Selective Inhibitory Activity Research Articles

Synthesis of New Glucose-containing 5-Arylisoxazoles and their Enzyme Inhibitory Activity

Abstract: Carbohydrates are an important group of biomolecules that have received special attention due to their significant role in the design and synthesis of new bioactive compounds. In this study, a new class of 5-arylisoxazole-glucose hybrids was designed and synthesized for evaluation of their inhibitory effects on α-glucosidase, α-amylase, and tyrosinase. The target compounds depicted selective α-glucosidase inhibitory activity over α-amylase, which is an important factor in reducing probable gastrointestinal problems in the treatment of type 2 diabetes. In this respect, compound 9a, possessing the phenylisoxazole group, was found to be the most potent α-glucosidase inhibitor (IC50 = 292.2 ± 0.1 μM) compared to acarbose (IC50 = 750.2 ± 0.1 μM) as the positive control. All compounds were also evaluated for their anti-tyrosinase effect, and among them, compound 9j, containing a fluoroaryl moiety, showed potent activity (IC50 = 50.1 ± 6.3 μM) in comparison to kojic acid (IC50 = 23.6 ± 2.6 μM). Also, docking studies were performed to investigate the probable mode of action, which indicated the construction of important H-bonding interactions between the sugar moiety and the enzyme’s active sites. According to the results, hybrids containing heterocycles attached to glucose can be used to inhibit α-glucosidase.

Discovery of Potent Selective HDAC6 Inhibitors with 5-Phenyl-1H-indole Fragment: Virtual Screening, Rational Design, and Biological Evaluation.

Among the HDACs family, histone deacetylase 6 (HDAC6) has attracted extensive attention due to its unique structure and biological functions. Numerous studies have shown that compared with broad-spectrum HDACs inhibitors, selective HDAC6 inhibitors exert ideal efficacy in tumor treatment with insignificant toxic and side effects, demonstrating promising clinical application prospect. Herein, we carried out rational drug design by integrating a deep learning model, molecular docking, and molecular dynamics simulation technology to construct a virtual screening process. The designed derivatives with 5-phenyl-1H-indole fragment as Cap showed desirable cytotoxicity to the various tumor cell lines, all of which were within 15 μM (ranging from 0.35 to 14.87 μM), among which compound 5i had the best antiproliferative activities against HL-60 (IC50 = 0.35 ± 0.07 μM) and arrested HL-60 cells in the G0/G1 phase. In addition, 5i exhibited better isotype selective inhibitory activities due to the potent potency against HDAC6 (IC50 = 5.16 ± 0.25 nM) and the reduced inhibitory activities against HDAC1 (selective index ≈ 124), which was further verified by immunoblotting results. Moreover, the representative binding conformation of 5i on HDAC6 was revealed and the key residues contributing 5i's binding were also identified via decomposition free-energy analysis. The discovery of lead compound 5i also indicates that virtual screening is still a beneficial tool in drug discovery and can provide more molecular skeletons with research potential for drug design, which is worthy of widespread application.

Synthesis and In vitro evaluation of bichalcones as novel anti-toxoplasma agents

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, an apicomplexan parasite that infects approximately a third of the world’s human population. This disease can cause serious complications during pregnancy and can be fatal in immunocompromised hosts. The current treatment options for toxoplasmosis face several limitations. Thus, to address the urgent medical need for the discovery of novel anti-toxoplasma potential drug candidates, our research focused on exploring a series of monomeric and dimeric chalcones, polyphenolic molecules belonging to the class of flavonoids. Chalcones 1aa—1bg and axially chiral A-A′-connected bichalcones 2aa—2bg were evaluated in vitro against the proliferation of the parasite in a cell-based assay. A comparison of the efficacy demonstrated that, in several cases, bichalcones exhibited increased bioactivity compared to their corresponding monomeric counterparts. Among these compounds, a bichalcone with a phenyl substituent and a methyl moiety 2ab showed the most potent and selective inhibitory activity in the nanomolar range. Both enantiomers of this bichalcone were synthesized using an axially chiral biphenol building block. The biaryl bond was forged using Suzuki cross-coupling in water under micellar catalysis conditions. Separation of the atropisomers of this biphenol building block was conducted by chiral HPLC on a preparative scale. The biological evaluation of the enantiomers revealed that the (Ra)-enantiomer (Ra)-2ab is the eutomer. These studies suggest that bichalcones may be important drug candidates for further in vivo evaluations for the discovery of anti-toxoplasma drugs.

3-Benzylaminomethyl Lithocholic Acid Derivatives Exhibited Potent and Selective Uncompetitive Inhibitory Activity Against Protein Tyrosine Phosphatase 1B (PTP1B)

3-Benzylaminomethyl Lithocholic Acid Derivatives Exhibited Potent and Selective Uncompetitive Inhibitory Activity Against Protein Tyrosine Phosphatase 1B (PTP1B)

Cryptic Cerulenin Rearrangement in Ketosynthase Covalent Inhibition.

The antibiotic cerulenin is a fungal natural product identified as a covalent inhibitor of ketosynthases within fatty acid and polyketide biosynthesis. Due to its selective and potent inhibitory activity, cerulenin has found significant utility in multidisciplinary biochemical, biomedical, and clinical studies. Although its covalent inhibition profile has been confirmed, cerulenin's mechanism has not been fully determined at a molecular level, frustrating the drug development of related analogues. Herein, we describe the use of stable isotopic tracking with NMR and MS methods to unravel the covalent mechanism of cerulenin against type II fatty acid ketosynthases. We detail the discovery of a unique C2-C3 retro-aldol bond cleavage and a structural rearrangement upon covalent inhibition of cerulenin at the active cysteine residue in E. coli type II fatty acid ketosynthases FabB and FabF.

Exploring the Neuroprotective Potential of Desmodium Species: Insights into Radical Scavenging Capacity and Mechanisms against 6-OHDA-Induced Neurotoxicity.

In this study, we collected seven prevalent Taiwanese Desmodium plants, including three species with synonymous characteristics, in order to assess their antioxidant phytoconstituents and radical scavenging capacities. Additionally, we compared their inhibitory activities on monoamine oxidase (MAO) and 6-hydroxydopamine (6-OHDA) auto-oxidation. Subsequently, we evaluated the neuroprotective potential of D. pulchellum on 6-OHDA-induced nerve damage in SH-SY5Y cells and delved into the underlying neuroprotective mechanisms. Among the seven Desmodium species, D. pulchellum exhibited the most robust ABTS radical scavenging capacity and relative reducing power; correspondingly, it had the highest total phenolic and phenylpropanoid contents. Meanwhile, D. motorium showcased the best hydrogen peroxide scavenging capacity and, notably, D. sequax demonstrated remarkable prowess in DPPH radical and superoxide scavenging capacity, along with selective inhibitory activity against MAO-B. Of the aforementioned species, D. pulchellum emerged as the frontrunner in inhibiting 6-OHDA auto-oxidation and conferring neuroprotection against 6-OHDA-induced neuronal damage in the SH-SY5Y cells. Furthermore, D. pulchellum effectively mitigated the increase in intracellular ROS and MDA levels through restoring the activities of the intracellular antioxidant defense system. Therefore, we suggest that D. pulchellum possesses neuroprotective effects against 6-OHDA-induced neurotoxicity due to the radical scavenging capacity of its antioxidant phytoconstituents and its ability to restore intracellular antioxidant activities.

Rational Design of Novel Allosteric EYA2 Inhibitors as Potential Therapeutics for Multiple Brain Cancers.

The Eyes Absent (EYA) family of developmental proteins, often in partnership with the sine oculis (SIX) homeobox proteins, promote cancer metastasis and recurrence in numerous tumor types. In addition to being a transcriptional coactivator, EYA2 is a Tyr phosphatase that dephosphorylates H2AX which leads to repair instead of apoptosis upon DNA damage and ERβ which inhibits the anti-tumor transcriptional activity of ERβ. The SIX members of the EYA-SIX complex are difficult to target, therefore, we targeted the EYA2 to promote cell death and prevent cancer progression. We conducted structural optimization of a previously discovered allosteric inhibitor of EYA2, 9987, using the combination of in silico modeling, biochemical and cell-based assays. A new series of compounds was developed with significantly improved cellular activity and physiochemical properties desirable for brain targets. Specifically, compound 2 e showed >30-fold improvement in the medulloblastoma cell line D458, relative to 9987, while maintaining potent and selective inhibitory activity against EYA2 Tyr phosphatase activity and a good multiparameter optimization score for central nervous system drugs.

Metabolomic Analysis and Antiviral Screening of a Marine Algae Library Yield Jobosic Acid (2,5-Dimethyltetradecanoic Acid) as a Selective Inhibitor of SARS-CoV-2.

Current small-molecule-based SARS-CoV-2 treatments have limited global accessibility and pose the risk of inducing viral resistance. Therefore, a marine algae and cyanobacteria extract library was screened for natural products that could inhibit two well-defined and validated COVID-19 drug targets, disruption of the spike protein/ACE-2 interaction and the main protease (Mpro) of SARS-CoV-2. Following initial screening of 86 extracts, we performed an untargeted metabolomic analysis of 16 cyanobacterial extracts. This approach led to the isolation of an unusual saturated fatty acid, jobosic acid (2,5-dimethyltetradecanoic acid, 1). We confirmed that 1 demonstrated selective inhibitory activity toward both viral targets while retaining some activity against the spike-RBD/ACE-2 interaction of the SARS-CoV-2 omicron variant. To initially explore its structure-activity relationship (SAR), the methyl and benzyl ester derivatives of 1 were semisynthetically accessed and demonstrated acute loss of bioactivity in both SARS-CoV-2 biochemical assays. Our efforts have provided copious amounts of a fatty acid natural product that warrants further investigation in terms of SAR, unambiguous determination of its absolute configuration, and understanding of its specific mechanisms of action and binding site toward new therapeutic avenues for SARS-CoV-2 drug development.

Diorganotin (IV) amino acid complexes as potential anticancer agents. Synthesis, structural characterization, and in vitro assays

Nine new organotin (IV) derivatives from L-amino acids (l-lysine, L-ornithine, L-glutamic acid, and L-aspartic acid) were synthesized by one-pot ultrasound-assisted methodology. All compounds were characterized by ATR-FTIR (Attenuated Total Reflectance-Fourier Transform Infrared), LRMS (Low-Resolution Mass Spectrometry), and solution NMR (1H, 13C, 119Sn Nuclear Magnetic Resonance) spectroscopies. Complexes Bu2Sn(Lys) (1), Ph2Sn(Lys) (2), Bu2Sn(Orn) (3), and Ph2Sn (Glu-OMe) (6a) were crystallized, and the structures were established by single-crystal X-ray diffraction analysis. Diffraction results evidenced that complexes 1 to 3 were five-coordinated mononuclear species while the phenyl substituted derivative Ph2Sn (Glu-OMe) (6a) forms a polymeric network via Sn–O–Sn bridging whereby the tin atom is six-coordinated. In turn, 119Sn NMR results revealed that all tin complexes exist as mononuclear penta-coordinated species in solution. The tin derivatives were screened for ADME (Adsorption, Distribution, Metabolism, and Excretion) properties via the freely available tools SWISS ADME, and the results were analyzed hereafter. The antiproliferative activity of the complexes was tested against three human cancer cell lines: colorectal adenocarcinoma HT-29, breast adenocarcinoma MDA-MB-231, and chondrosarcoma SW-1353 using a non-tumoral cell line of human osteoblast as control, demonstrating selective inhibitory activities against cancer cells. Hence, these compounds could be a promising alternative to classical chemotherapy agents.

Prenylated indole diketopiperazine alkaloids as phosphatase inhibitors from the marine-derived fungus Talaromyces purpureogenus

Six previously undescribed prenylated indole diketopiperazine alkaloids, talaromyines A-F (1−6), were isolated from the marine-derived fungus Talaromyces purpureogenus SCSIO 41517. Their structures including absolute configurations were elucidated on the basis of comprehensive spectroscopic data including NMR, HR-ESI-MS, and electronic circular dichroism calculations, together with chemical analysis of hydrolysates. Compounds 1−5 represent the first example of spirocyclic indole diketopiperazines biosynthesized from the condensation of L-tryptophan and L-alanine. Compounds 2 and 4−5 showed selective inhibitory activities against phosphatases TCPTP and MEG2 with IC50 value of 17.9–29.7 μM, respectively. Compounds 4−5 exhibited mild cytotoxic activities against two human cancer cell lines H1975 and HepG-2.

Design, synthesis and neuroprotective biological evaluation of novel HDAC6 inhibitors incorporating benzothiadiazinyl systems as cap groups.

Histone deacetylase 6 (HDAC6), as the key regulatory enzyme, plays an important role in the development of the nervous system. More and more studies indicate that HDAC6 has become a promising therapeutic target for CNS diseases. Herein we designed and synthesized a series of novel HDAC6 inhibitors with benzothiadiazinyl systems as cap groups and evaluated their activity invitro and invivo. Among them, compound 3 exhibited superior selective inhibitory activity against HDAC6 (IC50 = 5.1 nM, about 30-fold selectivity over HDAC1). The results of docking showed that compound 3 can interact well with the key amino acid residues of HDAC6. Compound 3 showed lower cytotoxicity (20 μM to SH-SY5Y cells, inhibition rate = 25.75%) and better neuroprotective activity against L-glutamate-induced SH-SY5Y cell injury model invitro. Meanwhile, compound 3 exhibited weak cardiotoxicity (10 μM hERG inhibition rate = 17.35%) and possess good druggability properties. Especially, compound 3 could significantly reduce cerebral infarction from 49.87% to 32.18%, and similar with butylphthalide in MCAO model, indicating potential clinical application prospects for alleviating ischemic stroke-induced brain infarction.

Nitrification inhibitors impose distinct effects on comammox bacteria and canonical ammonia oxidizers under high N fertilization regimes

The response of complete ammonia-oxidizing (comammox) bacteria to variable nitrogen (N) inputs and nitrification inhibitors (NIs) remains poorly understood. We conducted two experiments, in a soil where both clade A and B comammox Nitrospira were present, to identify the type (ammonium sulphate vs. urea) and the level of N-fertilization (0–500 mg-N-kg‐1d.w. soil) that are conducive to the growth of comammox, compared to canonical ammonia-oxidizing microorganisms (AOM), and subsequently to assess their response to three commercial (DCD, nitrapyrin, DMPP) and a new potent (ethoxyquin) NI under these favourable N fertilization regimes. The highest concentration of ammonium sulphate stimulated comammox compared to canonical AOM which showed a dose-dependent increase by both N sources. DMPP primarily, and DCD, imposed a more persistent inhibition on nitrification than nitrapyrin and ethoxyquin, a result which did not concur with the soil persistence of NIs (DMPP was the least persistent among the commercial NIs) but was mostly driven by their selective inhibitory activity on ammonia-oxidizing bacteria (AOB) that prevailed in the studied soil. The inhibition of AOB by DMPP and DCD was complemented by the proliferation of comammox and ammonia-oxidizing archaea (AOA). NIs induced significant changes on AOB and comammox communities. Comammox Nitrospira clade B were enriched by DCD and DMPP but depleted by nitrapyrin and ethoxyquin. We showed that (i) releasing comammox Nitrospira and AOA from AOB competition allows them to proliferate efficiently, even under high inorganic ammonium conditions; (ii) comammox clade B are more responsive than clade A to high inorganic N input and NIs.

Five new meroterpenes from the seeds of Psoralea corylifolia and their diacylglycerol acyltransferase activity

Five new meroterpenes (1−5), have been isolated and elucidated from the seeds of Psoralea corylifolia together with four known bakuchiol analogues (6−9). The structures of these compounds were elucidated by extensive spectroscopic analysis (NMR, HRESIMS and CD). Among them, compounds 1–9 were found to exhibit selective inhibitory activity on DGAT1 with IC50 values ranging from 54.4 ± 1.3 to 98.2 ± 1.1 μM.

Design, semi-synthesis of soft coral-derived Aspergillus sp. secondary metabolite geodin derivatives and their antibacterial activities

A series of novel ester derivatives 2 − 7, of natural product geodin 1, isolated from the soft coral-derived fungus Aspergillus sp., were designed and semi-synthesised through one step reaction with high yield. Compound 5 showed strong antifouling inhibitory activities with MIC of 4.80 μM while compound 4 showed selective inhibitory activities with MICs values 8.59 μM against Aeromonas salmonicida and Pseudomonas aeruginosa (Sea-Nine 211, MIC = 0.27 μM). Compounds 3, 4 and 6 showed potent anti-pathogenic inhibitory activities with MICs of 2.29 μM, 4.29 μM and 4.56 μM respectively against Staphylococcus aureus (Ciprofloxacin, MIC = 0.156 μM). Compound 2 showed weak inhibitory activity against A. salmonicida with MIC 18.75 μM (Sea-Nine 211, MIC = 0.27 μM) and with MICs 9.38 μM against S. aureus (ciprofloxacin, MIC = 0.156 μM). However, compound 7 showed very low antibacterial activities with MIC = >20 μM. The preliminary structure-activity relationships of compounds 2 − 7 further prove that the modification of 4-OH group of natural product geodin 1 improves the antibacterial activities such as antifouling and anti-pathogenic activities.

Potent, selective and reversible hMAO-B inhibition by benzalphthalides: Synthesis, enzymatic and cellular evaluations and virtual docking and predictive studies

Monoaminooxidases (MAOs) are important targets for drugs used in the treatment of neurological and psychiatric disorders and particularly on Parkinson's Disease (PD). Compounds containing a trans-stilbenoid skeleton have demonstrated good selective and reversible MAO-B inhibition. Here, twenty-two (Z)-3-benzylidenephthalides (benzalphthalides, BPHs) displaying a trans-stilbenoid skeleton have been synthesised and evaluated as inhibitors of the MAO-A and MAO-B isoforms. Some BPHs have selectively inhibited MAO-B, with IC50 values ranging from sub-nM to μM. The most potent compound with IC50 = 0.6 nM was the 3′,4′-dichloro-BPH 16, which showed highly selective and reversible MAO-B inhibitory activity. Furthermore, the most selective BPHs displayed a significant protection against the apoptosis, and mitochondrial toxic effects induced by 6-hydroxydopamine (6OHDA) on SH-SY5Y cells, used as a cellular model of PD. The results of virtual binding studies on the most potent compounds docked in MAO-B and MAO-A were in agreement with the potencies and selectivity indexes found experimentally. Additionally, related to toxicity risks, drug-likeness and ADME properties, the predictions found for the most relevant BPHs in this research were within those ranges established for drug candidates.

Microascones, Decahydrofluorene-Class Alkaloids from the Marine-Derived Fungus Microascus sp. SCSIO 41821.

Eight new decahydrofluorene-class alkaloids, microascones A and B (1 and 2), 2,3-epoxyphomapyrrolidone C (3), 14,16-epiascomylactam B (4), 24-hydroxyphomapyrrolidone A (5), and microascones C-E (6-8), along with five known analogs (9-13) were isolated from the marine-derived fungus Microascus sp. SCSIO 41821. Compounds 1 and 2 have an unprecedented complex macrocyclic alkaloid skeleton with a 6/5/6/5/6/5/13 polycyclic system. Their structures and absolute configurations were determined by spectroscopic analysis, quantum chemical calculations of ECD spectra, and 13C NMR chemical shifts. Compounds 10-13 showed selective enzyme inhibitory activity against PTPSig, PTP1B, and CDC25B, and 4, 9, and 10 exhibited strong antibacterial activity against seven tested pathogens. Their structure-bioactivity relationship was discussed, and a plausible biosynthetic pathway for 1-8 was also proposed.

Structural optimization and biological evaluation of ML364 based derivatives as USP2a inhibitors

Ubiquitination is a representative post-translational modification that tags target proteins with ubiquitin to induce protein degradation or modify their functions. Deubiquitinating enzymes (DUBs) play a crucial role in reversing this process by removing ubiquitin from target proteins. Among them, USP2a has emerged as a promising target for cancer therapy due to its oncogenic properties in various cancer types, but its inhibitors have been limited. In this study, our aim was to optimize the structure of ML364, a USP2a inhibitor, and synthesize a series of its derivatives to develop potent USP2a inhibitors. Compound 8v emerged as a potential USP2a inhibitor with lower cytotoxicity compared to ML364. Cellular assays demonstrated that compound 8v effectively reduced the levels of USP2a substrates and attenuated cancer cell growth. We confirmed its direct interaction with the catalytic domain of USP2a and its selective inhibitory activity against USP2a over other USP subfamily proteins (USP7, 8, or 15). In conclusion, compound 8v has been identified as a potent USP2a inhibitor with substantial potential for cancer therapy.

Design, synthesis, and activity evaluation of novel multitargeted l-tryptophan derivatives with powerful antioxidant activity against Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurological disease, and the multitarget directed ligand (MTDL) strategy may be an effective approach to delay its progression. Based on this strategy, 27 derivatives of l-tryptophan, 3a-1-3d-1, were designed, synthesized, and evaluated for their biological activity. Among them, IC50 (inhibitor concentration resulting in 50% inhibitory activity) values of compounds 3a-18 and 3b-1 were 0.58 and 0.44 μM for human serum butyrylcholinesterase (hBuChE), respectively, and both of them exhibited more than 30-fold selectivity for human serum acetylcholinesterase. Enzyme kinetics studies showed that these two compounds were mixed inhibitors of hBuChE. In addition, these two derivatives possessed extraordinary antioxidant activity in OH radical scavenging and oxygen radical absorption capacity fluorescein assays. Meanwhile, these compounds could also prevent β-amyloid (Aβ) self-aggregation and possessed low toxicity on PC12 and AML12 cells. Molecular modeling studies revealed that these two compounds could interact with the choline binding site, acetyl binding site, and peripheral anionic site to exert submicromolar BuChE inhibitory activity. In the vitro blood-brain barrier permeation assay, compounds 3a-18 and 3b-1 showed enough blood-brain barrier permeability. In drug-likeness prediction, compounds 3a-18 and 3b-1 showed good gastrointestinal absorption and a low risk of human ether-a-go-go-related gene toxicity. Therefore, compounds 3a-18 and 3b-1 are potential multitarget anti-AD lead compounds, which could work as powerful antioxidants with submicromolar selective inhibitory activity for hBuChE as well as prevent Aβ self-aggregation.

Visible-Light-Promoted α-C(sp3)-H Amination of Ethers with Azoles and Amides.

A visible-light-induced highly efficient C(sp3)-H amination of ethers with amides and azoles has been presented under mild conditions via a nitrogen- and carbon-centered radical coupling process. This protocol successfully utilizes 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and tert-butyl nitrite (TBN) as cocatalysts to deliver the aminated products of ethers under aerobic conditions. Notably, the developed reaction features the corresponding products in good yields (up to 93%) with a wide substrate scope. The mechanistic study indicates that C-N bond formation proceeds via a direct radical cross-coupling process. Preliminary biological activity analysis indicates that the resulting products have good and selective inhibitory activity on osteosarcoma (OS) cell lines and are promising for use as hits for drug discovery.

Hybrids of selective COX-2 inhibitors and active derivatives of edaravone as COX-2 selective NSAIDs with free radical scavenging activity: Design, synthesis and biological activities

Novel hybrids of selective COX-2 inhibitors (coxibs) and active derivatives of free radical scavenger edaravone were designed to overcome the risk of cardiovascular events and stroke increased by NSAIDs (nonsteroidal anti-inflammatory drugs) in this study. All the hybrids were assayed for the COX-2 inhibitory and DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging activities in vitro. Finally, we found a series of hybrids with good inhibitory activity and selectivity of COX-2 and excellent free radical scavenging activity in vitro. The most promising compound 6a (WYZ90) exhibited very potent COX-2 inhibitory activity (COX-2, IC50 = 75 nM), weak COX-1 inhibitory activity (COX-1, IC50 = 5734 nM), better free radical scavenging activity (DPPH, IC50 = 19.9 μM) than edaravone, moderate drug-likeness and ADME properties in silico, acceptable pharmacokinetic properties (T1/2 = 4.16 h, 10 mg/kg, o.p.) and oral bioavailability (F% = 36.03 %) in mice. In addition, compound WYZ90 showed similar analgesic activity to the selective COX-2 inhibitor celecoxib in acetic acid-induced mice and better antioxidant activity in Fe2+-induced lipid peroxidation in mouse liver tissue homogenate than edaravone. In conclusion, this study provided a novel class of coxibs containing edaravone moiety as COX-2 selective NSAIDs with free radical scavenging activity and the candidate compound WYZ90 showed not only similar selective COX-2 inhibitory and analgesic activity to celecoxib but also better free radical scavenging and antioxidant activity than edaravone.