One-pot heterogeneous synthesis of Δ(3)-tetrahydrocannabinol analogues and xanthenes showing differential binding to CB(1) and CB(2) receptors

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive cannabinoid in hemp (Cannabis sativa L.) and responsible for many of the pharmacological effects mediated via cannabinoid receptors. Despite being the major cannabinoid scaffold in nature, Δ(9)-THC double bond isomers remain poorly studied. The chemical scaffold of tetrahydrocannabinol can be assembled from the condensation of distinctly substituted phenols and monoterpenes. Here we explored a microwave-assisted one pot heterogeneous synthesis of Δ(3)-THC from orcinol (1a) and pulegone (2). Four Δ(3)-THC analogues and corresponding Δ(4a)-tetrahydroxanthenes (Δ(4a)-THXs) were synthesized regioselectively and showed differential binding affinities for CB1 and CB2 cannabinoid receptors. Here we report for the first time the CB1 receptor binding of Δ(3)-THC, revealing a more potent receptor binding affinity for the (S)-(-) isomer (hCB1Ki = 5 nM) compared to the (R)-(+) isomer (hCB1Ki = 29 nM). Like Δ(9)-THC, also Δ(3)-THC analogues are partial agonists at CB receptors as indicated by [(35)S]GTPγS binding assays. Interestingly, the THC structural isomers Δ(4a)-THXs showed selective binding and partial agonism at CB2 receptors, revealing a simple non-natural natural product-derived scaffold for novel CB2 ligands.

Synthesis, crystallography and biological activity of myo-inositol phosphates

The antioxidant property of myo-inositol hexakisphosphate is important in the prevention of hydroxyl radical formation which may allow it to act as a 'safe' carrier of iron within the cell. Here, the hypothesis that the recently discovered natural product, myo-inositol 1,2,3-trisphosphate represents the simplest structure to mimic phytate's antioxidant activity has been tested. The first synthesis of myo-inositol 1,2,3-trisphosphate has been completed, along with its X-ray structure determination and that of key synthetic intermediates. Iron binding studies of myo-inositol 1,2,3-trisphosphate demonstrated that phosphate groups with the equatorial-axial-equatorial conformation are required for complete inhibition of hydroxyl radical formation. myo-Inositol monophosphatase is a key enzyme in recycling myo-inositol from its monophosphates in the brain and its inhibition is implicated in lithium's antimanic properties. Current synthetic strategies require inositol compounds to be protected (often with more than one group), resolved, phosphorylated and deprotected to produce the desired optically active myo-inositol phosphates. Here, the synthesis of myo-inositol 3-phosphate has been achieved in only 4 steps from myo-inositol. The stereoselective addition of the chiral phosphorylating agent (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphospholidin-2-one to a protected inositol intermediate allowed separation of diastereoisomers and easy deprotection to myo-inositol 3-phosphate. This strategy also allows the possible introduction of labels of oxygen and sulphur to give a thiophosphate of known stereochemistry at phosphorus which would be useful for the analysis of the stereochemical course of phosphate hydrolysis catalysed by inositol monophosphatase.

Design, synthesis and evaluation of cyclothialidine analogues as DNA gyrase inhibitors

Cyclothialidine, a natural product isolated from Streptomyces .filipinensis NR0484, has been proven to be a potent and selective inhibitor of the bacterial enzyme DNA gyrase. Gyrase inhibition results in cell death, the enzyme being the target of several currently used antibiotics. Cyclothialidine showed poor activity against whole bacterial cells, highlighting scope for improvement regarding cell membrane pemeability in order for the full potential of this new class of antibiotics to be realised, Structurally, cyclothialidine contains a 12-membered lactone ring which is partly integrated into a pentapeptide chain, with a substituted aromatic moiety bordering the lactone, Retrosynthetically it can be traced back to cis-3-hydroxyproline, 3,5-dihydroxy-2,6-dimethylbenzoic acid and four commercially available amino acids; two serine, one cysteine and one alanine. In this work, a model of cyclothialidine was synthesised in order to establish the methodology for more complex compounds. Analogues with hydroxy, dihydroxy and dihydroxymethyl substituted aromatic moieties were then prepared to ensure successful protection methods could be performed and the pharmacophore synthesised. The key aromatic moiety, 2,6-dimethyl-3,5-dihydroxybenzoic acid was produced via two successive Mannich reaction/reduction steps. Acid protection using 4-nitrobenzyl bromide and TBDMS hydroxyl protection followed by bromination of one methyl afforded the desired intermediate. Reaction with a serine/cysteine dipeptide, followed by deprotection and cyclisation under Mitsunobu conditions lead to the 12-membered lactone. An amine substituted aromatic analogue and also replacement of the cysteine sulphur by oxygen were attempted but without success. In an effort to improve cell permeability, a conjugate was synthesised between the pharmacophore and a cholesterol moiety. It was hoped the steroid fragment would serve to increase potency by escorting the molecule through the lipid environment of the cell membrane. The pharmacophore and conjugate were tested against a variety of bacterial strains but the conjugate failed to improve activity.

Design, Synthesis, and Biological Characterization of Largazole Analogues

Histone deacetylases (HDACs) have been shown to play key roles in tumorigenesis, and

have been validated as effective enzyme target for cancer treatment. Largazole, a marine natural

product isolated from the cyanobacterium Symploca, is an extremely potent HDAC inhibitor that

has been shown to possess high differential cytotoxicity towards cancer cells along with excellent

HDAC class-selectivity. However, improvements can be made in the isoform-selectivity and

pharmacokinetic properties of largazole.

In attempts to make these improvements and furnish a more efficient biochemical probe

as well as a potential therapeutic, several largazole analogues have been designed, synthesized,

and tested for their biological activity. Three different types of analogues were prepared. First,

different chemical functionalities were introduced at the C2 position to probe the class Iselectivity profile of largazole. Additionally, docking studies led to the design of a potential

HDAC8-selective analogue. Secondly, the thiol moiety in largazole was replaced with a wide

variety of othe zinc-binding group in order to probe the effect of Zn2+ affinity on HDAC

inhibition. Lastly, three disulfide analogues of largazole were prepared in order to utilize a

different prodrug strategy to modulate the pharmacokinetic properties of largazole.

Through these analogues it was shown that C2 position can be modified significantly

without a major loss in activity while also eliciting minimal changes in isoform-selectivity. While

the Zn2+-binding group plays a major role in HDAC inhibition, it was also shown that the thiol

can be replaced by other functionalities while still retaining inhibitory activity. Lastly, the use of

a disulfide prodrug strategy was shown to affect pharmacokinetic properties resulting in varying

functional responses in vitro and in vivo.

v

Largazole is already an impressive HDAC inhibitor that shows incredible promise.

However, in order to further develop this natural product into an anti-cancer therapeutic as well as

a chemical probe, improvements in the areas of pharmacokinetics as well as isoform-selectivity

are required. Through these studies we plan on building upon existing structure–activity

relationships to further our understanding of largazole’s mechanism of inhibition so that we may

improve these properties and ultimately develop largazole into an efficient HDAC inhibitor that

may be used as an anti-cancer therapeutic as well as a chemical probe for the studying of

biochemical systems.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Ellipticine is a natural product which possesses multimodal anti-cancer activity. This thesis encompasses the synthesis and biological evaluation of novel ellipticine and isoellipticine derivatives as anti-cancer agents. Expanding on previous work within the group utilising vinylmagnesium bromide, derivatisation of the C5 position of ellipticine was accomplished by reaction of a key ketolactam intermediate with Grignard reagents. Corresponding attempts to introduce diverse substitution at the C11 position were unsuccessful, although one novel C11 derivative was produced using an alkyllithium reagent. A panel of novel ellipticinium salts encompassing a range of substitutions at the N2, C9 and N6 positions were prepared. Extensive derivatisation of the N10 position of isoellipticine was undertaken for the first time. Novel substitution in the form of acid and methyl ester functionalities were introduced at the C7 position of isoellipticine while novel C7 aldehyde and alcohol derivatives were synthesised. A large panel of isoellipticinium salts were prepared with conditions adjusted for the reactivity of the alkyl halide. Novel coupling reactions to increase the yield of isoellipticine were attempted but proved unsuccessful. A panel of 54 novel derivatives was prepared and a multimodal analysis of their anti-cancer activity was conducted. The NCI 60-human tumour cell lines screen was a primary source of information on the in vitro activity of compounds with derivatives found to exert potent anticancer effects, with mean GI50 values as low as 1.01 μM across the full range of cancer types and as low as 16 nM in individual cell lines. A second in vitro screen in collaboration with researchers in the University of Nantes identified derivatives which could potently inhibit growth in a p53 mutant NSCLC cell line. The cell cycle effects of a selected panel of isoellipticines were studied in leukaemia cell lines by researchers in the Department of Biochemistry and Cell Biology, UCC. Emerging from this, the therapeutic potential of one of the derivatives in AML was then assessed in vivo in an AML xenograft mouse model, with tumour weight reduced by a factor of 7 in treated mice relative to control.

Towards the total synthesis of amphidinolides C and F

The amphidinolides are marine macrolides extracted from dinoflagellates of the genus Amphidinium. To date, 37 amphidinolides have been isolated and identified, most of them possessing cytotoxicity against human cancer cell lines. Among these, amphidinolides C, F, C2 and C3 represent synthetic targets of interest owing to their scarcity, structural complexity and promising biological activities. This thesis describes the work realised towards the total synthesis of amphidinolides C and F, with a focus on the different strategies investigated and the key fragments synthesised. In the first approach, the C18−C29 fragment of amphidinolide F was prepared using an intramolecular etherification of an epoxide under acidic catalysis to produce the 2,5-trans-disubstituted tetrahydrofuran ring featured in the natural product. Unfortunately, dithiane alkylation with the C1−C17 iodide counterpart generated the desired coupling product in low yield. A second approach proposing to build the C17−C18 bond by a silicon-tethered RCM proved unsuccessful, because the requisite diene could not be obtained. It was then envisioned to form the C18−C19 bond by displacement of a triflate with an alkyne and install the ketone at C18 by a protoborylation/oxidation sequence. To this end, the C19−C29 triflate precursor was synthesised. Displeasingly, the C1−C18 alkyne counterpart (work by Dr Filippo Romiti) could not be prepared and coupling of the two fragments was not attempted. In the latest approach, the C10−C29 fragment of amphidinolide F was obtained employing a boron-mediated aldol condensation and a dithiane alkylation to form the C13−C14 and C18−C19 bonds. Several endgame strategies were examined including the successful Yamaguchi esterification of the C13-epi C10−C29 fragment and the C1−C9 acid. A challenging Stille crosscoupling was then effected to close the macrocycle but only yielded the desired macrolactone in trace amounts after global desilylation.

Aryne Reactions in the Synthesis of Heterocyclic Molecules

Reactive intermediates play an important the within the realm of chemical synthesis. Their high energy and transient nature make them difficult to observe and characterize, but it is these same properties that empower them to form bonds traditionally seen as difficult to prepare and unusual architectures quickly and efficiently. Herein, two reactive intermediates, arynes and transitient (2azaaryl)-cuprates, are exploited for their abilities to prepare important chemical motifs. Both serve as an avenue into the functionalization of arenes to provide products which hold value in a variety of fields including natural product total syntethis, pharmaseuticals and ligand design.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Discrimination of alpha-Amino Acids Using Green Tea Flavonoid (-)-Epigallocatechin Gallate as a Chiral Solvating Agent

We report a special, hitherto-unexplored property of (-)-epigallocatechin gallate (EGCG) as a chiral solvating agent for enantiodiscrimination of alpha-amino acids in the polar solvent DMSO. This phenomenon has been investigated by H-1 NMR spectroscopy. The mechanism of the interaction property of EGCG with alpha-amino acids has been understood as arising out of hydrogen-bonded noncovalent interactions, where the -OH groups of two phenyl rings of EGCG play dominant roles. The conversion of the enantiomeric mixture into diastereomers yielded well-resolved peaks for D and L amino acids permitting the precise measurement of enantiomeric composition. Often one encounters complex situations when the spectra are severely overlapped or partially resolved hampering the testing of enantiopurity and the precise measurement of enantiomeric excess (ee). Though higher concentration of EGCG yielded better discrimination, the use of lower concentration being economical, we have exploited an appropriate 2D NMR experiment in overcoming such problems. Thus, in the present study we have successfully demonstrated the utility of the bioflavonoid (-)-EGCG, a natural product as a chiral solvating agent for the discrimination of large number of alpha-amino acids in a polar solvent DMSO. Another significant advantage of this new chiral sensing agent is that it is a natural product and does not require tedious multistep synthesis unlike many other chiral auxiliaries.

Z-selective olefin metathesis using chelating ruthenium alkylidene catalysts

Publications about olefin metathesis will generally discuss how the discovery and development of well-defined catalysts to carry out this unique transformation have revolutionized many fields, from natural product and materials chemistry, to green chemistry and biology. However, until recently, an entire manifestation of this methodology had been inaccessible. Except for a few select examples, metathesis catalysts favor the thermodynamic trans- or E-olefin products in cross metathesis (CM), macrocyclic ring closing metathesis (mRCM), ring opening metathesis polymerization (ROMP), and many other types of reactions. Judicious choice of substrates had allowed for the direct synthesis of cis- or Z-olefins or species that could be converted upon further reaction, however the catalyst controlled synthesis of Z-olefins was not possible until very recently.

Research into the structure and stability of metallacyclobutane intermediates has led to the proposal of models to impart Z-selectivity in metathesis reactions. Having the ability to influence the orientation of metallacyclobutane substituents to cause productive formation of Z- double bonds using steric and electronic effects was highly desired. The first successful realization of this concept was by Schrock and Hoveyda et al. who synthesized monoaryloxide pyrolidine (MAP) complexes of tungsten and molybdenum that promoted Z-selective CM. The Z-selectivity of these catalysts was attributed to the difference in the size of the two axial ligands. This size difference influences the orientation of the substituents on the forming/incipient metallacyclobutane intermediate to a cis-geometry and leads to productive formation of Z-olefins. These catalysts have shown great utility in the synthesis of complicated natural product precursors and stereoregular polymers. More recently, ruthenium catalysts capable of promoting Z-selective metathesis have been reported by our group and others. This thesis will discuss the development of ruthenium-based NHC chelated Z-selective catalysts, studies probing their unique metathesis mechanism, and synthetic applications that have been investigated thus far.

Chapter 1 will focus on studies into the stability of NHC chelated complexes and the synthesis of new and improved stable chelating architectures. Chapter 2 will discuss applications of the highly active and Z-selective developed in Chapter 1, including the formation of lepidopteran female sex pheromones using olefin cross metathesis and highly Z- and highly E-macrocycles using macrocyclic ring closing metathesis and Z-selective ethenolysis. Chapter 3 will explore studies into the unique mechanism of olefin metathesis reactions catalyzed by these NHC chelated, highly Z-selective catalysts, explaining observed trends by investigating the stability of relevant, substituted metallacyclobutane intermediates.

Avaliação da intervenção terapêutica com produto natural na cicatrização de ferida cutânea: o uso de óleo de capivara

A cicatrização de feridas cutâneas visa restaurar a integridade da pele, objetivando um fechamento rápido da lesão e a formação de uma cicatriz funcional e esteticamente satisfatória. Diversos modelos in vivo têm sido estudados a fim de caracterizar diversos componentes e mecanismos envolvidos no reparo tecidual e avaliar os efeitos de potenciais compostos terapêuticos que aceleram o processo de cicatrização. O óleo extraído da gordura subcutânea de capivara possui certas propriedades, entre elas está o seu uso em ferimentos externos com o intuito de acelerar o processo cicatricial, embora tal evento não seja confirmado em pesquisas experimentais. O presente estudo teve como objetivo investigar os possíveis efeitos da aplicação tópica de óleo de capivara em feridas cutâneas induzidas em camundongos Swiss, avaliando sua interferência macro e microscópica no processo de cicatrização das lesões. A lesão foi realizada no dorso dos animais, que receberam aplicação tópica do óleo de capivara (0,1 ml) durante 3, 7, 14 e 21 dias de pós-operatório (PO), conforme protocolo estabelecido. Ao final dos dias, foi realizada eutanásia dos animais e fragmentos de lesão e pele adjacente foram coletados e processados para a microscopia de luz. Cortes histológicos da amostra foram corados com hematoxilina e eosina, azul de toluidina, picro sirius red, resorcina fucsina de Weigert, e imunomarcadas para detecção de antígeno nuclear de proliferação celular. Foram analisados macroscopicamente o aspecto geral da lesão, contração da lesão e reepitelização, e microscopicamente a espessura da neoepiderme, quantidade de leucócitos polimorfonucleares (PMN) e mastócitos, proliferação celular na epiderme e derme, e avaliação das fibras de colágeno e do sistema elástico-microfibrilar na matriz extracelular da derme cicatricial. Os animais tratados topicamente com o óleo de capivara mostraram, comparados ao grupo controle que não recebeu tratamento, melhor desenvolvimento da crosta de fibrina nas fases iniciais da cicatrização e desaparecimento da mesma antes do 14 dia PO; maior contração e reepitelização da área da lesão, bem como neoepiderme mais espessa em 7 dias PO; aumento do número de leucócitos PMN em 3 dias PO e sua gradativa redução nos dias subsequentes e diminuição do número de mastócitos em 21 dias PO. Além disso, no grupo tratado com óleo de capivara foi observada uma benéfica modulação das fibras colágenas e elásticas presentes na matriz extracelular da derme, apresentando aceleração da deposição de fibras de colágeno do tipo I e melhor organização dessas fibras no tecido, bem como aceleração da elastogênese com o aparecimento das fibras do sistema elástico-microfibrilar a partir do 7 dia PO. O presente trabalho demonstrou que o emprego tópico do óleo de capivara em feridas cutâneas de camundongos interfere favoravelmente no processo de cicatrização, acelerando o fechamento da ferida e a reparação da epiderme e derme

三氟甲基修饰苯并呋喃的合成研究及天然产物discodermolide片段的合成与修饰

苯并呋喃类化合物和吲哚类化合物在自然界中广泛存在，具有重要生理活性，近年来得到广泛的研究。三氟甲基在药物设计中有非常重要的地位，一般来说，三氟甲基引入药物分子中能提高药物的生物活性和减少用药量。合成三氟甲基修饰的苯并呋喃和吲哚在药物设计上很有意义，目前这方面的研究有限。本论文发展了一条合成三氟甲基修饰的苯并呋喃类化合物的方法。此方法从取代的水杨醛出发合成了一类关键中间体2-(3,3,3-三氟-2-氯丙烯基)酚氧酸酯，此中间体在碱的作用下，选用不同溶剂得到了2位三氟甲基取代的苯并呋喃类化合物和2-(3,3,3-三氟丙炔基)苯酚类化合物；后者在单质碘作用下环化得到2位三氟甲基3位碘取代的苯并呋喃类化合物。将此方法应用于三氟甲基修饰的吲哚的合成，发现了一个新的反应，此反应中酰基氧进攻不饱和键成环，得到一个苯并六员杂环体系，此体系水解开环得到N-(2-(3,3,3-三氟丙酰基)苯基)乙酰胺。 天然海洋产物discodermolide具有优异的抗癌效果，对其以及其类似物的合成与研究是当前研究的热点。本论文合成了discodermolide的C(17)-C(22)骨架片段和环氧化的C(19)-C(24)片段类似物，并且进行了氟原子修饰的尝试。

毛蕊异黄酮类似物的合成及其对JAK/STAT信号途径调控的构效关系研究

干扰素（IFNs）是最早发现的具有广泛用途的一类细胞因子，IFN-α通过JAK/STAT信号途径调控机体一系列生理和病理反应。至今尚未发现类干扰素的小分子。我们前期研究发现天然产物毛蕊异黄酮可激活干扰素诱导的JAK/STAT信号途径。为发现类干扰素小分子、获得小分子探针，本课题拟建立成熟的JAK/STAT信号途径的筛选模型，合成毛蕊异黄酮及其类似物，研究这些化合物的构效关系，进而尝试通过共价键标记生物素或香豆素来直接研究它们与相关受体的作用。 从异香草醛出发经7步合成反应得到了毛蕊异黄酮。采用平行合成策略得到异黄酮类化合物；采用分支式合成策略，以取代苯乙酸作为合成砌块，获得具有与异黄酮类似结构的香豆素、3-芳基喹诺酮。与分离得到的黄酮类化合物，构建了一个包括异黄酮、黄酮、香豆素、3-芳基喹诺酮在内的化合物库。 建立了包含IFN-α刺激反应元件 (ISRE)的荧光素酶报告基因体系，通过筛选化合物库中的化合物，发现异黄酮骨架为激活JAK/STAT信号途径必须结构、毛蕊异黄酮7-位酚羟基被取代后活性丧失。根据以上结果，对毛蕊异黄酮3′-位标记物的合成进行了初步尝试。 发现山茱萸科植物青荚叶(Helwingia japonica (Thunb.) Dietr.)有抑制蛋白酪氨酸磷酸酯酶1B（PTP1B）的活性。从其地上部分95%乙醇提取物的乙酸乙酯部分分离得到5个化合物,应用波谱方法及与已知品对照的手段鉴定它们为p-menth-2-en-1β, 4β, 8-triol (Z-1)、blumenol A (Z-2)、2′,3′,4′,5′,6′-五羟基查尔酮(Z-3)、洋芹素7-O-β-D-吡喃葡萄糖苷(Z-4)、木犀草素7-O-β-D-吡喃葡萄糖苷(Z-5). Interferons (IFNs) are one kind of cytokines with broad functions. IFN-α mediates series physiological and pathological changes of human body via JAK/STAT pathway. Untill now, no IFNs-like small molecules are discovered. In our preliminary experiment, the natural product calycosin has been observed to activate JAK/STAT pathway. Therefore, we establish a luciferase reporter gene system and synthesize calycosin and its analogues to reveal their structure-activity relationship (SAR). Besides, in order to prove that calycosin activates JAK/STAT pathway through IFN receptor, we attempted to tag it with biotin or coumarin by covalent bonding. Calycosin was synthesized from isovanillin via seven steps. Other isoflavones were obtained by parallel synthesis; coumarins and quinolones were prepared through divergent synthesis, using substituted phenylacetic acids as building blocks. Combing with natural flavones, a small molecule library was established. A luciferase reporter gene system, consisting of 5 copies of the ISRE (interferon-stimulated response element), was used for screening of small molecules from that library. We found that the core-structure of isoflavone was necessary, and if the 7-OH is substituted, the activity slumps. According to our observation, we tried to tag biotin or coumarin at 3′-OH of calycosin. The 95% ethanol extract of the aerial parts of Helwingia japonica (Thunb.) Dietr. showed protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Five compounds were isolated. On the basis of spectral data or by comparison with authentic samples, they were identified as p-menth-2-en-1β,4β,8-triol (1), blumenol A (2), 2′,3′,4′,5′,6′-pentahydroxychalcone (3), apigenin 7-O-β-D-glucopyranoside (4), and luteolin 7-O-β-D-glucopyranoside (5).