Synthesis and Dopaminergic Neuroprotective Activity of the Proposed Structure of Bassiamide A, a Caffeamide Alkaloid Derived from Bassia Indica Wight.

Herein, we describe the synthesis of the proposed structure of the caffeamide alkaloid bassiamide A. The amide moiety of bassiamide A was readily formed via an amide coupling reaction between caffeic acid and the known N-(3-aminopropyl)-3-methylbutanamide. However, the spectral data of the synthesized bassiamide A did not agree with that of a previous study. The structure of the synthesized bassiamide A was confirmed using combined two-dimensional NMR analysis. Extended analyses of the bioactivity of the synthesized bassiamide A revealed its efficacy in protecting dopaminergic neurons from MPP+-induced neurotoxicity in Caenorhabditis elegans. Additionally, treatment with bassiamide A notably ameliorated the impaired food-sensing ability and locomotion of Caenorhabditis elegans, suggesting a protective effect on the functionality of dopaminergic neurons.

Caffeic Acid Phenethyl Ester and Caffeamide Derivatives Suppress Oral Squamous Cell Carcinoma Cells.

Caffeic acid phenethyl ester (CAPE) contains antibiotic and anticancer activities. Therefore, we aimed to investigate the anticancer properties and mechanisms of CAPE and caffeamide derivatives in the oral squamous cell carcinoma cell (OSCC) lines SAS and OECM-1. The anti-OSCC effects of CAPE and the caffeamide derivatives (26G, 36C, 36H, 36K, and 36M) were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Cell cycle and total reactive oxygen species (ROS) production were analyzed using flow cytometry. The relative protein expression of malignant phenotypes was determined via Western blot analysis. The results showed that 26G and 36M were more cytotoxic than the other compounds in SAS cells. After 26G or 36M treatment for 48 h, cell cycle S phase or G2/M phase arrest was induced, and cellular ROS increased at 24 h, and then decreased at 48 h in both cell lines. The expression levels of cell cycle regulatory and anti-ROS proteins were downregulated. In addition, 26G or 36M treatment inhibited malignant phenotypes through mTOR-ULK1-P62-LC3 autophagic signaling activated by ROS generation. These results showed that 26G and 36M induce cancer cell death by activating autophagy signaling, which is correlated with altered cellular oxidative stress.

In Vitro Antimicrobial Potential of CAPE and Caffeamide Derivatives against Oral Microbes.

Caffeic acid phenethyl ester (CAPE) is a natural component isolated from propolis and used in traditional medicine. We aimed to investigate the antimicrobial properties and action mechanism of CAPE and caffeamide derivatives (26G and 36M) against oral disease microbes. We resolved the minimum inhibitory and bactericidal concentrations of 26G and 36M and their stability at different temperatures and pH. We also evaluated their effect on biofilm formation and antibiotic resistance gene expression in methicillin-resistant Staphylococcus aureus (MRSA). Our results revealed that 26G and 36M showed the best anticancer and antimicrobial activities, respectively, compared with the other four caffeamide derivatives. Both 26G and 36M showed heat-dependent decreases in antimicrobial activity. The 36M derivative was stable irrespective of pH, whereas 26G was not stable under high pH conditions. Biofilm formation and antibiotic resistance-related gene expression were consistent with their respective phenotypes. This study provides evidence for the potential application of CAPE and caffeamide derivatives in dental medicine to cure or prevent oral diseases.

Protective Effects and Mechanisms of N-Phenethyl Caffeamide from UVA-Induced Skin Damage in Human Epidermal Keratinocytes through Nrf2/HO-1 Regulation.

The skin provides an effective barrier against physical, chemical, and microbial invasion; however, overexposure to ultraviolet (UV) radiation causes excessive cellular oxidative stress, which leads to skin damage, DNA damage, mutations, and skin cancer. This study investigated the protective effects of N-phenethyl caffeamide (K36) from UVA damage on human epidermal keratinocytes. We found that K36 reduced UVA-induced intracellular reactive oxygen species (ROS) production and induced the expression of the intrinsic antioxidant enzyme heme oxygenase-1 (HO-1) by increasing the translocation of nuclear factor erythroid 2⁻related factor 2 (Nrf2). K36 could inhibit the phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinases (JNK) and reduce UVA-induced matrix metalloproteinase (MMP)-1 and MMP-2 overexpression; it could also elevate the expression of tissue inhibitors of metalloproteinases (TIMP). In addition, K36 ameliorated 8-hydroxy-2'-deoxyguanosine (8-OHdG) induced by UVA irradiation. Furthermore, K36 could downregulate the expression of inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6) and the subsequent production of nitric oxide (NO) and prostaglandin E2 (PGE2). Based on our findings, K36 possessed potent antioxidant, anti-inflammatory, antiphotodamage, and even antiphotocarcinogenesis activities. Thus, K36 has the potential to be used to multifunctional skin care products and drugs.

N-Propargyl Caffeamide Skews Macrophages Towards a Resolving M2-Like Phenotype Against Myocardial Ischemic Injury via Activating Nrf2/HO-1 Pathway and Inhibiting NF-ĸB Pathway.

BACKGROUND/AIMS: Macrophages exhibit dynamic pro-inflammatory and resolving activities in myocardial infarction. The present study investigated whether caffeic acid derivatives could induce macrophage polarization towards a resolving M2 phenotype against myocardial infarction injury. METHODS: Western blotting, RT-PCR and flow cytometry techniques are used to evaluate macrophage biomarkers expression and specific proteins in the related signaling pathways. Ligation of the left anterior descending artery induced rat model of myocardial infarction, TTC staining and immunohistochemical staining are used to examine cardioprotective effect in vivo. RESULTS: We initially evaluated the anti-inflammatory activity of four caffeic acid derivatives including n-propargyl caffeamide (PACA) in RAW264.7 macrophages. As result, PACA selectively suppressed the up-regulation of inducible nitric oxide synthase (iNOS) over cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated cells. We subsequently examined the effects of PACA on macrophage polarization by determining macrophage biomarkers. PACA down-regulated M1 biomarkers (e.g., iNOS, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 10 (CXCL10) and CD80) but up-regulated M2 biomarkers (e.g., Ym-1 and arginase-1). On the other hand, PACA suppressed macrophage chemotaxis while enhanced macrophage phagocytosis. We further examined the in vivo cardioprotective activity of PACA in a rat model of myocardial infarction. Following ligation of the left anterior descending artery, PACA treatment effectively reduced myocardial infarct size and promoted macrophage M2 polarization. We finally explored the underlying mechanisms. We found that PACA attenuated LPS-induced NF-ĸB activation while activated Nrf2/HO-1 pathway. HO-1 inhibitor SnPP attenuated the effects of PACA on iNOS expression in LPS-challenged macrophages, possibly by regulating the cross-talk between HO-1 and NF-ĸB pathways. CONCLUSIONS: The key finding from the present study was that PACA promoted timely switch of macrophage phenotypes from pro-inflammatory M1 to resolving M2. We anticipate that PACA is a potential drug candidate for the resolution of inflammation and cardiac repair after myocardial infarction.

Synthesis and biological evaluation of caffeic acid derivatives as potent inhibitors of α-MSH-stimulated melanogenesis.

We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin medication including a cosmetic skin-whitening agent. The SAR studies revealed that cyclohexyl ester and secondary amide derivatives of caffeic acid showed significant inhibitory activities.

Synthesis, biological evaluation, and metabolic stability of chlorogenic acid derivatives possessing thiazole as potent inhibitors of α-MSH-stimulated melanogenesis.

A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and evaluated for their inhibitory activity against α-MSH. The inhibitory activity was improved by replacing an α,ß-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, compound 7d, one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with an IC50 of 0.90µM. Further studies on metabolic stability and bioactivation potential were also accomplished.

N-(4-methoxyphenyl) caffeamide-induced melanogenesis inhibition mechanisms.

BACKGROUND: The derivative of caffeamide exhibits antioxidant and antityrosinase activity. The activity and mechanism of N-(4-methoxyphenyl) caffeamide (K36E) on melanogenesis was investigated. METHODS: B16F0 cells were treated with various concentrations of K36E; the melanin contents and related signal transduction were studied. Western blotting assay was applied to determine the protein expression, and spectrophotometry was performed to identify the tyrosinase activity and melanin content. RESULTS: Our results indicated that K36E reduced α-melanocyte-stimulating hormone (α-MSH)-induced melanin content and tyrosinase activity in B16F0 cells. In addition, K36E inhibited the expression of phospho-cyclic adenosine monophosphate (cAMP)-response element-binding protein, microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). K36E activated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK3ß), leading to the inhibition of MITF transcription activity. K36E attenuated α-MSH induced cAMP pathways, contributing to hypopigmentation. CONCLUSIONS: K36E regulated melanin synthesis through reducing the expression of downstream proteins including p-CREB, p-AKT, p-GSK3ß, tyrosinase, and TRP-1, and activated the transcription factor, MITF. K36E may have the potential to be developed as a skin whitening agent.

N-(4-bromophenethyl) Caffeamide Protects Skin from UVB-Induced Inflammation Through MAPK/IL-6/NF-κB-Dependent Signaling in Human Skin Fibroblasts and Hairless Mouse Skin.

Long-term exposure to ultraviolet (UV) irradiation causes skin inflammation and aging. N-(4-bromophenethyl) caffeamide (K36H) possesses antioxidant and antimelanogenic properties. The present study investigated the effects of K36H on UVB-induced skin inflammation in human skin fibroblasts and hairless mice and evaluated the underlying mechanisms. The in vitro results indicated that K36H reduced UVB-induced mitogen-activated protein kinase (MAP kinase) expression. Furthermore, K36H treatment reduced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression in UVB-irradiated fibroblasts by regulating IκB and nuclear factor-kappa B (NF-κB) expression. In the animal study, topically applied K36H markedly reduced inflammation and skin thickness and prevented photodamage to the skin of hairless mice. In addition, K36H inhibited the levels of UV-upregulated inflammation-related proteins levels such as IL-1, iNOS, and NF-κB in the dermis of hairless mice. Our findings demonstrated the antioxidant and anti-inflammatory properties of K36H in human skin fibroblasts and hairless mice. Therefore, K36H can be developed as an antiphotodamage and antiphotoinflammation agent.

Protection against Ultraviolet A-Induced Skin Apoptosis and Carcinogenesis through the Oxidative Stress Reduction Effects of N-(4-bromophenethyl) Caffeamide, A Propolis Derivative.

Ultraviolet A (UVA) is a major factor in skin aging and damage. Antioxidative materials may ameliorate this UV damage. This study investigated the protective properties of N-(4-bromophenethyl) caffeamide (K36H) against UVA-induced skin inflammation, apoptosis and genotoxicity in keratinocytes. The protein expression or biofactor concentration related to UVA-induced skin damage were identified using an enzyme-linked immunosorbent assay and western blotting. K36H reduced UVA-induced intracellular reactive oxygen species generation and increased nuclear factor erythroid 2-related factor 2 translocation into the nucleus to upregulate the expression of heme oxygenase-1, an intrinsic antioxidant enzyme. K36H inhibited UVA-induced activation of extracellular-signal-regulated kinases and c-Jun N-terminal kinases, reduced the overexpression of matrix metalloproteinase (MMP)-1 and MMP-2 and elevated the expression of the metalloproteinase-1 tissue inhibitor. Moreover, K36H inhibited the phosphorylation of c-Jun and downregulated c-Fos expression. K36H attenuated UVA-induced Bax and caspase-3 expression and upregulated antiapoptotic protein B-cell lymphoma 2 expression. K36H reduced UVA-induced DNA damage. K36H also downregulated inducible nitric oxide synthase, cyclooxygenase-2 and interleukin-6 expression as well as the subsequent generation of prostaglandin E2 and nitric oxide. We observed that K36H ameliorated UVA-induced oxidative stress, inflammation, apoptosis and antiphotocarcinogenic activity. K36H can potentially be used for the development of antiphotodamage and antiphotocarcinogenic products.

N-Propargyl Caffeamide (PACA) Ameliorates Dopaminergic Neuronal Loss and Motor Dysfunctions in MPTP Mouse Model of Parkinson's Disease and in MPP+-Induced Neurons via Promoting the Conversion of proNGF to NGF.

Insufficient production of nerve growth factor (NGF) is implicated in Parkinson's disease (PD). We recently discovered that caffeic acid derivative N-propargyl caffeamide (PACA) not only potentiated NGF-induced neurite outgrowth but also attenuated 6-hydroxydopamine neurotoxicity in neuronal culture. The aim of the present study was to investigate whether PACA could increase NGF levels against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) neurotoxicity in a mouse PD model. We induced parkinsonism in mice by intraperitoneal injection of MPTP for seven consecutive days. Animal motor functions were assessed by rotarod test and pole test. Our results showed that PACA ameliorated motor impairments in MPTP-challenged mice. Based on Western blot analysis and/or immunofluorescence staining of NGF and tyrosine hydroxylase (TH), PACA preserved TH levels in the midbrain substantia nigra pars compacta. PACA also increased NGF expression while it decreased proNGF accumulation. Interestingly, NGF was widely induced in the midbrains including astrocytes. To elucidate the mechanisms by which PACA induces NGF, we focused on the effects of PACA on two neurotrophic signaling pathways, the PI3K and MEK pathways. We found that PACA induced the phosphorylation of Akt, ERK, and CREB against MPTP-mediated alterations. Importantly, PACA increased NGF levels and subsequently induced TrkA activation in MPTP-treated mice. Consistently, PACA also increased NGF levels in dopaminergic PC12 cells and primary rat midbrain neurons against N-methyl-4-phenylpyridinium iodide (MPP+) toxicity. ERK and PI3K inhibitors attenuated the effects of PACA on NGF levels. Collectively, our results suggest that PACA may rescue NGF insufficiency via sequential activation of PI3K/Akt, ERK1/2, and CREB signaling pathways. Graphical Abstract ᅟ.

In Vivo Cardioprotective Effects and Pharmacokinetic Profile of N-Propyl Caffeamide Against Ischemia Reperfusion Injury.

Caffeic acid derivatives constitute a class of potent anti-inflammatory and cardioprotective drug candidates. We recently synthesized a new caffeic acid derivative N-propyl caffeamide (PCA). Our pilot experiments demonstrated that PCA enhanced the survival of rat cardiomyocyte H9c2 cells against oxygen glucose deprivation and reoxygenation challenge in a concentration-dependent manner. Interestingly, PCA exhibited better cardioprotective potential than caffeic acid phenethyl ester and propyl caffeate. Thus, we hypothesized that PCA could protect heart against ischemia reperfusion (I/R) injury in mice. We first determined the stability and pharmacokinetic profile of PCA in male Sprague-Dawley rats by ultra-performance liquid chromatography coupled with UV and MS/MS detections. The stability of PCA in rat plasma was defined by the half-life of 31.39, 7.19 and 1.37 h in rat plasma at 25, 37 and 60 °C, respectively. To study the pharmacokinetic profiles, PCA was injected into male SD rats at the dose of 15 mg/kg via intravenous bolus administration. PCA showed the elimination half-life of approximate 235 min in rats. We subsequently evaluated the cardioprotective potential of PCA in mice model of myocardial infarction. Our results demonstrated that PCA effectively reduced infarct size and release of myocardial enzymes (e.g., CK, CK-MB and LDH). Biochemical analyses suggested that PCA increased the activities of antioxidant enzymes (e.g., CAT and SOD) while attenuated lipid peroxidation. Moreover, PCA profoundly reduced the number of apoptotic cells in infarcted myocardium. Consistently, PCA increased the expression level of anti-apoptotic protein Bcl2 whereas suppressed the expression of pro-apoptotic protein Bax in cardiac tissues. Collectively, PCA appears to be a novel bioavailable and stable pharmacological treatment for myocardial infarction.

KS370G, a caffeamide derivative, attenuates unilateral ureteral obstruction-induced renal fibrosis by the reduction of inflammation and oxidative stress in mice.

Unilateral ureteral obstruction (UUO) is an established animal model used to study renal nephropathy. Caffeic acid phenethyl ester, a natural phenolic compound, possesses antifibrotic, anti-inflammation and anti-oxidative stress effects; however, rapid decomposition by esterases substantially decreases its bioavailability. The goal of this study was to investigate the beneficial effects of KS370G, a synthetic caffeamide derivative, on UUO-induced renal injury. Following the UUO, KS370G (10mg/kg) was administered by oral gavage once a day. Renal injury was analyzed at 14 days post-operation. Our results show that KS370G significantly attenuated collagen deposition in the obstructed kidney and inhibited UUO-induced renal fibrosis markers expression, including fibronectin, type I collagen, vimentin, and α-smooth muscle actin (α-SMA). KS370G significantly lowered the expression of renal inflammatory chemokines/adhesion molecules and monocyte cells marker (MCP-1, VCAM-1, ICAM-1 and CD11b). KS370G also reduced renal malondialdehyde levels and reversed the expression of renal antioxidant enzymes (SOD and catalase) after UUO. Furthermore, KS370G significantly inhibited UUO-induced elevated plasma AngII and TGF-ß1 levels, TGF-ß1 protein expression and Smad3 phosphorylation. These findings demonstrate that KS370G reduces renal obstructive nephropathy by possibly inhibiting AngII, TGF-ß and Smad3 signaling pathways.

N-Phenethyl caffeamide and photodamage: protecting skin by inhibiting type I procollagen degradation and stimulating collagen synthesis.

Skin is mainly damaged by genetic and environmental factors such as ultraviolet (UV) light and pollutants. UV light is a well-known factor that causes various types of skin damage and premature aging. Reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases that break down type I collagen. This study investigated the antioxidant and antiphotodamage activity and mechanisms of N-phenethyl caffeamide (K36) in human skin fibroblasts. The results indicated that K36 demonstrated strong 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, which dose-dependently reduced the production of UVB-induced intracellular ROS in human dermal fibroblasts. K36 prevented UVB-irradiation-induced type I collagen degradation by inhibiting the expression of matrix metalloproteins-1, -3, and -9 and the phosphorylation of mitogen-activated protein (MAP) kinases. Furthermore, K36 elevated collagen synthesis in skin fibroblasts by inhibiting UVB-induced Smad7 overexpression. K36 downregulated the expression of the transcription factor, activator protein-1 (AP-1). Our results indicated that K36 exhibited antioxidant properties and prevented skin collagen degradation caused by UV exposure and the stimulation of collagen synthesis, which suggests the potential use of K36 in preventing photodamage.