Leonurine hydrochloride-a new drug for the treatment of menopausal syndrome: Synthesis, estrogen-like effects and pharmacokinetics.

This research aimed to investigate the estrogen-like effects of Leonurine hydrochloride (Leo). First, we developed a total synthesis of Leo from 3,4,5-trimethoxy-benzoic acid and the structure was confirmed through 1H NMR and mass spectrometry (MS). Then the estrogenic activity of Leo in vitro and in vivo was studied. The proliferation and proliferation inhibitory effects of Leo on MCF-7 cells and MDA-MB-231 cells indicate that Leo exerts estrogen-like effects through estrogen receptor α (ERα) and estrogen receptor ß((ERß) in vitro. Uterotrophic assay in juvenile mice showed that Leo has an estrogen-like effect in vivo, as it can promote the development of the uterus of juvenile mice, increase its uterine coefficient and the size of the uterine cavity, as well as the increased number of uterine glands and the thickened uterine wall. For further research, cyclophosphamide (CTX) was used to establish a mouse model of ovarian function decline. Through this model, we found that Leo can restore the estrous cycle of mice, increase the number of primordial and primary follicles in the ovaries of mice, and regulate the disordered hypothalamic-pituitary-ovarian (HPOA) axis of mice. Finally, the pharmacokinetics of Leo was studied and oral bioavailability of Leo was calculated to be 2.21%. Leo was synthesized and the estrogen-like effect in vitro and in vivo was confirmed as well as its pharmacokinetics.

Polyhydroxybenzoic acid derivatives as potential new antimalarial agents.

With more than 200 million cases and 400,000 related deaths, malaria remains one of the deadliest infectious diseases of 2021. Unfortunately, despite the availability of efficient treatments, we have observed an increase in people infected with malaria since 2015 (from 211 million in 2015 to 229 million in 2019). This trend could partially be due to the development of resistance to all the current drugs. Therefore, there is an urgent need for new alternatives. We have, thus, selected common natural scaffolds, polyhydroxybenzoic acids, and synthesized a library of derivatives to better understand the structure-activity relationships explaining their antiplasmodial effect. Only gallic acid derivatives showed a noticeable potential for further developments. Indeed, they showed a selective inhibitory effect on Plasmodium (IC50 ~20 µM, SI > 5) often associated with interesting water solubility. Moreover, this has confirmed the critical importance of free phenolic functions (pyrogallol moiety) for the antimalarial effect. Methyl 4-benzoxy-3,5-dihydroxybenzoate (39) has, for the first time, been recognized as a potential lead for future research because of its marked inhibitory activity against Plasmodium falciparum and its significant hydrosolubility (3.72 mM).

Synthesis and in vitro antileishmanial efficacy of benzyl analogues of nifuroxazide.

Leishmaniasis is a vector-borne parasitic disease that mostly affects populations in tropical and subtropical countries. There is currently no vaccine to protect against and only a handful of drugs are available to treat this disease. Leishmaniasis is curable, but its eradication and elimination are hindered by the emergence of multidrug resistant strains of the causative pathogens, accentuating the need for new and effective antileishmanial drugs. In search for such agents, nifuroxazide, a clinical antibiotic, was evaluated through investigation of its benzyl analogues for in vitro antileishmanial efficacy against promastigotes of various Leishmania (L.) strains. The monobenzylated analogues 1 and 2 were the most potent of all, possessing nanomolar activities up to 10-fold higher than the parent drug nifuroxazide against all three tested Leishmania strains. Both analogues stand as antipromastigote hits for further lead investigation into their potential to act as new antileishmanial agents.

Thymidine phosphorylase and prostrate cancer cell proliferation inhibitory activities of synthetic 4-hydroxybenzohydrazides: In vitro, kinetic, and in silico studies.

Over-expression of thymidine phosphorylase (TP) plays a key role in many pathological complications, including angiogenesis which leads to cancer cells proliferation. Thus in search of new anticancer agents, a series of 4-hydroxybenzohydrazides (1-29) was synthesized, and evaluated for in vitro thymidine phosphorylase inhibitory activity. Twenty compounds 1-3, 6-14, 16, 19, 22-24, and 27-29 showed potent to weak TP inhibitory activities with IC50 values in the range of 6.8 to 229.5 µM, in comparison to the standards i.e. tipiracil (IC50 = 0.014 ± 0.002 µM) and 7-deazaxanthine (IC50 = 41.0 ± 1.63 µM). Kinetic studies on selected inhibitors 3, 9, 14, 22, 27, and 29 revealed uncompetitive and non-competitive modes of inhibition. Molecular docking studies of these inhibitors indicated that they were able to interact with the amino acid residues present in allosteric site of TP, including Asp391, Arg388, and Leu389. Antiproliferative (cytotoxic) activities of active compounds were also evaluated against mouse fibroblast (3T3) and prostate cancer (PC3) cell lines. Compounds 1, 2, 19, and 22-24 exhibited anti-proliferative activities against PC3 cells with IC50 values between 6.5 to 10.5 µM, while they were largely non-cytotoxic to 3T3 (mouse fibroblast) cells proliferation. Present study thus identifies a new class of dual inhibitors of TP and cancer cell proliferation, which deserves to be further investigated for anti-cancer drug development.

Phenolic acid phenethylesters and their corresponding ketones: Inhibition of 5-lipoxygenase and stability in human blood and HepaRG cells.

5-lipoxygenase (5-LO) catalyzes the biosynthesis of leukotrienes, potent lipid mediators involved in inflammatory diseases, and both 5-LO and the leukotrienes are validated therapeutic targets. Caffeic acid phenethyl ester (CAPE) is an effective inhibitor of 5-LO and leukotriene biosynthesis but is susceptible to hydrolysis by esterases. In this study a number of CAPE analogues were synthesized with modifications to the caffeoyl moiety and the replacement of the ester linkage with a ketone. Several new molecules showed better inhibition of leukotriene biosynthesis than CAPE in isolated human neutrophils and in whole blood with IC50 values in the nanomolar (290-520 nmol/L) and low micromolar (1.0-2.3 µmol/L) ranges, respectively. Sinapic acid and 2,5-dihydroxy derivatives were more stable than CAPE in whole blood, and ketone analogues were degraded more slowly in HepaRG hepatocyte cultures than esters. All compounds underwent modification consistent with glucuronidation in HepaRG cultures as determined using LC-MS/MS analysis, though the modified sinapoyl ketone (10) retained 50% of its inhibitory activity after up to one hour of incubation. This study has identified at least one CAPE analogue, compound 10, that shows favorable properties that warrant further in vivo investigation as an antiinflammatory compound.

Preparation and Characterization of Protocatechuic Acid Sulfates.

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) is a phenolic acid present in plants as a secondary metabolite and is also produced in the human organism as a metabolite from the degradation of polyphenols by the intestinal microbiota, particularly of flavonoids. However, PCA, like most polyphenols, is biotransformed in the human body to different conjugates as sulfates, which are found circulating in blood and could be involved in the bioactivity of the original compound. This paper describes a simple process for the preparation of PCA monosulfates with satisfactory yields. Two compounds were obtained that were identified as PCA-3-sulfate and PCA-4-sulfate by mass spectrometry and ¹H and 13C nuclear magnetic resonance using one- and two-dimensional techniques (heteronuclear single-quantum coherence and heteronuclear multiple-bond correlation). Differential MS fragmentation behavior and UV spectra were observed for each compound, which could be used for their identification in samples of unknown composition. The described procedure can be used for the preparation of these polyphenol metabolites in view of their use in in vivo and in vitro studies, as well as standards for their analysis in biological fluids, to contribute to the elucidation of biological effects of dietary polyphenols.

Anti-Diarrheal Drug Repositioning in Tumour Cell Cytotoxicity.

BACKGROUND: Drug repositioning is becoming an ideal strategy to select new anticancer drugs. In particular, drugs treating the side effects of chemotherapy are the best candidates. OBJECTIVE: In this present work, we undertook the evaluation of anti-tumour activity of two anti-diarrheal drugs (nifuroxazide and rifaximin). METHODS: Anti-proliferative effect against breast cancer cells (MDA-MB-231, MCF-7 and T47D) was assessed by MTT analysis, the Brdu incorporation, mitochondrial permeability and caspase-3 activity. RESULTS: Both the drugs displayed cytotoxic effects on MCF-7, T47D and MDA-MB-231 cells. The lowest IC50 values were obtained on MCF-7 cells after 24, 48 and 72 hours of treatment while T47D and MDA-MB-231 were more resistant. The IC50 values on T47D and MDA-MB-231 cells became significantly low after 72 hours of treatment showing a late cytotoxicity effect especially of nifuroxazide but still less important than that of MCF-7 cells. According to the IC50 values, the non-tumour cell line HEK293 seems to be less sensitive to cytotoxicity especially against rifaximin. Both the drugs have shown an accumulation of rhodamine 123 as a function of the rise of their concentrations while the Brdu incorporation decreased. Despite the absence of a significant difference in the cell cycle between the treated and non-treated MCF-7 cells, the caspase-3 activity increased with the drug concentrations rise suggesting an apoptotic effect. CONCLUSION: Nifuroxazide and rifaximin are used to overcome the diarrheal side effect of anticancer drugs. However, they have shown to be anti-tumour drugs which make them potential dual effective drugs against cancer and the side effects of chemotherapy.

Design of Antibacterial Agents: Alkyl Dihydroxybenzoates against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 µM), 16 (80 µM) and 28 (88 µM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.

Lichen Acids May Be Used as A Potential Drug For Cancer Therapy; by Inhibiting Mitochondrial Thioredoxin Reductase Purified From Rat Lung.

BACKGROUND: Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (Trx) in many cellular events such as DNA synthesis, DNA repair, angiogenesis, antioxidative defense, and regulating apoptosis. Although TrxR is indispensible in protecting cells against oxidative stress, the overexpression of TrxR is seen in many aggressive tumors. Therefore, targeted inhibition of TrxR has been accepted as a new approach for chemotherapy. OBJECTIVE: In this study, in vitro inhibition effect of the lichen acids (diffractaic, evernic, lobaric, lecanoric, and vulpinic acid) on mitochondrial TrxR purified from rat lung was investigated. METHOD: It was the first time the enzyme was purified from rat lungs by using 2', 5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was checked with SDS-PAGE. In vitro inhibition effect of the lichen acids was investigated spectrophotometrically. To emphasize the importance of the obtained data, the commercial anticancer drugs cisplatin and doxorubicin were used as positive controls. RESULTS: Molecular mass of the enzyme was calculated as approximately 52.4 kDa. The enzyme was purified with a 63.6% yield, 208.3 fold, and 0.5 EU/mg proteins specific activity. The IC50 values of five lichen acids were significantly lower than IC50 values of anticancer drugs. CONCLUSION: All of the lichen acids, especially lecanoric and vulpinic acid, exhibited much stronger inhibitory effect on TrxR than the anticancer drugs cisplatin and doxorubicin. These lichen acids have pharmacological potential as effective natural antioxidants, antimicrobials, and anticancer agents.

Synthesis and cytotoxic activity of 4-O-ß-D-galactopyranosyl derivatives of phenolic acids esters.

The glycosylation of naturally occurring phenolic acids has a significant impact on their solubility, stability and physiochemical properties. D-Galactose residue was found to form a part of glycoconjugates in several tissues and involved in a variety of physiological process. To the best of our knowledge, we have noticed a little information about the glycosylation of the phenolic acids with galactose residue. In this work, we describe the glycosylation of methyl vanillate and methyl ferulate with peracetylated-ß-D-galactopyranose in the presence of BF3·OEt2. The coupling reaction yielded efficiently and selectively only the acetylated ß-D-galactopyranosides 3 and 6. Removal of the acetyl groups using sodium methoxide afforded the corresponding ß-D-galactopyranosides 4 and 7 in good yields. Anticancer activity in vitro was evaluated against two human cancer cell lines (MCF-7 breast cancer cell lines and PC-3 prostate cancer cell lines). ß-D-galactopyranosides 4 and 7 demonstrated improved cytotoxic activity compared to the parental esters.

Ultrasound irradiation promoted enzymatic alcoholysis for synthesis of monoglyceryl phenolic acids in a solvent-free system.

Monoglyceryl phenolic acids (MPAs) were known as the natural hydrophilic antioxidants which could be used in different fields such as food, pharmaceutical, cosmetic etc. A novel enzymatic route of MPAs synthesis by the alcoholysis of phenolic acid ethyl esters with glycerol under ultrasound irradiation in solvent free system was developed. Optimization of reaction parameters shows that a high conversion of above 97.4% can be obtained under the following conditions: phenolic acid ethyl esters to glycerol molar ratio of 1:10, with 6% catalyst (Novozym 435), at 60°C and 200rpm, with ultrasound input of 250W, at 20kHz frequency. Compared to the conventional stirring method, the activation energy for phenolic acid ethyl esters conversion was decreased from 65.0kJ/mol to 32.1kJ/mol under ultrasound promotion; the apparent kinetic constant (Vm/Km) increased above 1.2-folds; the lipase amount decreased to 50%; the time required for the maximum conversion reduced up to 3-folds without damaging the lipase activity, which is the fastest report for enzymatic synthesis of MPAs.

Study on the Anticoagulant or Procoagulant Activities of Type II Phenolic Acid Derivatives.

In this study, three type II phenolic acids (caffeic acid, p-hydroxycinnamic acid, and ferulic acid) were used to synthesize a total of 18 phenolic acid derivatives. With molecular docking for molecule design and target protein (factors) screening, in combination with the confirmation of target proteins (factors) by surface plasmon resonance, and the evaluation of haemostatic and anticoagulant activities with five blood assays (plasma recalcification time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thrombin time), the data indicated that caffeic acid derivatives showed certain anticoagulant or procoagulant activities and that two other series contained compounds with the best anticoagulant activities. Using Materials Studio analysis, particular functional groups that affect anticoagulant or procoagulant activities were revealed, and these conclusions can guide the discovery of compounds with better activities.

Structure Properties and Mechanisms of Action of Naturally Originated Phenolic Acids and Their Derivatives against Human Viral Infections.

BACKGROUND: A great effort has been made to develop efficacious antiviral drugs, but many viral infections are still lack of efficient antiviral therapies so far. The related exploration of natural products to fight viruses has been raised in recent years. Natural compounds with structural diversity and complexity offer a great chance to find new antiviral agents. Particularly, phenolic acids have attracted considerable attention owing to their potent antiviral abilities and unique mechanisms. The aim of this review is to report new discoveries and updates pertaining to antiviral phenolic acids. METHODS: The relevant references on natural phenolic acids were searched. The antiviral phenolic acids were classified according to their structural properties and antiviral types. Meanwhile, the antiviral characteristics and structure-activity relationships of phenolic acids and their derivatives were summarized. RESULTS: The review finds that natural phenolic acids and their derivatives possessed potent inhibitory effects on multiple virus in humans such as human immunodeficiency virus, hepatitis C virus, hepatitis B virus, herpes simplex virus, influenza virus and respiratory syncytial virus. In particular, caffeic acid/gallic acid and their derivatives exhibited outstanding antiviral properties by a variety of modes of action. CONCLUSION: Naturally derived phenolic acids especially caffeic acid/gallic acid and their derivatives may be regarded as novel promising antiviral leads or candidates. Additionally, scarcely any of these compounds has been used as antiviral treatment in clinical practice. Therefore, these phenolic acids with diverse skeletons and mechanisms provide us an excellent resource for finding novel antiviral drugs.

Tetramer as efficient structural mode for organizing antioxidative carboxylic acids: The case in inhibiting DNA oxidation.

To overcome the problem on the relationship of antioxidative effect with the branch number in a tetramer, we herein designed a series of antioxidants with pentaerythritol, glycerol, and ethylene glycol as the cores, and gallic, ferulic, caffeic, and p-hydroxybenzoic acids as the antioxidative moieties. In the case of DNA oxidation mediated by 2,2'-azobis(2-amidinopropane hydrochloride, AAPH), it was found that the stoichiometric factor (n) of a carboxylic acid increased rapidly when the acid was esterified with ethylene glycol, glycerol, and pentaerythritol to form a dimer, trimer, and tetramer, respectively. Interestingly, the coefficient in the equation of n∼{branch} ({branch} referred to the number of branches) was higher than one, indicating that the antioxidative effect was enhanced more promptly than the increase of the number of branches. Meanwhile, tetramer exhibited high intercalation effect with DNA strand. Therefore, additionally antioxidative effect was ascribed to the tethering effect resulting from tetrameric structure and strong intercalation with DNA strand generated by tetramer.

Development of hydroxybenzoic-based platforms as a solution to deliver dietary antioxidants to mitochondria.

Oxidative stress and mitochondrial dysfunction have been associated with metabolic and age-related diseases. Thus, the prevention of mitochondrial oxidative damage is nowadays a recognized pharmacological strategy to delay disease progression. Epidemiological studies suggested an association between the consumption of polyphenol-rich diet and the prevention of different pathologies, including diseases with a mitochondrial etiology. The development of mitochondrial-targeted antioxidants based on dietary antioxidants may decrease mitochondrial oxidative damage. Herein, we report the design and synthesis of two new mitochondriotropic antioxidants based on hydroxybenzoic acids (AntiOxBENs). The results obtained showed that the novel antioxidants are accumulated inside rat liver mitochondria driven by the organelle transmembrane electric potential and prevented lipid peroxidation, exhibiting low toxicity. Some of the observed effects on mitochondrial bioenergetics resulted from an increase of proton leakage through the mitochondrial inner membrane. The new derivatives present a higher lipophilicity than the parent compounds (protocatechuic and gallic acids) and similar antioxidant and iron chelating properties. AntiOxBENs are valid mitochondriotropic antioxidant prototypes, which can be optimized and used in a next future as drug candidates to prevent or slow mitochondrial oxidative stress associated to several pathologies.

Production of verbascoside and phenolic acids in biomass of Verbena officinalis L. (vervain) cultured under different in vitro conditions.

Methanolic extracts from the biomass of Verbena officinalis cultured under continuous artificial light and in darkness on 12 variants of the Murashige and Skoog medium containing different concentrations (0.5-3.0 mg/L) of plant growth regulators: 6-benzyladenine, kinetin, 1-naphthaleneacetic acid and indole-3-butyric acid, were analysed for the amounts of verbascoside and phenolic acids, before and after acid hydrolysis, using the HPLC-DAD method. The amounts of verbascoside were very high (max. 2454.12 mg/100 g DW - light, and 2135.59 mg/100 g DW - darkness). The total amounts of phenolic acids reached a maximum of 46.02 mg/100 g DW (free phenolic acids) and 141.05 mg/100 g DW (bound compounds). The main metabolites were: ferulic, o-coumaric and caffeic acids. The maximum amount of verbascoside was 3.28 times higher than in extracts from the herb of the parent plant. The cultures could be proposed as a potential biotechnological source for selected biologically active compounds.

A Bio-Chemosynthetic Approach to Superparamagnetic Iron Oxide-Ansamitocin Conjugates for Use in Magnetic Drug Targeting.

A combination of mutasynthesis using a mutant strain of A. pretiosum blocked in the biosynthesis of amino-hydroxybenzoic acid (AHBA) and semisynthesis relying on a Stille cross-coupling step provided access to new ansamitocin derivatives of which one was attached by a thermolabile linker to nanostructured iron oxide particles. When exposed to an oscillating electromagnetic field the resulting iron oxide/ansamitocin conjugate 19 heats up in an aqueous suspension and the ansamitocin derivative 16 is released by means of a retro-Diels-Alder reaction. It exerts strong antiproliferative activity (IC50 =4.8 ng mg-1 ) in mouse fibroblasts. These new types of conjugates have the potential for combating cancer through hyperthermia and chemotherapy using an electromagnetic external trigger.

Synthesis and in vitro antioxidant and antimicrobial studies of novel structured phosphatidylcholines with phenolic acids.

Novel phenoylated phosphatidylcholines were synthesized from 1,2-dipalmitoyl phosphatidylcholine/egg 1,2-diacyl phosphatidylcholine and phenolic acids such as ferulic, sinapic, vanillic and syringic acids. The structures of phenoylated phosphatidylcholines were confirmed by spectral analysis. 2-acyl-1-lyso phosphatidylcholine was synthesized from phosphatidylcholine via regioselective enzymatic hydrolysis and was reacted with hydroxyl protected phenolic acids to produce corresponding phenoylated phosphatidylcholines in 48-56% yields. Deprotection of protected phenoylated phosphatidylcholines resulted in phenoylated phosphatidylcholines in 87-94% yields. The prepared compounds were evaluated for their preliminary in vitro antimicrobial and antioxidant activities. Among the active derivatives, compound 1-(4-hydroxy-3,5-dimethoxy) cinnamoyl-2-acyl-sn-glycero-3-phosphocholine exhibited excellent antioxidant activity with EC50 value of 16.43µg/mL. Compounds 1-(4-hydroxy-3-methoxy) cinnamoyl-2-acyl-sn-glycero-3-phosphocholine and 1-(4-hydroxy-3,5-dimethoxy) cinnamoyl-2-palmitoyl-sn-glycero-3-phosphocholine exhibited good antioxidant activity with EC50 values of 36.05 and 33.35µg/mL respectively. Compound 1-(4-hydroxy-3-methoxy) cinnamoyl-2-palmitoyl-sn-glycero-3-phosphocholine exhibited good antibacterial activity against Klebsiella planticola with MIC of 15.6µg/mL and compound 1-(4-hydroxy-3-methoxy) benzoyl-2-acyl-sn-glycero-3-phosphocholine exhibited good antifungal activity against Candida albicans with MIC of 15.6µg/mL.

Protocatechuic acid grafted onto chitosan: Characterization and antioxidant activity.

In this study, protocatechuic acid (PA) was grafted onto chitosan (CS) by a carbodiimide mediated cross-linking reaction. The structural characterization, physical property and antioxidant activity of PA grafted CS (PA-g-CS) was investigated. As results, three copolymers with different grafting ratios (61.64, 190.11 and 279.69mg PAE/g) were obtained by varying the molar ratios of reaction substrates. PA-g-CS showed the same UV absorption peaks as PA at 258 and 292nm. As compared to CS, PA-g-CS exhibited a decreased band at 1596cm(-1) and a new band at 1716cm(-1), suggesting the formation of amide and ester linkages between PA and CS. New proton signals at δ6.77-7⋅33ppm were observed on (1)H NMR spectrum of PA-g-CS, assigning to the methine protons of PA. Signals at δ 150.8-116.6 ppm on (13)C NMR spectrum of PA-g-CS was assigned to the aromatic ring carbon of PA moieties. All the structural information confirmed the successful grafting of PA onto CS. SEM observation showed CS had a smooth surface, while PA-g-CS had a rough surface. TGA revealed the thermal stability of PA-g-CS was lower than CS. Antioxidant activity assays further verified the reducing power and DDPH radical scavenging activity of PA-g-CS was much higher than CS.

Synthesis and SAR Studies of Neuritogenic Gentiside Derivatives.

Tetradecyl 2,3-dihydroxybenzoate (ABG-001) has been designed and synthesised as a lead compound to treat Alzheimer's disease, based on structure-activity relationships of gentisides. In this paper, the alkyl chain and ester linkage group of ABG-001 were modified. Consequently, several series of novel gentiside derivatives were designed and synthesised, and their neuritogenic activity was evaluated in PC12 cells. Among all the tested compounds, S-dodecyl 2,3-dihydroxybenzothioate (15d, named as ABG-199) was the most potent; the compound induced significant neurite outgrowth at 0.1 µM, which was comparable to that of nerve growth factor at the optimal concentration of 40 ng/mL and ABG-001 at 1 µM. A brief study on the mechanism of action of ABG-199 revealed that extracellular signal-regulated kinase phosphorylation was involved in ABG-199-induced neurite outgrowth in PC12 cells.