Morpholins anti-cancer drugs - Dissertation Example

?Graphical Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com Morpholins anti-cancer drugs Given Sur a , Given Surnamea, ? and Given-name Surnameb aAffiliation bAffiliation 1. Introduction 1.1. Glycosidases functions and classification Cell’s surface is composed of lipids, carbohydrates and proteins. Compared to other surface molecules glycolipids and glycoproteins are the longest. For this reason they are often take part in interactions with substrates or other cells, consequently carbohydrates are of paramount importance in cellular interactions and disease processes such as cancer, infections or inflammations. Because there is a huge variety of difficult to study carbohydrates, until recently, these molecules have attracted no particular attention (Oppenheimer, 2004; 2006). It was noticed that abnormal cells manifest unusual carbohydrate pattern composing its surface. Formed glycoproteins and glycolipids structure is unique for each type of cells. It is formed through a series of enzyme catalysed processes within cells. By inhibition of any of the enzyme that takes part in the formation of these cell specific glycoproteins and glycolipids will inhibit the growth of undesired cells. The concept opens opportunities for the development of new drugs and treatment of a number of diseases including diabetes and cancer. Formation of the described oligosaccharide pattern occurs with the aid of enzymes called glycosidases. This type of enzymes is widespread and takes active part in the cleavage of glycosidic bonds. Usually, from 1 to 3% of a typical genome is used to store information regarding the construction of glycosidases (Davies, et al., 2005; Asano, et al., 2000). This value serves as an indication of the importance if this class of enzymes. Because they actively participate in the construction of carbohydrates pattern in every type of cell even a small dysfunction can affect a number of disease states. Consequently, glycosidases inhibitors have potential medical applications (Asano, 2003; de Melo, et al., 2006; Davies, et al., 2003) Talking specifically about cancer, it was noticed that ?-N-acetylgalactosamine-O-serine/threonine (Tn) is a carbohydrate formed in high amounts in prostate cancer cells (Yip and Withers 2006). The compound can covalently attach to serine or threonine. The process leads to formation of clusters in which one monosaccharide is linked to one amino acid. The produced clusters are often the ideal targets for antitumor antibodies. Such antibodies can be generated by glycopeptides linked to clustered sialyted epitopes. The effectiveness of which is usually higher then single sialyted epitopes (Butters, et al., 2003). Glycosidases classification is based on the similarities in the sequence of their amino acids (Table 1) (Henrissat and Bairoch, 1993; Henrissat, 1991). Enzymes within the same group share the same structural features and perform their functions using the same mechanism (Rye and Withers, 2000) Usually, there are two mechanisms employed by enzymes to cleave glycosidic bonds. As a result, a free hydroxyl group is formed with retention or inversion of configuration (Scheme 1)(Sinnott, 1990; Zechel and Withers, 2000; Vasella, et al., 2002). In the mechanism (a) glycosidases cleave the required bonds using asparagine and glutamine 6A apart from each other. One carboxylic group is deprotonated an acts as a base by abstracting a proton from water during the formation of the intermediate (Withers and Umezawa, 2001; Davies, et al., 2005; Hoj, et al., 1992). Table 1. Type of carbohydrate-active enzyme and its function Carbohydrate-active enzyme Abbreviation Function Carbohydrate Esterases CE Carbohydrate esters hydrolysis Polysaccharide Lyases PL Non-hydrolytic cleavage of glycoside bonds GlycosylTransferases GT Glycosidic bonds formation Glycoside Hydrolases GH Glycosidic bonds rearrangement or hydrolysis The remaining carboxylic group protonates the oxygen atom from the anomeric centre and assists in its removal. Both bond formation and cleavage proceeds via a transition state similar in structure to the oxocarbenium ion. Initially produced sugar is a hemi-acetal with the opposite configuration compared to the starting material. An interesting feature of the mechanism is that no covalent intermediates are produced during the reaction. The enzymes that catalyse this process are called inverting glycosidases (Zechel and Withers, 2000). Retaining (b) glycosidases also have two carboxylic groups in their structure. But compared to inverting (a) glycosidases the distance between them is lower and makes 5.5A. During the first step one amino acid acts as a base while the second, deprotonated amino acid attacks the anomeric carbon. As in the first case, the reaction also proceeds through the formation of an oxocarbenium intermediate ion. The first step is called glycosylation and produces the transition state in which the anomeric configuration is opposite to the one found in the starting material. The second step is deglycosylation and involves the collapse of the intermediate. The amino acid that initially acted as an acid now performs the basic functions abstracting a proton from the water molecule. The anomeric centre is attacked by the water molecule and forms the second oxocarbenium ion transition state. The obtained product can be classified as a hemi-acetal the configuration of which is the same as in the starting material (Zechel and Withers, 2000; Atsumi, 1993, Stubbs, 2008). The described mechanism is not the only one (Knapp, et al., 1996). For example, the same result can be achieved employing a double displacement mechanism. In this mechanism the catalytic nucleophilic group is the oxygen from the substrate acetamide group (Mark, et al., 2001). Also, glycosidases are able to utilise the NAD+ dependent redox elimination followed by addition to catalyse the carbon-oxygen bond cleavage. These mechanisms are employed to much greater extend then the one described above (Macauley, et al., 2005; Koshland, 1953; Kapferer, 2003; Shie, 2006; Watts, 2004; Quaroni, 1979; He, 1997). There are two classes of glycosidase inhibitors: covalent/irreversible and noncovalent (Legler, 1990). Each class is unique in applications of its members. Noncovalent inhibitors can temporarily attach to glycosidases and are more promising from the medical point of view. This class of compounds is well studied and has been a subject of many reviews. Covalent inhibitors, in contrast to noncovalent, lower the activity of the enzyme by introducing extra functional groups covalently linked to the enzyme. In most cases the activity of the enzyme is lowered by either creating a physical abstraction near the enzyme active site or by altering the site’s structure (Lillelund, et. al., 2002, Numao, 2003). Glycosidase covalent inhibitors have a number of applications. One of the most widely used is the identification of the amino acids composing the enzyme’s active site (Withers and Aebersold, 1995). The obtained information is usually backed up with data produced by introducing mutations in the identified active site with subsequent kinetic studies. It is also possible to use covalent inhibitors in the identification of mechanisms by which enzymes operate. In order to do that extremely specific compounds have been synthesised which had the ability to selectively deactivate a required enzyme while detecting the effect of this deactivation in biological systems (Kitz, et al., 1965). Covalent inhibitors can be mechanism based or affinity labels. The letter contains groups that have the ability to change specificity of the affected enzyme or its functionality. These changes are realised through introduction of irreversible modifications in the enzyme. There are two classes of affinity labels: the compounds from the first class react with enzymes due to presence of functional groups incorporated in their structure and labels that require external activation in order to perform their functions. In contrast to affinity labels mechanism based inhibitors are activated by intermediates associated with the activity of the studied enzyme (Mosi and Withers, 2002; Fersht, 1999; Vodovozova, 2007; Sinclair, 2007). Figure 1: Action of inverting (a) and retaining (b) ?-glycosidase. 1.2. Inhibition of glycosidase A potent group of glycosidases inhibitors is the iminosugars. These compounds are usually extracted from microorganisms or plants. Excellent specificity and potency of iminosugars can be explained by their ability to simulate transition state furan and pyran units. Substantial glycosidase inhibition suggests that both electrostatic and conformational factors are important in the formation of the active site (Wicki, et al., 2002; Miao, 1994). Iminosugars can be defined as polyhydroxylated alkaloids which have the ability to inhibit glycosidase by forming the same charge and conformation as the intermediate ion (b) but not (a) which is usually generated during the carbon oxygen bond cleavage (Scheme 2). The following reaction leads to the product with inversion or retention of configuration via single or double nucleophilic displacement (Afarinkia and Bahar, 2005; Watson, et al., 2001) Figure 2: Conformational changes during glycosidases catalysed bond cleavage. Figure 3: Iminosugars inhibition mechanism As it is observed on scheme 2, cleavage of the carbon oxygen bong generates a positive charge on either carbon or oxygen of the natural substrate (Heightman and Vasella, 1999; Krasikov, et al., 2001; Lillelund, et al.,. 2002; Mikhaylova, 1996; Poon, 2007) Consequently, substitution either of these atoms by protonated nitrogen will produce a compound capable of competing with the cleaved hydrocarbon for the enzyme (Scheme 3). In order to manifest the required inhibitory activity the compound should contain at least two hydroxyls and one nitrogen atom. Such compounds can be mono or bicyclic and include rings like indolizidine, pyrrolizidine or pyrrolidine (Sears and Wong, 1999; Legler, G., 1990) Nojirimycin is the first discovered compound similar in structure to sugars (Scheme 3). This alkaloid includes a heterocyclic nitrogen atom which replaces oxygen in glucose. Nojirimycin was initially used as an antibiotic and was isolated from mulberry leaves. Later it was shown to manifest potent inhibitory properties towards glycosidases obtained from various sources. A significant disadvantage of the compound was the presence of a hydroxyl group attached to C-1 atom. This hydroxyl damages cells and makes the overall compound toxic. But, if reduced to 1-deoxynojirimycin the inhibitor becomes more stable and less harmful without losing its inhibitory properties. The compound manifested excellent results in the ex vivo assays, but in vivo results were much less optimistic (Romaniouk, 2004; McMahon, 1997; Sun, et al., 2005; Kopitz, et al., 1997; Clarke and Strating, 1989). For example, N-alkylated derivatives were more powerful iminosugars. As an example, miglitol (1) and N-butyl-1-deoxynojirimycin (2) can be put forward. Both compounds had anti-HIV activity, were useful against diabetes and manifested promising results in treatment of both B and C hepatitis. Reductive ammination between alkyl aldehydes and iminosugars is commonly used to synthesise both compounds. As a reducing agent sodium cyanoborohydride is usually employed. As it was established, the activity of 1-deoxynojirimycin both depends on pH and the nature of substituents attached to the nitrogen atom. Taking into account the presented information it was presumed that introduction of both oxygen and nitrogen into a six membered ring will lead do powerful glycosidase inhibitors making them potential anti-cancer drugs. The activity of these inhibitors could be altered using different substituents attached to the nitrogen atom. Figure 4: N-alkylated iminosugars Figure 5: Preparation of morpholine derivatives 2. Results and discussion As it was stated in the introduction part, morpholins are highly promising glycosidase inhibitors. This research takes advantage of reductive amination as a potent way of morpholine derivative production. It aims to synthesise a variety of N-alkylated morpholines and investigate the effects of different substituents on the inhibitory properties of the produced compounds. Both the employed reductive amination reaction which affords the desired compounds and examination of the inhibitory properties of the presented compounds were realised in the corresponding group’s research (Burland, 2011). Therefore, this work aims at gaining better understanding of the chemistry and functionality of morpolines and presents a further expansion of the research previously done in the group. Because glycosidase activity is associated with a range of diseases (Morjani, 2001, Schnabelrauch, 1994) synthesised morpolins were used for drug preparation and testing. 2.1. Production of morpolins The synthesis of morpolins was realised according to the scheme 5 and the mechanism of the reaction is presented on scheme 6. Regarding the reaction mechanism, first stages of the process are similar to imine formation. Initial protonation of one of the aldehyde oxygen is followed by the amine attacking the carbonyl carbon. The process leads to the formation of the intermediate compound with protonated nitrogen. The following stages can be described as proton migration with subsequent water elimination. Attack of the reduction agent on the produced protonated imine affords the intermediate 9. The compound is not isolated, all the describes stages are repeated again to afford the target morpoline derivative (Scheme 6). As it was mentioned earlier it was possible to change inhibitory properties of iminosugars by introducing various alkyl groups attached to the nitrogen atom. In particular this method can be used to enhance deoxynojirimycin as glycosidase inhibitor. To investigate effects of alkylation the research aimed at producing compounds 5 and 8 and compare their inhibitory properties. Looking at the NMR spectrum of compound 5 several distinct peaks are seen. Triplet at 1.25 ppm, singlet at 2.1 ppm and quadruplet at 4.2 ppm all correspond to EtOAc which did not evaporate. The signal at 7.3 ppm corresponds to traces of CHCl3 present in CDCl3. Other required signals are also present and the formation of the desired product is obvious. Unfortunately, despite repeating the reaction two times, it was not possible to produce benzyl derivative 8. Ideally, the compound should have manifested multiple peaks at the benzene region of the proton NMR spectrum (6.5-8.5), but in that region only one singlet at 7.3 is observed. As in the previous case, this is undeuterated chloroform. The origin of the negative result is unclear. It can possibly be attributed to changes in basic properties shifting from butylamine to benzylamine. Or by the fact that the reaction was not conducted in one pot, the intermediate 4 was purified by flash column chromatography before entering in the reaction with the amine. Compound 6 and 7 possess the same structural features and were synthesised to investigate the effect of adding the ester functional group. Moreover, it would have been possible to draw conclusions regarding the presence of one hydroxyl group in the benzene ring. NMR spectrums of both compounds suggest successful formation of the desired products. Clearly, formation of the desired product was observed due to the presence of multiple peaks at 6.5-7.7 ppm, which correspond to the benzene ring. The structure of 6 is similar to 7, consequently their proton NMR results are also mostly the same. Because of the para position of the OH group, protons in the benzene ring form two distorted doublets in the region 6.5-7.0 ppm (Clayden, 2001). Reductive elimination proved to be a potent way of synthesis of the required morpoline derivatives. Compared to one pot synthesis, purification of the intermediate compound also affords the target product in the moderate yield. The fact introduces more variety to the method. Synthesis and purification of the three morpoline derivatives concluded the first part of the research. Figure 6: Reductive amination mechanism on the example of morpoline derivatives. 2.2. Evaluating the produced morpolins as glycosidase inhibitors Having produced a series of morpolins, these compounds were intended to be tested as inhibitors of glycosidase in the enzymatic cleavage presented on scheme 7. Because p-nitrophenol is accumulated during the course of the reaction, it was possible to calculate its speed based on the amount of p-nitrophenol produced which can be determined using Beer-Lambert-Bouguer law. In order to use this procedure a series of p-nitrophenol solutions were prepared, absorption of each was measured and the corresponding calibration plot was constructed (Scheme 6). Talking about enzymes capable of carbon-oxygen bond cleavage, glucosidase is a general term referred to types of enzymes only capable to hydrolyse glucose derivatives while glycosidase referred hydrolysis of any type of sugar. The activity of the investigated type of enzyme is associated with a variety of diseases therefore it is important to investigate its inhibition using the synthesised morpoline derivatives. Initially, the reaction using uninhibited enzyme was investigated. Because it is followed by Michaelis-Menten kinetics, Lineweaver Burk plot can be constructed and the rate of the investigated reaction is given by: v0= where: v0 is the initial rate of the reaction; Vmax can be defined as the maximum rate of the reaction. If the amount of the enzyme is increased Vmax will also increase because of the following relationship: Vmax = kcat[enzyme] [S] – concentration of the substrate; Km-Michaelis constant, used to characterise all enzymes; The reaction in this case proceeds according to the following mechanism: In order to acquire the necessary amount of data for the described equations the required amount of the substrate (p-nitrophenyl-?–D-galactopyranoside) was dissolved in a buffer solution. The reaction was initiated by the addition of the enzyme. The cuvette was introduced into a spectrometer to measure the absorbance at fixed time intervals. The reaction rate (?substrate/second) was calculated in three steps: 1) Preparation of absorbance versus time plots for each substrate concentration 2) Calculation of the slope of line (?absorbance/second). This step was done because v0 can be expressed as absorbance changes per unit of time. 3) Divide the slope by the molar absorption of the substrate. If v0 is known for several substrate concentrations it is possible to calculate Km and Vmax. The reaction can be described by Michaelis-Menten kinetics, for this reason the dependence between v0 and [S] should take the form of a rectangular hyperbola. On the plot (v0/[S]), increase in the concentration of [S] the rate approaches Vmax. Also, Km is equal to substrate concentration at which v0=1/2 Vmax. Concequently, Vmax and Km can be calculated using the plot. This method allows only approximate determination of Vmax and Km. To achieve more precise results a Lineweaver-Burke plot must be constructed (1/v0 versus 1/[S]). Taking into account the equation for Michaelis-Menten kinetics and taking its reciprocals the following equation can be produced: The equation is used to construct the Lineweaver-Burke plot. All the values can be calculated in three steps: 1) Take the values for the most linear region from the dependence between absorbance and time v0 = ?abs/?time; v0 = (0.296-0.2942)/(300-0) = 0.000006/sec 2) Calculation of the rate using Beer-Lambert equation: A= ??c?l Therefore, c = A/(??l) where A = measured absorbance, ? = extinction coefficient, (17800 M-1 cm-1) c = concentration, l = the cuvette thickness, cm c =  The result indicates 3.37 ? 10-10 M = 0.000337 ? 10-6 M = 0.000337 ?M this indicates that means 0.000337 micromoles per liter or 0.000337 nanomoles/mL are produced in one second in the total volume of 790?L = 790 ? 10-6 L = 0.790 mL. The whole amount of product formed in the total volume of the reaction mixture is required not the concentration. Considering this information, the rate can be calculated as: 0.000337 nanomoles/mL ? 0.790 mL = 0.0004276 nanomoles produced in one second or 0.0004276 nanomoles ? 60 sec = 0.0256 nanomoles formed in one minute. Figure 7: Glycosidase catalysed cleavage of the carbon-oxygen bond. Figure 8: Changes in absorbance with time. Both 6-Methoxy-4-Butylmorpholin-2-yl-methanol (5) and 2-(2-Hydroxymethyl-6-methoxymerpholi-4-yl)-9-(4-hydroxyphenyl)-propionic acid methyl ester (7) were used to prepare drugs for enzyme inhibition. Amount of drug required to test their inhibition property was calculated as presented below: The concentration of p-nitrophenyl-?–D-galactopyranoside (0.000115 M) was obtained from standard curve of p-nitrophenol by using it’s absorbance (0.3). 600?L of substrate contains 6.9x10-8 moles of substrate, since the ratio of release of 4-nitrophenol by enzymic reaction from p-nitrophenyl-?–D-galactopyranoside is 1:1 therefore the number of moles of p-nitrophenol is 6.9x10-8 moles. In order to find out the mass of 6.9x10-8 moles of each drug to be calculated, the RMM of 6-Methoxy-4-Butylmorpholin-2-yl-methanol (5) (203g/mol) and 2-(2-Hydroxymethyl-6-methoxymerpholi-4-yl)-9-(4-hydroxyphenyl)-propionic acid methyl ester (7) (325g/mol) were achieved using their structures. Mass of 6-Methoxy-4-Butylmorpholin-2-yl-methanol, (5) required to inhibit the enzyme is 1.40x10-5 g and mass of 2-(2-Hydroxymethyl-6-methoxymerpholin-4-yl)-9-(4-hydroxyphenyl)-propionic acid methyl ester, (7) is 2.24x10-5 g. 0.014 g of 6-Methoxy-4-Butylmorpholin-2-yl-methanol (5) was dissolved in 10 ml of disodium buffer so by taking 10 ?L of the solution the required mass of the drug was achieved. The final drug concentration was produced to be 1.4 g/L. 0.0224 g of 2-(2-Hydroxymethyl-6-methoxymerpholi-4-yl)-9-(4-hydroxyphenyl)-propionic acid methyl ester (7) was dissolved in 10ml of the buffer to achieve the required mass of the drug. The final drug concentration was produced to be 22.4 g/L. Since the enzyme was denatured, the inhibitory action of drugs was not tested. But, taking into account inhibitory properties of these compounds towards a range of glucosidases and galactosidases only small inhibition was expected. 3. Methods 3.1. General information Unless stated otherwise all reagents and solvents are obtained from commercial sources, used without extra purification and are for use in research and development only. Manipulations and reactions were performed in fume cupboard the inert environment of dry argon, in flame dried glassware which were washed with acetone and dried using pressured air and under magnetic stirring. Flash chromatography was performed on Merck or Fisher silica gel 60 (230-400 mesh) using compressed air to maintain the required pressure. Merck silica 60 F254 aluminium backed plates (0.2 mm depth) were used to perform TLC analysis. Compounds were detected using ninhydrin on TLC plates, then the plates were treated with with hair dryer to give brightly coloured pink to purple spots by means of UV light (254 nm), 5% ethanol/sulphuric acid or ninhydrin. NMR spectra were obtained at 400 MHz (H1), 125 MHz (C13) on a Bruker nanobay AVIII 400 spectrometer in CDCl3 or CD3OD. Chemical shifts(?) are reported in parts per million (ppm) relative to TMS and the coupling constants (J) are described in hertz and s, d, t, dd and m represents singlet, doublet, triplet, doublet of doublets and multiplet respectively. IR spectrums were obtained using Perkin Elmer Spectrum 100 FT-IR spectrometer, wavenumbers (?) in cm-1. Absorbance was measured using Dynatech MR 5000 spectrometer. HRMS was performed on a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer. Molecular ion fragments and molecular ions are reported in m/z, mass/charge ratios. 3.2. 6-Methoxy-4-Butylmorpholin-2-yl-methanol, (5) Step 1: Synthesis of the dialdehyde To a stirring solution of methyl ?-D-glucopyranose (3) (0.1g., 0.0005 mol.) in methanol at 00C under argon a solution of sodium periodate (0.55 g, 0.0025 mol.) in distilled water was added dropwise. The reaction mixture was left stirring for 15 hours at room temperature. Magnesium sulphate was added as a drying agent and the reaction mixture was concentrated in vacuo. To the produced white solid EtOAc was added to extract the required dialdehyde. The solution was subsequently filtrated through Celite and concentrated in vacuo to afford 0.13 g. of the crude dialdehyde as a colourless oil (4). Step 2: Reductive amination reaction to produce morpholine To a stirring solution of the butyl amine (0.026 ml., 0.00026 mol) sodium cyanoborohydride (0.084 g., 0.0013 mol.) was added along with dialdehyde from step 1 (4) (0.13 g., 0.0008 mol) and 10A molecular sieves in methanol. A solution of 2M HCl in water was used to adjust the pH to 7. The reaction mixture was stirred for 24 hours at room temperature. Concentrated in vacuo and the remaining residue partitioned between water and EtOAc. The organic phase was separated and the aqueous layer was washed with EtOAc four times. Produced organic layers were combined, dried with magnesium sulphate and concentrated in vacuo. The produced crude morpoline was purified by flash column chromatography (EtOAc/MeOH 10/1) to afford 0.13 g (5) (0.1015 g if 100% yield, conclusion, solvent remained or not all impurities were removed) of the final product. IR ?max (KBr): 3495.77, 2984.53, 2937.75, 2325.62, 2325.62, 2298.94, 2173.94, 1733.52, 1447.41, 1373.98, 1239.67, 1131.71, 1096.96, 1043.04, 970.69, 937.88, 894.14, 847.96, 787.37, 634.54, 608.96, 598.04, 507.12, 503.00 cm-1. H1 NMR (400 MHz, CDCl3): ? 0.85-1.00 (m, 3H), 1.25 (t, J=7.12 Hz, 2H), 1,42 (m, 2H), 2.04 (s, 3H), 2.17 (s, 6H), 2.9 (s, 2H), 3.0 (s, 1H), 3.4 (s, 1H), 3.7 (m, 1H); C13 NMR (100 MHz): ? 171.19, 97.21, 68.85, 64.22, 60.42, 58.87, 55.90, 55.05, 54.19, 28.31, 21.07, 20.76, 14.20, 14.04. 3.3. 2-(2-Hydroxymethyl-6-methoxymorpholin-4-yl)-9-phenyl propionic acid methyl ester, (6) Step 1: Synthesis of the dialdehyde To a stirring solution of methyl ?-D-glucopyranose (3) (0.1g., 0.0005 mol.) in methanol at 00C under argon a solution of sodium periodate (0.55 g, 0.0025 mol.) in distilled water was added dropwise. The reaction mixture was left stirring for 15 hours at room temperature. Magnesium sulphate was added as a drying agent and the reaction mixture was concentrated in vacuo. To the produced white solid EtOAc was added to extract the required dialdehyde. The solution was subsequently filtrated through Celite and concentrated in vacuo to afford 0.1 g. of the crude dialdehyde as a colourless oil (4). Step 2: Reductive amination reaction to produce morpholine To a stirring solution of the L-phenylalanine (0.044g., 0.0002 mol) sodium cyanoborohydride (0.065 g., 0.001 mol.) was added along with dialdehyde from step 1 (0.1 g., 0.00062 mol) and 10A molecular sieves in methanol. A solution of 2M HCl in water was used to adjust the pH to 7. The reaction mixture was stirred for 24 hours at room temperature. Concentrated in vacuo and the remaining residue partitioned between water and EtOAc. The organic phase was separated and the aqueous layer was washed with EtOAc four times. Produced organic layers were combined, dried with magnesium sulphate and concentrated in vacuo. The produced crude morpoline was purified by flash column chromatography to afford 0.056 g (37%) of the final product (6). H1 NMR (400 MHz, CDCl3): ? 1.1-1.7 (m, 5H), 2.0 (m, 2H), 4.1 (s, 3H), 4.7 (s, 3H), 5.1 (s, 1H), 5.8 (m, 2H), 6.6 (m, 5H) 3.4. 2-(2-Hydroxymethyl-6-methoxymerpholin-4-yl)-9-(4-hydroxyphenyl)-propionic acid methyl ester, (7) Step 1: Synthesis of the dialdehyde To a stirring solution of methyl ?-D-glucopyranose (3) (0.1g., 0.0005 mol.) in methanol at 00C under argon a solution of sodium periodate (0.55 g, 0.0025 mol.) in distilled water was added dropwise. The reaction mixture was left stirring for 15 hours at room temperature. Magnesium sulphate was added as a drying agent and the reaction mixture was concentrated in vacuo. To the produced white solid EtOAc was added to extract the required dialdehyde. The solution was subsequently filtrated through Celite and concentrated in vacuo to afford 0.13 g. of the crude dialdehyde as a colourless oil (4). Step 2: Reductive amination reaction to produce morpholine To a stirring solution of the Tyrosine-OMe (0.052 g., 0.00026 mol) sodium cyanoborohydride (0.084 g., 0.0013 mol.) was added along with dialdehyde from step 1 (0.13 g., 0.0008 mol) and 10A molecular sieves in methanol. A solution of 2M HCl in water was used to adjust the pH to 7. The reaction mixture was stirred for 24 hours at room temperature. Concentrated in vacuo and the remaining residue partitioned between water and EtOAc. The organic phase was separated and the aqueous layer was washed with EtOAc four times. Produced organic layers were combined, dried with magnesium sulphate and concentrated in vacuo. The produced crude morpoline was purified by flash column chromatography (EtOAc/MeOH 20/1) to afford 0.366 g (0.16 if 100% yield) (7) of the final product. H1 NMR (400 MHz, CD3OD): ? 1.1-1.7 (m, 5H), 2.0 (m, 2H), 4.1 (s, 3H), 4.7 (s, 3H), 5.1 (s, 1H), 5.1 (s, 1H), 5.8 (m, 2H), 6.6 (d, J=8 Hz, 2H), 6.9 (d, J=8 Hz, 2H); C13 NMR (100 MHz): ? 157.16, 131.26, 129.54, 116.16, 98.37, 71.36, 70.60, 64.17, 55.06, 54.52, 51.76, 51.52, 49.67, 49.45, 49.24, 49.03, 48.81, 48.60, 48.39, 35.59. 3.5. (4-N-Benzyl-(6S)-methoxy-morpholin-(2S)-yl)-methanol, (8) Step 1: Synthesis of the dialdehyde To a stirring solution of methyl ?-D-glucopyranose (3) (0.1g., 0.0005 mol.) in methanol at 00C under argon a solution of sodium periodate (0.55 g, 0.0025 mol.) in distilled water was added dropwise. The reaction mixture was left stirring for 15 hours at room temperature. Magnesium sulphate was added as a drying agent and the reaction mixture was concentrated in vacuo. To the produced white solid EtOAc was added to extract the required dialdehyde. The solution was subsequently filtrated through Celite and concentrated in vacuo to afford 0.086 g. of the crude dialdehyde as a colourless oil (4). Step 2: Reductive amination reaction to produce morpholine To a stirring solution of the benzylamine (0.019 ml., 0.00053 mol) sodium cyanoborohydride (0.056 g., 0.00088 mol.) was added along with dialdehyde from step 1 (0.086 g., 0.00053 mol) and 10A molecular sieves in methanol. A solution of 2M HCl in water was used to adjust the pH to 7. The reaction mixture was stirred for 24 hours at room temperature. Concentrated in vacuo and the remaining residue partitioned between water and EtOAc. The organic phase was separated and the aqueous layer was washed with EtOAc four times. Produced organic layers were combined, dried with magnesium sulphate and concentrated in vacuo. The reaction did not produce the required product (8). H1 NMR (400 MHz, CD3OD): the reaction did not proceed. 3.6. Standard curve for para-nitrophenyl Five standard solutions of p-nitrophenyl were prepared. Each solution had different hue of yellow and was mixed with borate buffer, the absorbance in each case was measured. The following procedure was used to prepare the borate buffer (pH 9.6). To 50 ml of a solution containing 0.6189 g. of boric acid and 0.7456 g. of potassium chloride 36 ml of 0.2 M of sodium hydroxide was added. The potassium hydroxide solution was added till the required pH of 9.6 was obtained. The produced buffer was used to prepare p-nitrophenyl solutions. Using information from the table and the results obtained from B,C,D,E, and F a calibration plot was constructed 3.7. Preparation of disodium hydrogen phosphate (0.2 M) Buffer Disodium hydrogen phosphate (28.4 g) was dissolved in 1000 ml deionised water in a conical flask. 3.8. Enzyme preparation Enzymes from a vile containing 445970 units of enzyme were initially dissolved in 100 ml of disodium hydrogen phosphate (0.2 M) buffer to produce a solution containing 4459.7 units (A). The concentration of the solution A was further reduced by 100 times to give solution B containing 445.97 units of enzyme. The final solution contained 44.59 units of enzyme, and it was produced by reducing the concentration of the solution B by 100 times. For the inhibition experiments only the solution A was used. 3.9. Preparation of substrate (p-nitrophenyl-?–D-galactoside) 10 mM The substrate, p-nitrophenyl-?–D-galactoside 0.301 g, was dissolved in 100 ml of disodium buffer solution in order to obtain 10 mM solution of the substrate. 800 micro litre cuvettes were used to obtain different concentrations of the substrate and the enzyme. Enzyme catalysed the formation of p-nitrophenol the amount of which was determined using the absorbance measurements at set time intervals. The highest absorbance was 0.3 during 25 mins. 4. Conclusion and future work To conclude, a series of morpholine derivatives, as possible glycosidase inhibitors, was synthesised. Reductive amination proved to be an effective method capable of producing excellent yield of the target compounds. Unfortunately, due to denatured nature of the studied enzyme it was not possible to study the inhibitory properties of the achieved morpolines. But it is known that similar compounds are extremely useful in cancer treatment, HIV, diabetes, viral infections or Gaucher’s disease. Combined with kinetic studies and protein crystallography the compounds were employed as chemical probes and provide useful information regarding glycosidase mechanisms. Consequently, such compounds have a number of possible therapeutic applications (Tsai, 2002; Tybulewicz, 1992; Xu, 2003; Tull, 1996; Lu, 2005; ). The method that is usually used in designing the described inhibitors is based on the simulation of the transition state forming during carbon oxygen bond cleavage. For this reason a significant amount of compounds in which a nitrogen atom substitutes oxygen or carbon have been produced. In living organisms the described inhibitors produce a positive charge via protonation, and reproduce the positive charge that can be found in the transition state in glycosidase hydrolysis reactions. Synthesised in this work morpolines are not the only compound that fall under the above description. Substantial research has been made in the synthesis of pyrrolidines and piperidines as the compounds able to manifest the sugar similar conformations. For these reasons the described research can be expanded on the synthesis of azocanes and azepanes. Such compound can be synthesised by following a pattern similar to the one used for morpoline derivatives and afford compounds with an additional hydroxymethyl group which can lead to extra favourable interactions in the produced glycosidase active site (Scheme 7). Both compounds 16 and 17 are incapable of adopting the configuration of D-galactopyranose, but able to demonstrate potential inhibitory properties by imitating ?-D-galactose conformation in the active site. The properties of the compound 18 are also worth investigating. It was established to be a weak inhibitor of ?-galactosidase, but a powerful inhibitor of ?-glucosidase. Compounds 19 and 20 are also promising examples of ?-D-galactosidase derived from Bovine kidney with 19 capable to inhibit the enzyme by 95%. All these compounds are subjects for future work. Table 2. Concentrations of standard solutions of p-nitrophenol Concentration, M Para-nitrophenyl solution 4.20?10-4 A 2.10?10-4 B 1.75?10-4 C 1.40?10-4 D Table 3. Absorbance of standard p-nitrophenol solutions Solution Volume of the solution, ml Volume of the borate buffer, ml Absorbance water 300 600 0 A 2.0401 B 0.8024 C 0.6346 Figure 6: p-nitrophenol standard curve Table 4. Concentrations of standard solutions of p-nitrophenol Number of enzyme units Solution 445970 Initial vial 4459.70 A 445.970 B 44.5970 C Table 5. p-nitrophenyl-?–D-galactoside solutions Buffer, ?L Substrate, ?L Enzyme, ?L Glycine, ?L Absorbance 460 300 10 10 0.0800 260 500 10 10 0.1989 500 260 10 10 0.1989 Buffer, ?L Substrate, ?L Enzyme, ?L Glycine, ?L Absorbance Table 6. Changes in absorbance with time Time Absorbance 0 0.2942 5 0.2959 10 0.2960 15 0.2961 Figure 7: Azepanes proposed for future work 5. References 1. Afarinkia, K., and Bahar, A., 2005. Recent advances in the chemistry of azapyranose sugars. Tetrahedron: Asymmetry, 16, pp. 1239–1287. 2. Asano, N., 2003. Glycosidase inhibitors: Update and perspectives on practical use. Glycobiology, 13, pp. 93–104. 3. 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Cellular and Molecular Life Sciences, 59, pp. 694–707. Read More