Norharman Synthesis Essay


NNMT (nicotinamide N-methyltransferase, E.C. catalyses the N-methylation of nicotinamide to 1-methylnicotinamide. NNMT expression is significantly elevated in a number of cancers, and we have previously demonstrated that NNMT expression is significantly increased in the brains of patients who have died of Parkinson's disease. To investigate the cellular effects of NNMT overexpression, we overexpressed NNMT in the SH-SY5Y cell line, a tumour-derived human dopaminergic neuroblastoma cell line with no endogenous expression of NNMT. NNMT expression significantly decreased SH-SY5Y cell death, which correlated with increased intracellular ATP content, ATP/ADP ratio and Complex I activity, and a reduction in the degradation of the NDUFS3 [NADH dehydrogenase (ubiquinone) iron–sulfur protein 3] subunit of Complex I. These effects were replicated by incubation of SH-SY5Y cells with 1-methylnicotinamide, suggesting that 1-methylnicotinamide mediates the cellular effects of NNMT. Both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone by reversing their effects upon ATP synthesis, the ATP/ADP ratio, Complex I activity and the NDUFS3 subunit. The results of the present study raise the possibility that the increase in NNMT expression that we observed in vivo may be a stress response of the cell to the underlying pathogenic process. Furthermore, the results of the present study also raise the possibility of using inhibitors of NNMT for the treatment of cancer.

Abbreviations: CGC, cerebellar granule cell; CS, citrate synthase; CxI, Complex I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MeN, 1-methylnicotinamide; MPP+, 1-methyl-4-phenylpyridinium ion; NDUFS3, NADH dehydrogenase (ubiquinone) iron–sulfur protein 3; NNMT, nicotinamide N-methyltransferase; PD, Parkinson's disease; qPCR, quantitative PCR; RT, reverse transcription; UPL, Universal Probe Library

  • © The Authors Journal compilation © 2011 Biochemical Society

1. Introduction

Plant pathogenic microorganisms can infect crops, causing local or whole plant disease and leading to significant economic losses. How to control them in modern agriculture is still a big challenge. Many kinds of fungicides are used to prevent and cure the diseases caused by fungi; however, these chemical agents cannot fully protect the crops or completely cure the crops’ tissues from fungal infection under field conditions. Therefore, novel and more practical fungicidal reagents are urgently needed.

Plants can produce some secondary metabolites with insecticidal, antifungal, or antibacterial biological activity; therefore, natural products can be used as ideal lead structures to develop agrochemicals. The β-carboline alkaloids are a large group of natural and synthetic indole alkaloids that possess a common tricyclic pyrido [3,4-b] indole ring structure (Figure 1) [1,2,3]. Harmine, harman, harmol, harmaline, and harmalol, which are β-carboline and dihydro-β-carboline alkaloids, are four representative harmala alkaloids. Harmine was originally isolated from Peganum harmala L. [4], and found to exhibit a cytotoxic effect on HL60 and K562 leukemic cell lines [5]. Harmane has DNA intercalation ability, leading to not only intercalation into DNA [6,7] and formation DNA adducts [8], but also inhibition of Topo I [7,9], Topo II [9], and MAO-A activity [10,11]. Harmol can induce autophagy and suppression of survivin expression, subsequently induce apoptotic cell death in U251MG human glioma cells [12] and apoptosis by caspase-8 activation independently from Fas/Fas ligand interaction in human non-small cell lung cancer (NSCLC) H596 cells [13], and significantly inhibit the dioxin-mediated induction of CYP1A1 at mRNA, protein, and activity levels in a concentration-dependent manner in human and murine hepatoma cells [14]. Harmaline can inhibit DNA excision repair [15], human DNA Topo I activity [7], PKC activity [16], and TMV [17] and against the amastigote stage of Leishmania [16]. Harmalol is able to induce melanogenesis through p38 MAPK signaling [18] and can act as an agent for preventing dioxin-mediated effects [19].

Figure 1. Chemical structures of β-carboline alkaloids.

Figure 1. Chemical structures of β-carboline alkaloids.

β-Carboline and its structural analogues in the medical and pharmaceutical are a research focus. However, there is limited information about these chemicals in agricultural areas and they lack system development and application. The β-carboline amides, containing amides and a carboline structure, represent a new direction for the development of plant-derived bio-pesticides. In addition, the antifungal activity of β-carboline will change when the 3-position of β-carboline was substituted [17,20]. In this study, their fungicidal activities were systematically evaluated. To investigate the biological activities of the substituents, β-carbolines containing different substituents were synthesized and their fungicidal activities were also systematically evaluated.

2. Results and Discussion

2.1. Synthesis

Compounds harmine, harmane, harmaline, and harmalol were obtained from Sigma-Aldrich, St. Louis, MI, USA. Their chemical structures are shown in Figure 1. Previous structure–activity relationship studies had demonstrated the influence of substituents in positions-1, -3, and -9 of the β-carboline skeleton for a variety of synthetic β-carboline derivatives [21,22,23,24]. In order to study the effect of main structure and the substituent groups at position 1 and 3 on their herbicidal activity, we synthesized a series of 30 novel β-carboline derivatives bearing a substituted amide group at C-3 and substituted groups at C-1 (Figure 2 and Scheme 1). All these compounds were characterized by their melting point, mass, infrared, IR, and 1H-NMR spectra, which confirmed the proposed structures of the new compounds. Tryptophan, which has an electron-rich indole ring, was used as the parent material when applying Pictet–Spengler or Bischler–Napieralski reactions [25] with a variety of aromatic aldehyde cyclization to give tetrahydrocarboline compounds, then oxidizing to obtain β-carboline compounds by using DMF as a solvent and KMnO4 as an oxidant. Pictet–Spengler reactions that used acetic acid as the catalyzed solvent produced a reaction that was refluxed at 80 °C to obtain a higher yield (above 80%) of tetrahydro-β-carboline compounds. Since the reaction temperature was moderate and the by-product generated was less, the product could be obtained with a purity of more than 90% by suction filtration and washing. Then the product could be used directly in the next reaction after drying. The carboxyl on the 3-position of tetrahydro-β-carboline must be protected by esterification, due to the fact that a carboxyl with high reaction activity could be easily decarboxylated, thereby losing carbonyl in the potassium permanganate conditions. Using KMnO4 to oxidize tetrahydro-β-carboline derivatives produced a lower yield, but the reagent was relatively inexpensive, and the reaction was easy to operate in the laboratory. The acylation reaction of amide synthesis used acid halide with ammonia or amine. Step 1: β-carboline-3-carboxylic acid reacted with an excess of thionyl chloride to become the corresponding acid chloride in the situation of catalyzer MDF of 1‰. The excess of thionyl chloride was both reactant and reaction solvent in the reaction, which may also remove water to reduce moisture in the system requirements of dry operation. HCl gas and SO2 gas were generated by the reaction; we utilized lye to absorb them. Step 2: acid chloride reacted with the corresponding amine. The reaction requires adding week base to neutralize the HCl so as to avoid amine reacting with HCl to generate the amine hydrochloride, which does not participate in the reaction. Because the reaction is intense, the product of the first step should be dissolved firstly in methylene chloride, and added slowly dropwise to the mixture of amine and triethylamine that was placed on an ice bath, and then the mixture stirred at room temperature for half an hour.

Figure 2. Design of target compounds.

Figure 2. Design of target compounds.

2.2. Fungicidal Activities

Fungicide Screening. Compounds F1–30 were evaluated in a series of in vitro fungicidal tests, against a range of phytopathogenic species. The resulting data (Table 1) revealed that these alkaloids and their derivatives displayed potential fungicidal activity against six kinds of plant fungi including F. oxysporum f.sp.cubense, C. gloeosporioides (Penz.), R. solani, P. litchii, P. nicotianae, and O. citriaurantii ex Persoon. When the R1 was phenyl, the compounds exhibited higher activities than those with 4-nitrophenyl, 4-methoxyphenyl,3,4,5-trimethoxyphenyl,4-trifluoromethylphenyl, or 4-chlorophenyl as the R1. When the skeleton was β-carboline, the types of the substituents on the R2 and R3 had a significant influence on the fungicidal activities. For instance, compound F4 (N-(2-pyridyl)-1-phenyl-9H-pyrido[3,4-b]indole-3-formamide) exhibited excellent fungicidal activity against most of the tested fungi, whereas compound F2 (N,N-diethyl-1-phenyl-9H-pyrido[3,4-b]indole-3-formamide) exhibited only moderate activity; compound F5, containing a 4-trifluoromethylphenyl group, exhibited higher fungicidal activity than the compound F1 and the group containing 4-chlorophenyl. Several of these compounds selectively exhibited fungicidal activities against some fungi. For example, the activities of harmine (against F. oxysporum f.sp.cubense and R. solani), compound F16 (against O. citriaurantii ex Persoon), and compound F25 (against C. gloeosporioides (Penz.)) were much higher than that against other fungi and were higher than the other compounds against some fungi, as shown in Table 1.

Scheme 1. Synthesis of target compounds.

Scheme 1. Synthesis of target compounds.

Table 1. Fungicidal activities of harmine, harmane, harmaline, harmalol, and compounds F130 against six kinds of fungi (percent inhibition, %; 100 mg/L).

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