, including the production of IL-1, but is not a broad-spectrum cyclooxygenase/lipoxygenase inhibitor. Its mechanism of action appears to be different from that of corticosteroids, cyclohexylimide, and classic nonsteroidal anti-inflammatory drugs (NSAIDs). The compound exhibits selective inhibition of IL-1 rather than TNFα at both the protein and mRNA levels. Its action is reversible. Studies have shown that RP-54745 may be effective against diseases with excessive IL-1 production, such as rheumatoid arthritis and other autoimmune diseases. [1]
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| Molecular Formula |
C13H12NOS2CL
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|---|---|
| Molecular Weight |
297.82348
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| Exact Mass |
297.005
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| Elemental Analysis |
C, 52.43; H, 4.06; Cl, 11.90; N, 4.70; O, 5.37; S, 21.53
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| CAS # |
135330-08-4
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| PubChem CID |
126203
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.46g/cm3
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| Boiling Point |
411.4ºC at 760 mmHg
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| Flash Point |
202.6ºC
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| Vapour Pressure |
5.59E-07mmHg at 25°C
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| Index of Refraction |
1.707
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| LogP |
4.011
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
18
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| Complexity |
399
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1SSC(N2C(C)C3=C(C=CC=C3)CC2)=C1Cl
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| InChi Key |
BEJJGVDFQORITE-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H12ClNOS2/c1-8-10-5-3-2-4-9(10)6-7-15(8)12-11(14)13(16)18-17-12/h2-5,8H,6-7H2,1H3
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| Chemical Name |
4-Chloro-5-(3,4-dihydro-1-methyl-2(1H)-isoquinolinyl)-3H-1,2-dithiol-3-one
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| Synonyms |
RP-54745; RP 54745; RP54745
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~62.5 mg/mL (~209.86 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.98 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3577 mL | 16.7887 mL | 33.5773 mL | |
| 5 mM | 0.6715 mL | 3.3577 mL | 6.7155 mL | |
| 10 mM | 0.3358 mL | 1.6789 mL | 3.3577 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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