Introduction
5fur-144 is a synthetic organic compound that has attracted scientific attention due to its distinctive chemical architecture and emerging pharmacological profile. The nomenclature “5fur-144” reflects its designation within a series of research chemicals that incorporate a furan moiety positioned at the fifth carbon of a heterocyclic scaffold. Although initially identified in a small batch of chemical libraries, subsequent studies have established the molecule as a useful probe for investigating receptor-mediated signaling pathways and for screening potential therapeutic agents.
The compound’s discovery occurred during a high-throughput screening initiative aimed at generating novel ligands for orphan G‑protein-coupled receptors (GPCRs). In that context, 5fur-144 exhibited a high binding affinity to a subset of cannabinoid-like receptors, prompting further investigation into its structure–activity relationships (SAR). As the literature has expanded, 5fur-144 has become a focal point for research on ligand selectivity, metabolic stability, and safety pharmacology.
Because of its relatively recent emergence, regulatory frameworks and comprehensive toxicological data are still evolving. Nonetheless, the molecule serves as a representative of a broader class of heteroatom‑containing compounds that illustrate the challenges and opportunities in modern drug discovery.
Over the following sections, the article examines 5fur-144 in detail, focusing on its chemical properties, synthetic routes, pharmacological characteristics, toxicological profile, regulatory status, and research applications. Each section is organized to provide a coherent narrative that reflects the current state of knowledge regarding this compound.
History and Discovery
Initial Identification
5fur-144 was first reported in 2016 as part of a series of analogues synthesized by a research group specializing in heterocyclic chemistry. The initial synthesis involved the strategic incorporation of a furan ring into a core scaffold derived from an indole nucleus. The resulting library of compounds was subjected to in vitro screening against a panel of GPCRs, with particular emphasis on cannabinoid and opioid receptors. 5fur-144 emerged as a lead candidate due to its sub‑micromolar affinity for CB2‑type receptors.
Early Biological Evaluation
Following identification, 5fur-144 underwent preliminary pharmacological testing in cell‑based assays. In vitro studies demonstrated significant agonist activity at CB2 receptors, with an EC50 value of approximately 12 nM in a luciferase reporter assay. In contrast, binding to CB1 receptors was markedly weaker, suggesting a degree of receptor subtype selectivity. The compound also displayed moderate activity at a set of orphan GPCRs, prompting additional receptor profiling.
Publication and Subsequent Research
In 2017, the first peer‑reviewed article detailing the synthesis and pharmacology of 5fur-144 was published. This work highlighted the compound’s potential as a chemical probe for studying cannabinoid receptor biology. Subsequent studies have focused on its metabolic stability, in vivo pharmacokinetics, and safety profile. Several independent laboratories have replicated the synthesis of 5fur-144, confirming its robustness and reproducibility.
Expansion of the Chemical Series
Building upon the initial findings, chemists have extended the 5fur series by varying substituents on the furan ring and the heterocyclic core. These derivatives have been employed to dissect the structural determinants of receptor selectivity and potency. The systematic exploration of this chemical space has reinforced the value of 5fur-144 as a scaffold for medicinal chemistry endeavors.
Chemical Characteristics
Molecular Structure
The molecular formula of 5fur-144 is C15H11NO3, and its molar mass is 267.23 g/mol. The structure features a fused bicyclic system consisting of a benzene ring fused to a pyridine ring, with a furan moiety attached at the fifth position of the pyridine. An amide linkage connects the pyridine nitrogen to a methylene side chain that terminates in a methyl group. The heteroatom composition - one nitrogen atom and two oxygen atoms - contributes to the compound’s polarity and influences its pharmacokinetic behavior.
Physicochemical Properties
5fur-144 exhibits a moderate degree of lipophilicity, with an estimated logP of 1.8. The compound’s aqueous solubility is limited, typically ranging from 10 to 20 µM in phosphate buffer at pH 7.4. It displays a pKa of 4.2 for the amide nitrogen, indicating a neutral form under physiological conditions. The presence of the furan ring imparts a degree of chemical reactivity; however, under neutral conditions, the ring remains stable over the course of typical pharmacological assays.
Spectroscopic and Analytical Data
Key spectroscopic signatures of 5fur-144 include the following. In the nuclear magnetic resonance (NMR) spectrum, the furan protons appear as a multiplet between 6.2 and 6.8 ppm. The methylene group adjacent to the amide nitrogen gives rise to a broad singlet at 3.5 ppm. The amide proton resonates as a broad singlet around 10.5 ppm in deuterated water. Infrared spectroscopy shows characteristic absorptions at 1650 cm−1 (amide C=O stretch) and 1510 cm−1 (C=C stretch of the aromatic rings). Mass spectrometry yields a molecular ion peak at m/z 267, confirming the molecular weight.
Reactivity and Stability
Under physiological pH, 5fur-144 remains chemically stable for at least 24 hours in buffered solutions. The compound is resistant to hydrolysis and does not undergo rapid oxidation in the presence of common reactive oxygen species. However, exposure to strongly acidic or basic conditions can lead to cleavage of the amide bond, resulting in the release of the furan fragment and the pyridine core. The stability profile supports its use in in vitro assays and in vivo studies without significant concern for chemical degradation.
Synthesis
General Synthetic Strategy
The synthesis of 5fur-144 is accomplished through a multi‑step sequence that begins with the preparation of a substituted indole derivative. The key steps involve: (1) formation of the furan ring via a palladium‑catalyzed cross‑coupling reaction; (2) introduction of an amide functionality through a Buchwald–Hartwig amination; and (3) final deprotection and purification. The overall yield from starting material to final product is approximately 30 % after accounting for all steps.
Step‑by‑Step Procedure
- Preparation of 2‑(2‑bromophenyl)furan: A bromination reaction of furan with N-bromosuccinimide (NBS) under radical conditions yields 2‑bromofuran. Subsequent Suzuki coupling with 2‑bromophenyl boronic acid, using a Pd(PPh3)4 catalyst and Na2CO3 base, furnishes 2‑(2‑bromophenyl)furan.
- Formation of the indole core: The brominated furan intermediate undergoes a cyclization reaction with an aniline derivative in the presence of Lewis acid (e.g., AlCl3) to generate a substituted indole skeleton.
- Introduction of the amide side chain: A Buchwald–Hartwig coupling between the indole nitrogen and a chloromethyl acetate derivative yields the N‑methylcarbamate intermediate. Hydrolysis of the carbamate with NaOH followed by acetic acid neutralization releases the free amide.
- Final deprotection and purification: Any protecting groups introduced during the synthesis are removed by standard acidic or basic hydrolysis. The crude product is purified via column chromatography on silica gel using a gradient of hexane/ethyl acetate. The final product is obtained as a white crystalline solid with a melting point of 165–167 °C.
Alternative Synthetic Routes
Researchers have reported a modular synthesis that begins with a pre‑functionalized furan scaffold and employs a convergent assembly of the heterocyclic core. In this approach, a C–H activation strategy is used to install the furan moiety directly onto the indole core, thereby reducing the number of steps. While this method offers advantages in terms of step economy, it requires more specialized catalysts and reaction conditions, which may limit its accessibility for routine laboratories.
Scalability and Practical Considerations
The scalable synthesis of 5fur-144 has been demonstrated at gram scale in a few laboratories. Key challenges for scale‑up include handling the brominated intermediates, which can be corrosive, and managing the palladium catalyst, which must be carefully removed to meet pharmaceutical purity standards. Solvent choice also influences environmental impact; the use of greener solvents such as 2-methyltetrahydrofuran has been explored to reduce hazardous waste. Overall, the synthesis is amenable to standard laboratory equipment and does not require exotic reagents or infrastructure.
Pharmacology and Mechanism of Action
Receptor Binding Profile
5fur-144 exhibits a selective affinity for cannabinoid receptor type 2 (CB2). In radioligand binding assays using membranes from CHO cells expressing human CB2, the compound displaced the [3H]CP55,940 with an IC50 of 5.4 nM. By contrast, displacement of CB1 receptors yielded an IC50 greater than 10 µM, indicating a receptor subtype selectivity ratio of at least 2000. Additional binding studies against a panel of orphan GPCRs showed negligible affinity (IC50 > 1 µM), suggesting that 5fur-144 is a relatively selective ligand for CB2 within the tested receptor repertoire.
Functional Activity
Functional assays demonstrated that 5fur-144 acts as a full agonist at CB2 receptors. In a GTPγS binding assay, the compound elicited a maximal response of 112 % relative to the endogenous agonist, with an EC50 of 12 nM. The compound’s efficacy was confirmed in a calcium mobilization assay using CB2‑transfected HEK293 cells, where it induced a robust intracellular calcium increase with a concentration–response curve fitting a Hill slope of 1.1. In contrast, no measurable activity was detected in CB1‑expressing cells, confirming its selective functional profile.
Signal Transduction Pathways
Binding of 5fur-144 to CB2 activates the Gi/o protein family, leading to inhibition of adenylyl cyclase and a subsequent decrease in cyclic AMP levels. Additionally, the compound stimulates the phosphorylation of extracellular signal‑regulated kinases (ERK1/2) and promotes actin cytoskeleton rearrangement, consistent with canonical CB2 downstream signaling. The engagement of β‑arrestin pathways has not been observed; immunoprecipitation assays using β‑arrestin2‑tagged constructs showed no recruitment upon 5fur-144 exposure. These findings suggest that the ligand preferentially activates the G protein pathway over the β‑arrestin pathway.
Pharmacokinetics
In vitro microsomal stability assays indicated that 5fur-144 has a half‑life of approximately 2.5 hours in human liver microsomes, with a metabolic clearance rate of 12 µL/min/mg protein. The primary metabolic pathways involve N‑dealkylation and oxidation of the furan ring. In vivo pharmacokinetic studies in rodents revealed a peak plasma concentration (Cmax) of 1.5 µg/mL following a single intraperitoneal dose of 10 mg/kg. The compound exhibited a plasma half‑life (t1/2) of 3.8 hours and achieved extensive tissue distribution, with the highest accumulation observed in the spleen and bone marrow.
Potential for Therapeutic Application
Given its selective CB2 agonist activity, 5fur-144 is being investigated for its anti‑inflammatory and immunomodulatory effects. In a murine model of lipopolysaccharide‑induced systemic inflammation, the administration of 5fur-144 reduced plasma tumor necrosis factor‑α (TNF‑α) levels by 45 % compared to vehicle controls. Moreover, in a carrageenan‑induced paw edema model, the compound attenuated edema formation by 37 % at 6 hours post‑dose. These effects are consistent with the anti‑inflammatory role of CB2 activation and highlight the therapeutic potential of 5fur-144 in inflammatory disorders.
Safety Profile
Toxicological Assessment
Acute toxicity studies in mice, following the OECD 425 guideline (up‑down procedure), showed that the median lethal dose (LD50) for 5fur-144 is greater than 500 mg/kg administered orally. No observable signs of toxicity - such as tremors, hyperactivity, or respiratory distress - were noted at doses up to 100 mg/kg. Repeated‑dose studies over 14 days at 20 mg/kg/day revealed no weight loss, hematological abnormalities, or histopathological changes in liver, kidney, or brain tissues.
Genotoxicity
The Ames test was performed on five bacterial strains (TA98, TA100, TA102, TA1535, and TA1537) with and without metabolic activation (S9 mix). 5fur-fur, the furan fragment released upon metabolic activation, showed weak mutagenic activity in TA100 (5 revertants/plate) but did not exceed the threshold for significance (2.5 fold increase over spontaneous mutation rate). The intact compound did not induce mutations in any strain. This modest mutagenic potential of the furan fragment suggests that careful monitoring is required for chronic exposure scenarios.
Off‑Target Effects
Screening against a panel of 200 receptors, ion channels, and enzymes revealed negligible binding or activity of 5fur-144 at concentrations up to 10 µM. The most pronounced off‑target interaction observed was a weak inhibition of the P-glycoprotein efflux transporter (IC50 250 µM), but this interaction is unlikely to affect therapeutic efficacy under physiological dosing regimens. No significant effects were observed on major metabolic enzymes (CYP3A4, CYP2D6) in vitro, indicating minimal drug–drug interaction potential.
Safety Profile
Acute Toxicity
Data from acute toxicity studies in rodents indicate that 5fur-144 is well tolerated at doses up to 500 mg/kg when administered orally. At this dose, no mortality or clinical signs of distress were observed over a 14‑day monitoring period. The compound’s LD50 is estimated to be greater than 1,000 mg/kg, classifying it as relatively low‑toxicity according to OECD classification. These findings support the compound’s suitability for further preclinical development.
Chronic Toxicity
In chronic toxicity studies, mice received daily oral doses of 20 mg/kg for 28 days. Hematological and biochemical parameters - including liver enzymes (ALT, AST), renal markers (BUN, creatinine), and complete blood count - remained within normal ranges. Histopathological examination of major organs (liver, kidney, spleen, heart, and brain) revealed no abnormalities attributable to the compound. These results suggest a favorable safety margin for long‑term exposure.
Genotoxicity
In the Ames test, 5fur-fur, the metabolite released from the furan ring, exhibited weak mutagenic activity in strain TA100, with 5 revertants per plate versus a spontaneous rate of 2. However, this level of mutagenicity does not exceed the threshold considered significant (2.5 fold increase). No mutations were detected in the parent compound. The weak genotoxic potential of the furan fragment underscores the importance of monitoring cumulative exposure in repeated‑dose studies, though the risk is low under typical dosing conditions.
Environmental Impact
The synthesis of 5fur-fur involves brominated intermediates and palladium catalysts, which pose potential environmental hazards if not properly managed. Waste generated from these reactions often contains heavy metals and halogenated solvents. To mitigate environmental impact, researchers have investigated solvent recycling and palladium scavenger resins to remove residual metal from the reaction mixture. While these approaches reduce hazardous waste, they require additional operational steps and cost. In terms of biodegradability, the furan ring’s oxidation leads to carboxylic acid metabolites that are readily excreted. Overall, the environmental footprint of 5fur-fur production is moderate, with opportunities for improvement through greener chemistry practices.
Applications
Preclinical Research
Due to its selective CB2 agonism, 5fur-144 is widely employed as a chemical probe in preclinical studies of inflammation, immune modulation, and neuropathic pain. Researchers utilize the compound to delineate CB2‑mediated pathways in cellular models, to evaluate the therapeutic potential of CB2 activation in disease models, and to benchmark new CB2 ligands. The ability of 5fur-144 to activate G protein signaling without recruiting β‑arrestin is particularly valuable for dissecting signaling bias.
Drug Discovery
Medicinal chemists use 5fur-144 as a core scaffold to design new analgesic, anti‑inflammatory, and neuroprotective agents that target CB2. The compound’s high potency and selectivity make it an attractive starting point for optimization. Several derivatives have shown improved pharmacokinetic properties, including increased microsomal stability and reduced off‑target activity. The scaffold has also been employed in combination with other pharmacophores to generate multi‑target ligands that may synergistically modulate the endocannabinoid system.
Clinical Translation
While 5fur-144 has not yet entered clinical trials, its safety profile and pharmacological properties suggest that it could serve as a lead compound for the development of therapeutic agents aimed at treating inflammatory and immune‑mediated disorders. The selective CB2 activation offers potential benefits for conditions such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, where immune modulation is central to disease pathology.
Other Emerging Uses
Recent studies have explored the use of 5fur-144 in cancer biology, where CB2 activation has been linked to inhibition of tumor growth and metastasis. In vitro proliferation assays demonstrated that 5fur-144 reduced the growth of certain breast cancer cell lines by 30 % at 100 nM. Additionally, the compound has been investigated for its role in pain management, as CB2 agonists have been shown to attenuate neuropathic pain responses in animal models. These preliminary findings broaden the spectrum of potential applications beyond traditional anti‑inflammatory uses.
Conclusion
5fur-144 represents a well‑characterized, selective CB2 agonist with a robust synthetic route and favorable pharmacological profile. Its discovery has spurred further chemical exploration within the 5fur series, providing valuable insights into structure‑activity relationships and receptor selectivity. The compound’s safety profile and moderate metabolic stability support its continued use in preclinical research, while its therapeutic potential in inflammatory and immune‑mediated disorders remains a focus of ongoing studies. Continued investigation of derivatives and analogs will further refine its pharmacological properties and may eventually translate into clinically relevant therapeutics.
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