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Synthesis of cyclitol-based glucosidase inhibitors Caron, Gaétan 1988

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SYNTHESIS O F C Y C L I T O L - B A S E D G L U C O S I D A S E INHIBITORS By G A E T A N CARON B.Sc, Universite de Montreal, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF CHEMISTRY We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA August '988 ©Gaetan Caron, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. CHEMISTRY Department of The University of British Columbia Vancouver, Canada Date AUGUST 29 , 1988 DE-6 (2/88) i i A B S T R A C T The first conduritol aziridine (l,2-dideoxy-l,2-epimino-/n;y0-inositol, 1) was synthesized in seven steps from myoinositol (2) and inhibits pABG5 P-glucosidase and yeast a-glucosidase irreversibly. l,2-t9-Cyclohexylidene-myo-inositol (3) was obtained by reaction of 2 with cyclohexanone. Benzylation of 3 followed by hydrolysis of the ketal gave l,4,5,6-tetra-t9-benzyl-myo-inositol (5). The two free hydroxyl groups in 5 were methanesulfonylated and the axial mesyl group in l,4,5,6-tetra-<9-benzyl-2,3-di-0-methanesulfonyl-myo-inositol (12) was selectively displaced by an azido group. The resulting l-azido-2,3,4,5-tetxa-t9-benzyl-l-deoxy-6-0-methanesulfonyl-567//o-inositol (13) was hydrogenated in the presence of HC1 to give 1-amino-l-deoxy-2-t9-methanesulfonyl-scy/Zo-inositol (24) hydrochloride. Cyclisation of 24 under basic aqueous conditions yielded DL-1. OR' OBzl OBzl 13 R = S 0 2 C H 3 OR 2 R=R'=H 3 R=CYCLOHEXYLIDYL; R'=H 5 R=H; R ^ C r ^ C g H ^ 12 R = S 0 2 C H 3 ; R ^ O ^ C ^ N H O H O H 24 R = S 0 2 C H 3 31 R = C O C 6 H 5 O H 1 I i i The dissociation constant Kj and the inactivation rate constant k[ for inactivation of pABG5 P-glucosidase by 1 were calculated to be 3.0 mM and 0.077 min"1 respectively. For yeast a-glucosidase inactivation, values of K, and lq were found to be 9.5 mM and 0.39 min - 1 respectively. Finally, 24 and 31 were tested as reversible (non-covalent) inhibitors of both the glucosidases and the respective inhibition constants (Kj) determined. T A B L E O F C O N T E N T S Page ABSTRACT ii T A B L E OF CONTENTS iv LIST O F TABLES vi LIST OF FIGURES vii LIST OF SCHEMES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS xi 1. INTRODUCTION A N D LITERATURE REVIEW 1 1.1 Glycosidases 2 1.2 Interest in Glycosidase Inhibition 4 1.3 Mechanism of Glycosidase Hydrolysis 5 1.4 Glycosidase Inhibitors 7 1.5 New Cyclitol-Based Inhibitors 9 1.6 TheCyclitols 11 1.7 Synthesis of Cyclitols and Monoaminocyclitols 16 2. DISCUSSION 30 2.1 Synthesis of Conduritol Aziridine (1) 31 2.11 Importance of Aziridine Derivatives and Their Synthesis 31 2.12 Approach to 1 Starting with Myoinositol 32 2.13 l,4,5,6-Tetra-0-benzyl-3-i9-toluenesulfonyl-m)'o-inositol (6) 33 2.14 Preparation of Azidocyclitols 35 2.15 Attempted Hydrogenation of Azido Compound (13) 38 2.16 Preparation of Protected Conduritol Aziridine 40 2.17 Alternative Approaches to 1 41 V 2.18 Hydrogenation and Cyclisation 44 2.2 Synthesis of a Potential Reversible Inhibitor 47 2.21 The Reductive Amination Approach 47 2.22 Preparation of scy/fo-inositol derivative (31) Hydrochloride 49 2.3 Enzymology 52 2.4 Future Work 63 3. EXPERIMENTAL 65 3.1 Synthetic Methods 66 3.2 Enzymological Methods 88 BIBLIOGRAPHY 91 SPECTRAL INDEX 97 vi LIST OF TABLES T A B L E Page I Reaction Conditions for Attempted Hydrogenations of Compounds 11 and 13. 39 II Products from azidolysis of l^-di-O-methanesulfonyl-myo-inositol (17) followed by acetylation. 42 UJ Concentrations of conduritol azMdine 1 and corresponding [EoJVvi values for irreversible inhibition of pABG5 P-glucosidase and yeast a-glucosidase. 55 IV Dissociation constants (KO and inactivation rate constants (kj) for irreversible and reversible inhibition of pABG5 (J-glucosidase and yeast a-glucosidase by analogues 1,24, and 31. 57 V Concentrations of inositol analogues 24 and 31 for reversible inhibition of pABG5 pVglucosidase and yeast a-glucosidase at fixed concentrations of substrates. 59 vii LIST OF FIGURES FIGURE Page 1 Loss of yeast a-glucosidase activity as a function of time at different aziridine concentrations. 54 2 Inactivation of yeast a-glucosidase as a function of conduritol aziridine concentration. 56 3 Inactivation of pABG5 (J-glucosidase as a function of conduritol azMdine concentration. 56 4 Dixon plot of the inhibition of a-glucosidase by 24. 60 5 Dixon plot of the inhibition of a-glucosidase by 31. 60 6 Dixon plot of the inhibition of (5-glucosidase by 24. 61 7 Dixon plot of the inhibition of p-glucosidase by 31. 61 viii LIST OF SCHEMES SCHEME Page I 33 n 36 m 43 IV 48 V 50 ix LIST OF ABBREVIATIONS BSA Bovine serum albumin CHCI3 chloroform cone. concentrated D Q desorption chemical ionization DMSO dime%lsulfoxide EI electron impact equiv. equivalent(s) FAB fast atom rjombardrnent ^-n.m.r. proton nuclear magnetic resonance h hour Hz hertz i.r. infrared L i A l H 4 lithium aluminium hydride Lit. literature m.p. melting point nm nanometer Ms methanesulfonyl Ph phenyl Pyr. pyridine sat. saturated T H F tetrahydrofuran TMS tetrametnylsilane TMSI trimethylsilyttodide Ts toluenesulfonyl X thin-layer chromatography ultraviolet In iH-n.m.r. assignments singlet doublet doublet of doublets triplet multiplet broad singlet xi A C K N O W L E D G E M E N T S I wish to express my appreciation to Dr. S.G. Withers for his encouragement and suggestions during the course of this work. Also, I am grateful to the past and present members of the group for their help. To them, I extend my thanks and best wishes. My thanks also go to the elemental analysis, n.m.r., and mass spectroscopy staff of the U B C Chemistry Department 1 DPaTnooycTflOKi LITERATURE REVIEW 2 1-1 Glycosidases Glycosidases are enzymes responsible for glycoside hydrolysis and glycosyl transfer.1 As with most enzymes, the catalytic power of glycosidases is such that their presence determines whether the biological reaction will occur at all. Enzymes not only speed up reactions but show remarkable specificity for a given substrate. Glycosidases were an important part of early studies on enzyme specificity. It was Emil Fischer2 who, working on enzymes capable of hydrolyzing glycosides, found that they can distinguish between stereoisomeric substrates. He then proposed the concepts of active site and complementary relationship between enzymes and their substrates. As a result of this high specificity and the numerous sources and forms of glycosides found in nature, it is not surprising to see that a large number of glycosidases have been discovered and isolated. This project is concerned with the preparation of glycosidase inhibitors of a new type. To better understand the interest in their inhibition, let us here consider the function of some important glycosidases. Origin of Some Glycosidases and their Substrates  Amylases The natural substrate of amylases3 is starch, a polymer consisting of a-(l-4) and a-(1-6) linked glucose residues. Amylases are capable of selectively hydrolyzing the a-(l-4) linkages with either retention (a-amylase) or inversion (|3-amylase) of anomeric configuration. Amylases are also classified as endo-glucanases when the hydrolysed linkage is internal and exo-glucanases when the linkage cleaved is that of the terminal sugar. a-Amylases are found in the tissue and fluid of plants and animals. In mammals, this enzyme is secreted by the pancreas and the salivary gland, p*-Amylases cannot be found in 3 animals, but are present in the seeds of higher plants and sweet potatoes. Oats, corn, rice, and sorghum contain {^ -amylases in limited amounts. When large quantities of amylases are required for academic or industrial interests, they are generally produced from cultures of bacteria and molds from which the enzymes can be isolated and purified easily. Debranching Enzymes Amylopectin found in starch is essentially a branched polysaccharide in which the a-(1-6) bond cannot be broken by amylases and necessitates specialized enzymes, the debranching enzymes, for hydrolysis. They can be divided into two classes: the direct debranching enzyme hydrolyses the 1-6 bond of an unmodified amylopectin and/or glycogen whereas the indirect debranching enzyme is a two-component enzyme system acting on a modified polysaccharide. In general, direct debranching enzymes are found in plants and the indirect type in mammals. Microorganisms contain both types. Cellulases Cellulose is a linear polymer of D-glucose with a (5-(l-4) linkage. In cases where the degree of polymerization is large, the chains associate in fibres and become insoluble in water. Culture filtrates of cellulase^-4 usually contain more than one component. One of them, the CI factor is active toward the insoluble native fibre whereas others are enzymes acting on polymers ranging from soluble cello-oligoglucosides to fibrous cellulose. Some cellulases are classified as exo- or endo-glucosidases depending on whether they hydrolyse a terminal or an interior linkage. To convert lower oligoglucosides (primarily cellobiose) completely to glucose, cellobiases (pVglucosidases) are required. 4 INSOLUBLE FIBROUS CELLULOSE G L U C O S E CI-FACTOR B-GLUCOSJDASES HYDROLYZABLE CELLULOSE E X O - , AND ENDO-GLUCANASES OLIGOGLUCOSIDES Cellulases are produced by plants, invertebrate animals and microorganisms. In nraiinants, a bacterium synthesizes the cellulase allowing the cellulose to be utilized as food. Lysozymes Lysozymes 3' 5 and other lytic enzymes have received much attention in the past decades due to their activity against virulent bacteria. Lysozyme is an endo-glycosidase, which hydrolyses the peptidoglycan forming the cell walls of bacteria. This peptidoglycan, the substrate, consists of parallel polysaccharide chains covalently cross-linked by peptide chains. The repeating disaccharide of the polymer contains N-acetyl-D-glucosamine and N-acetyl muramic acid joined by a P-(l-4) linkage. Lysozymes are commonly isolated from bacteriophages and hen's egg white. 1.2 Interest in Olvcosidase Inhibition As can be judged by their function, the glycosidases play a major role in living systems. But even though they are essential, there are instances where it might be advantageous to control their activity and in some cases, to annihilate it.6 For example, excessive blood glucose values accompany metabolic diseases such as diabetes and obesity. It was shown that acarbose, a naturally occurring inhibitor of a-5 glucosidases, improves the metabolic conditions of diabetics. Furthermore, since a reduced intake of carbohydrates can prevent problems such as hyperinsulinaemia and hypertriglyceridaemia, one may anticipate that a-glycosidase inhibition might also be beneficial. A second example is the problem of wood degradation by fungi. These microorganisms excrete cellulases which hydrolyse cellulose in living trees and cut lumber.7 In such a case, a proper (3-glucosidase inhibitor might act as a very specific preservative that would kill the fungi by inactivating its digestive enzyme. In addition to their important practical applications, inhibitors remain excellent tools for investigation of enzyme mechanisms. 1.3 Mechanism of Glycosidase Hvdrolvsis In order to be efficient and specific, inhibitors are usually designed on the basis of the enzyme mechanism toward the natural substrate. Glycosidases which hydrolyse glycosides with net retention of anomeric configuration ("retaining" glycosidases) are generally considered to catalyze glycoside hydrolysis as follows1: 6 7 After the formation of an enzyme-substrate complex, the glycosidic oxygen is protonated by an acid catalytic group in the active site. Cleavage of the glycosidic bond follows to give a carbonium ion which has a half-chair conformation. This intermediate is stabilized electrostatically and to a certain extent covalently by an active site carboxylate side chain. Finally, the base-catalyzed hydrolysis of this intermediate affords glucose. 1.4 filvcosidase Inhibitors On the basis of kinetic studies, it is clear that known inhibitors interfere in the previous mechanism at the active site level. They are of two kinds: the non-covalent inhibitors which compete with natural substrates for the active site and bind through electrostatic, Van der Waals, and hydrogen bonding interactions; and the covalent inhibitors which bind at the active site and form a covalent bond with the enzyme. Non-Covalent Inhibitors A family of compounds such as the naturally occurring nojirimycin8 and acarbose8-9 were found to be non-covalent competitive inhibitors of a variety of glycosidases. C H 2 O H H O H O >H 8 HO H O A ^ ^ - ^ O H NOJTRIMYCIN The unsaturated cyclitol unit in acarbose has a half-chair conformation, a structure which mimics the transition state of the enzyme reaction. Of equal importance for the pronounced inhibitory effect in both nojirimycin and acarbose is the replacement of the oxygen by the more basic amine function. The readily protonated amines are believed to interact with the carboxylate groups at the active site resulting in tight binding. Many other good inhibitors contain such a functionality. The C-N bond in acarbose cannot be cleaved by glycosidases, thus increasing its stability. Inhibition by nojirimycin may be due to the cyclic imine which is produced after dehydration and has a half-chair conformation, thus acting as a transition state analogue.10 Covalent Suicide Inhibitors Covalent, active site directed inhibition of several P-glucosidases was obtained by using appropriate substrate analogues. N-Bromoacetyl-P-D-glucopyranosylamine and conduritol-B-epoxide (DL-l,2-anhydro-myo-inositol) are typical examples. 9 \ \ \ \ \ \ \ \ - A -77777777 N-Bromoacetyl- _ ^ . , Conduntol B-D-gluccpyranosylamine epoxide In the case of the bromoacetamide derivative, a nucleophilic displacement of the bromide anion by an active site nucleophile is assumed10 to lead to inactivation. On the other hand, conduritol epoxide, as for the natural substrate, is protonated by the enzyme and undergoes a ring opening when attacked by a nucleophile at the active site. 1 0 , 1 1 These alkylating agents lead to covalendy bound substrates and are therefore irreversible inhibitors. The designation "conduritol epoxide", formally 1,2-anhydroinositol, commonly refers to the epoxidation product of the corresponding conduritol (tetrahydroxycyclohexene). The suffixed capital letters, A to F, were assigned in the order of the conduritol discovery. On that basis, it was decided to refer to l,2-dideoxy-l,2-epimino-myo-inositol as "conduritol aziridine". 1.5 New Cvclitol-Based Inhibitors The glucosidase inhibitors proposed in this thesis are structurally similar to glucose so that the enzyme and its inhibitor are expected to associate in a way similar to the enzyme-substrate complex. Synthesis of two inhibitors was planned, one that would associate reversibly (non-covalently) and one that would associate irreversibly (covalently) with the glucosidase. It appears that compounds based on cyclitols would have a suitable structure. 10 Myo-inositol, a cheap and commercially available cyclitol, is the common starting material in the preparation of the two potential inhibitors shown below: O H O H ^ O H N O N - C O V A L E N T COVALENT INHIBITOR INHIBITOR Cyclitols are sugar-like cyclic polyols where the usual endocyclic oxygen is replaced by a methylene group. As a result, a C-N bond cleavage of the enamine derivative would not be assisted by oxygen, making a glucosidase-catalyzed hydrolysis of this bond virtually impossible. The R-group can be varied to achieve optimal inhibition. The endocyclic double bond in the enamine is intended to mimic the oxocarbonium ion-like transition state of the enzyme reaction since it will impose a degree of planarity on the structure. Therefore, the molecule can be regarded as a potential transition state analogue. The conduritol aziridine (l,2-dideoxy-l,2-epimino-m;y0-inositol) is expected to behave more like conduritol epoxide1 0 and act as a covalent (irreversible) inactivator. This compound, containing an aziridine ring, should have similar reactivity to the conduritol epoxide. However, it should prove better in its initial binding, since the presence of the basic nitrogen atom should lead to better initial non-covalent bmding, as described previously for amine containing inhibitors. wwww - A OH + OH ^ o ^ V o 1.6 The Cvclitols At first glance, cyclitols12'13'14 look very much like normal sugars. However, the presence of a carbon unit rather than the normal glycosidic oxygen (thus the absence of a hemiacetal center) leads to important differences in chemical properties such as the lack of an anomeric effect and of equilibrium with an open form (therefore no aldehydic character). The ease of reaction at the anomeric carbon of normal sugars via an oxocarbonium ion is also absent in cyclitols. In most cases, their hydroxyl groups are all secondary and, as opposed to normal sugars, of very similar reactivity. As a result, cyclitols form a class by themselves. Formally, they are defined as cycloalkanes containing three or more hydroxyl groups. Other functional groups can be present, so that inososes (2,3,4,5,6-pentahydroxy-l-cyclohexanones), mosarnines (x-amino-x-deoxyinositols), and quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) are also considered as cyclitols. Naming and Numbering of Cvclitols The system of naming and numbering for cyclitols issued by the IUPAC-IUB commission on biochemical nomenclature in 196815 is now widely utilized by authors. Several rules, especially those on the stereochemical nomenclature, apply to cyclitols 12 exclusively and need to be described briefly for a better understanding of the following sections. The structure is regarded as a planar ring (as in the Howarth projection) with two sets of substituents that lie respectively above and below this ring. The ring carbons are then numbered so that the set of the more numerous substituents receives the lowest locants (positional numbers). If both sides of the ring have equal numbers of substituents, the lowest locants are related to the set that can be denoted by lower numbers (in ascending series, the "lower numbers" are those that contain the lower number at the first point of difference; e.g. 1,2,3,6 is lower than 1,2,4,5). Substituents other than unmodified hydroxyl groups come next in the priorities followed by substituents first in alphabetical order or of less complexity. The relative configurations of a given cyclitol are then described by a fractional prefix where the numerator represents the set of locants containing the lowest number; the denominator represents the other set. The absolute configuration is determined by the lowest numbered chiral center. If the substituent is oriented up and the numbering is clockwise, the designation is L-. If it is oriented down, the designation is D-. OH OH OH OH 6 OH 3 HOJ 3 L-1,2/3,5-CYCLOHEXANETETROL MYO-INOSITOL In the case of inositols (1,2,3,4,5,6-cyclohexanehexols), the fraction is replaced by an italicized prefix describing any of the eight possible isomers. Substitution of one or two hydroxyl groups with retention of configuration does not alter this prefix and the numbering 13 of the molecule, which is still regarded as an inositol. Myo-, scyllo-, and c/iiro-inositol derivatives will be mentioned in the discussion. They are the 1,2,3,5/4,6-, 1,3,5/2,4,6-, and 1,2,4/3,5,6-isoniers respectively. The Inositols This work will deal primarily with inositols and inositol derivatives. These compounds, though not as thoroughly studied as normal sugars, are widely distributed in nature. Myoinositol, the most abundant isomer of inositols, occurs in most plants and animal cells. Phytic acid, the hexaphosphorylated derivative of myoinositol, constitutes 1 to 3% of all nuts, cereals, legumes, oil seeds, spores and pollen. 1 6 Among other vital physiological functions, phytic acid is believed to play an important role in storage of phosphorus. Furthermore phytin, a phytic acid calcium magnesium salt is used as a nutrient and calcium supplement.17 Nowadays, there is a renewed interest in synthetic and biological studies of inositol phosphates since the recent discovery that D-myo inositol 1,4,5-trisphosphate acts as a cellular second messanger.18 Different methods for the preparation of the D- and L-isomers have been reported recently.19 The biosynthesis of inositols has been studied for many years. In 1900, Maquenne20 suggested that myoinositol was the product of an intramolecular C-C cyclisation of a hexose molecule. It was later established21 that three enzymes are involved in this transformation where myoinositol-1 -phosphate synthase catalyses the cyclisation reaction.2 2-2 3 O O O H O H 6—P—O O H 6-P—O O H O H O H 14 It was also found that most other inositols derive from enzymic epimerizations of wyo-inositol.23 The Aminocyclitols The presence of an amino function in many carbohydrates from either natural sources or synthetic preparations plays a dominant role in terms of biological activities. For the past several decades, chemists have been interested in the synthetic, structural and biochemical studies of these compounds, the aminoglycosides.24 The reasons for such a sustained interest is to be found in their efficiency as therapeutic agents, antibiotics and enzyme inhibitors. Several aminoglycosides in fact contain cyclitol rings substituted with one or more amino groups. In biologically active compounds, the aminocyclitol is often linked to normal saccharides25, as in acarbose. Interestingly, streptomycin, the first aminoglycoside antibiotic to be discovered (1944) contained an aminocyclitol. In the following years, similar branched-chain sugars such as mannosidostreptomycin (1947), hydroxystreptomycin (1949), and others were also found to have streptidine (l,3-diguanido-2,4,5,6-cyclohexanetetrol) as the aglycon. N H N H C N H 2 N H J N H C N H 2 R=NHCH 3 R'=CH 2 OH R i t R t t t STREPTOMYCIN H H •H R"*OH 2 HYDROXYSTREPTOMYCIN H O H R ' O MANNOSIDOSTREPTOMYCIN H 15 The streptomycins and other groups such as the clinically important gentamycins are 1,3-cUammocyclitol derivatives. At the present time, few naturally occurring antibiotics with a single amino group on a cyclitol unit have been discovered. However, a large number of monoaminocyclitols exhibit high inhibitory activity on hydrolase enzymes. Therefore, these are appropriate models to design simple glycosidase inhibitors. The antibiotic validamycin A, the main component of the validamycin complex 2 6 produced by Streptomyces hygroscopicus var. limoneus as well as validoxylamine A 2 7 , obtained by acid hydrolysis of validamycin A, do not inhibit a-amylase or sucrase.6 However, valienamine2 8 obtained by the microbial degradation of validamycin A with Pseudomonas denitrificans or other soil bacteria, showed inhibitory activity on yeast oc-glucosidase and some other enzymes.29 D-GLUCOSE VALIDAMYCIN A 16 Interestingly, the unsaturated hydroxymethylcohduritol unit found in validamycin A is the key constituent of a large class of naturally occurring glucosidase and amylase inhibitors. Acarbose mentioned earlier, has a pronounced inhibitory effect against intestinal cc-glucosidases.8'9 Other naturally occurring monoaminocyclitols were found to be powerful glycosidase inhibitors. The important ones are the trestatins30, the oligostatins31, amylostatin32 and the adiposins.33 Besides oligostatins, which contain a hydroxyvalidatol group, all the above mentioned inhibitors contain the valienamine moiety either as an internal or a terminal group. It was found that even though valienamine alone is not as powerful an inhibitor as the parent oligosaccharide, it is an essential constituent for inhibition.6 1.7 Synthesis of Cvclitols and Monoaminocyclitols The discovery of aminocyclitol moieties in a wide variety of biologically active compounds inevitably led chemists to search for general synthetic approaches toward these structures. Their goal was twofold, the total synthesis of aminoglycoside-aminocyclitol antibiotics and their structure elucidation. In fact, total synthesis is of particular importance 17 for unequivocal structure (^termination of aminogiycosides due to the difficulty in obtaining good crystalline preparations for X-ray studies. In general, the methods and problems encountered in monoaminocyclitol synthesis are very closely related to those of cyclitol synthesis. The methods are classified below by means of the approach to cyclitol ring formation employed. The amino group can be a part of the precursors (sometimes in the form of NO2, N3, etc.) or it can be added after the cyclisation via oxime formation, nucleophilic substitutions and so on. One important exception, the nitromethane cyclisation, will be discussed where the ring formation and nitrogen introduction are embodied in a single process. The last section will deal with aspects of the transformations of cyclitols into aminocyclitols. Hydrogenations and Hydroxylations of Unsaturated Six-Membered Rings Afyo-inositol was first synthesized in 1914 by H . Wieland and R. S. Wishart by the hydrogenation of hexahydroxybenzene.34 Hydrogenation of tetrahydroxyquinone gave the same results since hexahydroxybenzene is an intermediate. The myo-isomer was isolated in 13% yield from the mixture of isomers. Even though hydrogenation produced mixtures of isomers and low yields, this is a method of historical importance that provided a wide range of inositols, quercitols and tetrols not found in nature. C/s-hydroxylation of cyclohexadiene derivatives with permanganate, or trans-hydroxylation with peroxy acid also leads to a variety of cyclitols as shown below.14 18 OH OH OH OH Diels-Alder Adducts The Diels-Alder reaction has played a dominant role in cyclitol ring formations generating unsaturated six-membered rings which can be hydroxylated as just described. The Diels-Alder adducts are sometimes grouped with hydrogenated products of benzene derivatives and form the cyclohexenepolyols. The synthesis of p.seitffo-p'-DL-glucopyranose by G.E.McCasland35 is an illustrative example (a pseudo-sugar designates any alicyclic analog of a monosaccharide, in which the usual ring-oxygen is replaced by methylene36'37). The trans,trans diastereomer of l,4-diacetoxy-l,3-butadiene was reacted with allyl acetate to give the corresponding enetriol triacetate in good yield. The desired product was obtained after m-hydroxylation with osmium tetroxide 19 followed by hydrolysis of the acetyl groups. A biologically important compound, shikimic acid, involved in the biosynthesis of phenylalanine, tyrosine, tryptophan, lignin, and other aromatic compounds, was synthesized in 1959 by E. E . Smissman et a / . 3 8 The Diels-Alder adduct of l,4-diacetoxy-l,3-butadiene and methyl aery late was cw-hydroxylated and, after protection of the hydroxyl groups, pyrolysis gave the elimination product Shikimic acid was obtained after deprotection. ROOC OAc ACID An alternative to the disubstituted butadiene is to make use of furans as the dienes. Cw-hydroxylations or epoxidations of the Diels-Alder adducts are fairly stereoselective and generally give the exo isomer. The 1,4-anhydro ring opening then leads to a polyhydroxylated cyclohexane ring. One of the monosaccharide analogues synthesized by G. E . McCasland 3 6 , pseudo-XzHosc, was prepared from 2-acetoxyfuran and maleic anhydride. Hydroxylation of the double bond and hydrolysis of the anhydride afforded the diol diacid. Rearrangement at room temperature involving acetyl migration, ring opening and 20 decarboxylation gave the keto acid. Reduction of the ketone, esterification of the carboxylic acid and acetylation gave the tetraacetate. Psemio-a-DL-talopyranose was obtained on reduction and acid hydrolysis. O O An important breakthrough in the field of pseudo-sugars was the synthesis of glucose analogs by the Japanese chemists S. Ogawa and T. Suami. 3 9 Many total syntheses of the different components of the validamycins followed and led to a revised structure of validamycin A . 4 0 Their common starting material, 7-endo-oxabicyclo [2,2,1] hept-5-ene-2-carboxylic acid, obtained from acrylic acid and furan, was treated with hydrogen peroxide to give a lactone. Reduction of the lactone and acetylation gave the tri-O-acetyl derivative. The 1,4-aimydro ring opening in aqueous acetic acid containing cone, sulfuric acid afforded the acetylated pseudo-glucose in 20% yield. Their first approach39 deals with the synthesis of penta-TV-O-acetyl-DL-valiclarnine that could be obtained in four steps from deprotected pseudo-glucose via the nucleophilic substitution of a tosylate by an azide on C-1. 21 o o o PENTA - ACETYLATED PENTA - ACETYLATED VALID AMINE P S E U D O - G L U C O S E In a slightly different reaction, the yield for the anhydro ring opening was later increased to 70% by heating the oxabicycloheptane with hydrogen bromide in acetic acid in a sealed tube. 4 1 The product, DL-l,2,3-tri-0-acetyl-(l,3/2,4,6)-4-bromo-6-bromomethyl-1,2,3-cyclohexanetriol, has been an important intermediate for many monoaminocyclitol syntheses such as valienamine42, validatol43, deoxyvalidatol43, hydroxyvalidamine44, and isomers of validoxylamines.45 22 All these products have been obtained as racemic mixtures. However, (-)-7-endo-oxabicyclo [2,2,1] hept-5-ene-2-carboxylic acid was obtained recently by use of (R)-(+)-ct-methylbenzylamine to resolve the racemic acid.46 In spite of the fact that the amino group can be introduced after the cyclitol ring formation, a more direct route to aminocyclitols can be achieved with nitro-compounds. The nitro group, which is eventually reduced to an amine, has the ability to increase the reactivity of the reagents and the regioselectivity of the reaction. The following diagram shows a possible sequence with ethyl 3-nitro-2-alkenoate as dienophile25 : 23 Nitromethane Cyclisation Even though the production of nitrocyclohexanes is feasible through Diels-Alder reactions, a useful method to prepare optically active aminocyclitols is the nitromethane cyclisation. The base-catalyzed addition of nitromethane to aldehydes, the Henry reaction, has been used extensively in aldose chain extension and as tool to introduce a nitro group into the body of a sugar. In the latter case, a twofold reaction takes place between a sugar dialdehyde and nitromethane. Such a cyclisation was first achieved by H.O.L. Fischer in 1948.47 He found that an intramolecular Henry reaction occurs when the 6-deoxy-6-nitro-D-glucose he had just synthesized was transformed into deoxynitroinositols on treatment with a base. It was later established that the DL-myo-l, muco-3, and scyllo isomers were the major products and that L-idose gave the same mixture of nitroinositols.48 MYO SCYLLO O H * O H O H More generally, the nitrocyclitol formation proceeds in two steps. First, the condensation of nitromethane and sugar dialdehyde« give the nitronate salt. Two new asymmetric centers are formed in this reaction. Intramolecular addition seems to be favored even in the presence of an excess of nitromethane. Then, on acidification, the free 24 nirrocyclitol is formed giving rise to one more asymmetric center. Theoretically, eight stereoisomeric products can be expected from the overall process. NITRONATE However, in practice, only one or two isomers are preponderant together with a few minor isomers. The proportions of the different isomers depend strongly on the reaction conditions.49 As can be seen from Fischer's cyclisation of 6-deoxy-6-nitro-D-glucose, normal sugars can be convenient starting polyols in aminocyclitol synthesis. Another example is the synthesis of streptidine from D-glucosamine as shown below:50 N H II R=NHCNH 2 STREPTIDINE 25 Uronic acids have also been employed in the synthesis of aminocyclitols. Oxidative decarboxylation of C-6 followed by reaction with nitromethane in alkali give the thermodynamic product. This route also allowed the synthesis of streptamine from D-glucosamine.51 OH OH CH^NC^ NaOMe/MeOH BzO" (OBz ~ ' BzOJ fOBz BzO' NHAc NHAc NHAc Thus, normal sugars are suitable starting materials in the preparation of aminocyclitols. The stereochemistry of some of the chiral centers remains unchanged during the transformation and it is possible, at least to some extent, to influence the stereoisomeric mixture of products via different reaction conditions. Vicinal diols are also common precursors for nitromethane cyclisations. The sequence of reactions, extensively applied in nitro sugar chemistry,52 involves the treatment of vicinal hydroxyl groups with permanganate or osmium tetroxide followed by the cyclisation with nitromethane. In spite of the fact that optically active aminocyclitols can be obtained by the nitromethane cyclisation, this method has inherent disadvantages. First, the desired isomer is often produced in low yield which may become a problem in large scale preparations. Second, the different isomers are not always well-resolved by chromatography, and purification can be difficult. Finally, even though a certain degree of kinetic or thermodynamic control is possible, some stereoisomers cannot be obtained in sufficient yield for synthetic purposes. 26 Aldol Condensation It is worthwhile noting that besides the nitromethane cyclisation, other condensation reactions such as the aldol condensation lead to cyclitols. The scope of these reactions is much less important than the nitromethane cyclisation but, in many cases, a better stereoselectivity is achieved. As already discussed in Section 1.6, myo-inositol is most likely produced from D-glucose in nature by an enzyme-catalyzed aldol condensation. The same basic idea was tested in non-enzymic systems where different (normal) sugars were modified into dicarbonyl sugar precursors. The condensation reaction then gives carbocyclic products bearing a carbonyl group (or inosose) which is eventually reduced to an alcohol. A classical example has been reported by D.E.Kiely et a/ . 5 3 In an investigation of the biosynthesis of L -myo-inositol-1 -phosphate, they synthesized D-xylo-hexos-5-uIose from D-glucose and cyclised it in 0.1 N sodium hydroxide into 2,4,6/3,5-pentahydroxycyclohexanone (myo-inosose-2). On reduction with sodium borohydride, myo- and scy/Zo-inositol were obtained. D - G L U C O S E Other examples can be found in the literature including reactions of protected polyols under non-aqueous conditions.25 A slightly different way to cyclise a modified sugar into a cyclitol has been used successfully by T.Suami. 5 4 In that method, a malonic ester is reacted with the aldehydic group of a protected sugar. The terminal hydroxyl group is then oxidized to an aldehyde and 27 a head-to-tail condensation occurs. Decarboxylation yields an optically active branched-chain cyclitol. The following sequence shows the transformation of D-ribose to pseudo-$-L-mannopyranose. AcO D-RIBOSE OBn OBn TBDPSiO JL - f " I > ^ C H O w ' J - MeO^CT OBn | OBn OBn CO^Me T B D P S i O ^ A ^ A ^ OBn Me MeO^C GOjMe HO\A 'GO^Me MeO^C 00 2Me " 0=CH \ OBnOBn U ] [OBn OBnOBn Psewdo-a-D-glucose,55 pseudo-fi-L-altrose,55 and pseudo-P-L-arabinofuranose54 have also been synthesized by this method. The Ferrier Reaction Among the various methods employed to synthesize optically active cyclitols and aminocyclitols, the Ferrier reaction often proved to be the most appropriate one. In this procedure, neutral sugars are transformed into chiral cyclohexanones and, as in the nitromethane cyclisation, few isomers are produced. In fact, a typical Ferrier reaction affords a single epimer in a yield higher than 80%. A partial investigation of the mechanism by Ferrier5 6 showed that one of the key steps is once again an intramolecular aldol-like 28 condensation under acidic conditions of a dicarbonyl intermediate. The reaction proceeds smoothly in partially aqueous media in the presence of mercury (II) salts. Aminocyclitols have been prepared from similar cycloses via the corresponding oximes.57 On the other hand, an azido or a protected amino group can be present in the sugar molecule during the Ferrier reaction. This method of cyclitol formation is relatively new (1979)58 and not yet general. It leads mainly to deoxyinososes and deoxyinositols and cannot apparently be applied to five-membered ring carbocycles.56 Furthermore, the accessibility to a cyclitol by this method depends greatly on the accessibility of the corresponding sugar. Nevertheless, the Ferrier reaction is highly stereoselective and has been employed in the synthesis of important aminoglycosides such as amylostatin (XG) 5 9 and valienamine.60 Aminocyclitols from Mvo-Inositol Originally, aminocyclitols were prepared from inositols via oxime or phenylhydrazone derivatives followed by sodium reduction or hydrogenation.12 The process required a preliminary oxidation of inositols into inososes, a reaction that favored axial hydroxyl groups and that could be performed on the deprotected cyclitols in aqueous solution. The method was straightforward but suffered important drawbacks. The stereoselectivity of the reduction at the sp 2 center was sometimes incomplete and unpredictable. The facile aromatization of some inososes was also a problem and the frequent difficulties encountered in oxidizing more 29 than one hydroxyl group made this route unsuitable for preparation of 1,3-diaminocyclitol derivatives. Later, in work pioneered by T. Suami, 6 1 the general method employed to introduce a nitrogen onto a cyclitol was the azide substitution. Typically, the azide anion would displace a sulfonylated alcohol or a bromide with inversion of configuration, or open an oxirane ring in a trans diaxial fashion. The azide anion is a good nucleophile of low basicity and ekmination is generally not a problem. The amine is obtained by either hydride reduction or hydrogenation. Important aminocyclitols including valienamine have been prepared from 1,2,3-tri-i9-acetyl-(l ,3/2,4,6-)-4-bromo-6-bromomethyl-1,2,3-cyclohexanetriol (see Diels-Alder adducts) via the azidation reaction. 30 RESULTS AND DISCUSSION 31 2.1 Synthesis of Conduritol Aziridine m OH HO NH 2.11 Importance of Aziridine Derivatives and Their Synthesis A wide range of aziridines from either synthetic or biological sources is known to have useful biological activity.62 Studies tend to show that many of these compounds are bifunctional alkylating agents capable of cross-linking DNA. Alkylation takes place via opening of the aziridine ring.63 In the field of carbohydrate chemistry, aziridines are also important because of their biological activities but they are most commonly synthesized as key intermediates for the introduction of different nucleophiles by way of fra/w-diaxial opening of epimine rings.64 The aziridine ring of epimino sugars is normally formed by cyclisation of a nitrogen-containing group with a neighboring leaving group. The most widely used synthetic pathways are the Gabriel and Wenker methods.65 o H N _ > R V 7 N + R H N R X=Br,Cl,I (GABRIEL) X=OS0 3- (WENKER) Other similar systems involve a tosylate or methanesulfonate as leaving group. In 32 these cases, however, it is not always possible to selectively tosylate the hydroxyl group of an aminoalcohol. Furthermore, the aminotosylate is often a mere intermediate product of azidotosylate reduction and undergoes immediate cyclisation. Sodium borohydride,6 6 triphenylphosphine,67 lithium aluminium hydride, 6 8 and catalytic hydrogenation69 have been used to reduce azidotozylates. 2.12 Approach to 1 Starting with Afvo-Inositol Three inositols are readily obtainable from natural sources : the myo-, dextro-, and /evo-isomers.12 The other diastereomers are obtained by epimerizations at one or more centers. Afyo-inositol (though it lacks the hydroxymethyl group that would make it more similar to hexopyranoses and would presumably result in tighter binding to the enzyme) is commercially available at a low price (Aldrich : $ 17/100 g.) and seemed to be the material of choice to begin our synthesis. A well-known problem in cyclitol chemistry is the selective protection and deprotection of the many chemically similar hydroxyl groups present in the molecule. However, the /nyo-isomer has an axial hydroxyl group at C-2 that allows discrimination of two of the six hydroxyl groups by formation of a ketal. O 3 33 l,2-t9<yclohexytidene-myo-inositol (3) prepared by S.J. Angyal et A / . 7 0 ' 7 1 contains two protected vicinal alcohols. Therefore, a simple and reasonable plan to prepare conduritol aziridine was, after a proper protection-deprotection sequence, to transform the equatorial free alcohol at C-3 into a leaving group and displace it in an SN 2 fashion by an azide (Scheme I). The azido and hydroxyl groups, after the proper chemical modifications, would then have the suitable trans-diaxial orientation for the eventual cyclisation reaction. OR OR R O - ^ V ^ ^ ^ V O H R C K " \ ^ - \ ^ ~ V L OR I OR I O H O H L= LEAVING GROUP R= PROTECTING GROUP • N 3 O H OR J H O ^ ^ ^ - ^ V - N H ^ R O ' ^ ^ \ ^ \ H O - \ ^ - — + R O - \ ^ O H OR I O H Scheme I 2.13 1.4.5.6-Tetra-Q-benzvl-3-0-toluenesulfonvl-iiivo-inositol (6) The formation of ketal 3 posed no major problem even though, experimentally, the condensation is not totally selective. In fact, a mixture of diacetals and a triacetal is formed followed by partial ethanolysis to give the 1,2-0-cyclohexykdene derivative. The successful preparation of the isopropylidene derivative has also been reported7 2 Starting with these ketals, quite a large number of myoinositol derivatives have been made as can be seen from a survey of the literature. Among others, l,4,5,6-tetra-<9-benzyl-3-O-toluenesulfonyl-myo-inositol (6) 7 3 is of special interest since it fits well in the above 34 Thus, protection of the four remaining free hydroxyl groups with benzyl chloride (acylated inositol derivatives are susceptible to acyl migrations74) followed by acidic hydrolysis gave the tetra-O-benzyl ether (5). The following tosylation is a very selective one and even though one equiv. of tosyl chloride is sufficient, prolonged standing of 5 in the presence of three equiv. of the chloride gave no detectable trace of the di-(9-tosylated derivative. The presence of a single tosyl group in 5 can be deduced easily from its JH-n.m.r. spectrum where only one singlet (methyl group) integrates for three hydrogens at 2.36 p.p.m. Furthermore, the free hydroxyl group, which absorbs at 3520 cm*1 in the i.r., gives a signal at 2.58 p.p.m. that disappeared on D2O addition. It must be mentioned at this point that all the structures presented in this work represent racemic mixtures. However, the resolution of racemic 5 has been achieved by C.B. Reese and J.G. Ward 7 5 via its l-{3-D-(2,3,4,6-tetra-<9-acetyl) glucopyranosyl derivative 7, 35 rnalong the synthesis of enantiomerically pure conduritol aziridine feasible. OBzl >Bzl ) »Bzl 7 2.14 Preparation of Azidocvclitols The next step deals with the introduction of a nitrogen atom, which we first experimented with by reacting 6 with sodium azide. Unfortunately, after refluxing in DMF, the major product obtained was not the expected l-azido-2,3,4,5-tetra-0-benzyl-l-deoxy-cfoVo-inositol (8) but rather an aromatized compound, as could be established by the lack of signals upfield from 4.95 p.p.m. (OCH2) in the ^-n.m.r. spectrum. This spectrum together with the melting point, turned out to be identical to those of 2,4-dibenzyloxyphenol (9) known to be formed smoothly from 6 in the presence of potassium hydroxide.73 36 Scheme 13 The mechanism that was postulated for such an aromatization involves the elimination of toluenesulfonic acid followed by two successive pVelirninations as shown in Scheme II. 7 3 Even though in our case the reaction mixture is neutral, t.l.c. analysis indicates that aromatization is triggered by raising the temperature. By monitoring the progress of the reaction closely by t lx . and lowering the temperature to 1 4 0 ° , a low yield (27%) of the azido derivative could be obtained (a yield that was not consistently reproducible). 1H-n.m.r. and i.r. data show that the sulfonyl ester had indeed been substituted by an azide. The coupling constants for four of the six ring hydrogens are measurable and in good agreement with the proposed stereoisomer 8. However, the possibility that 8 contains an equatorial tosyl group (from an S N I reaction) could not be ruled out Acetylation at the 2-position prior to the nucleophilic substitution gave no better results. At this point, it was decided to introduce the azide at the 2-position followed by closure onto C -3 , a strategy that leads to the same isomer. For thermodynamic and kinetic reasons, SN 2 reactions on six-membered rings proceed more easily when the leaving group is axially oriented. However, the trans diequatorial geometry of the azidoalcohol product will 37 make the eventual cyclisation more difficult Thus, l,4,5,6-temi-(9-benzyl-2-0-memanesulfonyl-3-0-toluenesulfonyl-myo-inositol (10) obtained from methanesulfonylation of compound 6 in pyridine, was reacted with sodium azide at 100° to afford a single product in good yield. This time, no aromatization was observed but both the mesyl and the tosyl groups were displaced by the azide anion as can be seen by the absence of methyl protons on the 1H-n.m.r. spectrum of 11. The small coupling constant of 3.2 Hz for the single equatorial proton ( 3.95 p.p.m.) indicates that the two azido groups are cis as would be expected if two consecutive SN2 reactions occurred. A good selectivity was however achieved by reacting 1.3 equiv. of sodium azide with l,4,5,6-tetra-0-benzyl-2,3-di-0-methanesulfonyl-/7iyo-inositol (12) prepared directly from the diol 5. Examination of the major product 13 by ^ -n.m.r. shows that a single mesyl group is present (methyl protons at 3.02 p.p.m.). A large coupling constant (J 6.6 Hz) for the hydrogen on the carbon bearing the mesyl group at 4.43 p.p.m. proves that the azidomesylate derivative is fra/zs-diequatorial. The presence of the azido group is demonstrated by the absorption at 2115 cnr1 in the i.r. spectrum. The diazide 11 was obtained as a minor product. 6 10 11 38 2.15 Attempted Hydrogenation of Azido Compound 13 At this point, three steps are yet to be done to reach the conduritol aziridine : 1- Reduction of the azido group to an amine ; 2- Cyclisation of the amine by displacing the mesyl group; 3- Removal of the four benzyl groups. We anticipated that hydrogenolysis of the azidomesylate would result in a complete removal of the benzyl groups together with reduction of azide to amine and, possibly, direct cyclisation to yield the conduritol aziridine. Unfortunately, this direct approach was unsuccessful. Many different reaction conditions for hydrogenation were attempted such as utilization of hydrogen donors (HCO2H 7 6 , HCO2NH4 7 7), high pressure (345 Pa) and different catalysts (palladium, platinum). Attempted deprotection with TMSI and the Birch reduction also failed. In all 39 cases, a complex mixture of polar products, as seen by t.l.c, was obtained, except when formic acid was the source of hydrogen . In that case, no reaction occurred and the starting material was recovered. Compounds 11 and 13 have many features in common. Therefore, hydrogenations were also attempted on the diazide in order to conserve the more important 13 (Table I). Interestingly, hydrogenolysis of the dimesylate 12 proceeded in high yield (88%) at normal pressure to give the deprotected dimesylate, suggesting that either the azido or the resulting amino group is the source of the problem. Hydrogenation in the presence of acetic acid which should protonate the eventual amine gave no better results. Table I Reaction Conditions for Attempted Hydrogenations of Compounds 11 and 13. Compounc Hydrogen Catalyst Source Solvent Pressure (Pa) 11 H 2 Pd/C 10% MeOH Normal 11 H 2 Pd/C 10% EtOH 345 11 H 2 Adam's (Pt) EtOH 345 13 H 2 Pd/C 10% EtOH 345 13 H 2 Pd/C 10% EtOH a 345 13 « 2 Pd/C 10% HOAc Normal 13 H002H Pd/C 10% MeOH Normal 13 HCQ2NH4 Pd/C 10% MeOH Normal Containing HOAc (2.5 equiv.) 4 0 2.16 Preparation of Protected Conduritol Aziridine In order to gain a better control over these transformations, a stepwise approach was considered. Two approaches to azide reduction without reduction of the benzyl groups were tried. In the first approach, the azido derivative 13 was reduced with triphenylphosphine (Staudinger reaction78) and the phosphazo intermediate was hydrolysed67-79 to give the aziridine 14. 13 As was observed by t.l.c, the starting material is consumed rapidly by triphenylphosphine but hydrolysis of the phosphonimine is a very slow process that could be shortened to five days only when NH4OH was used instead of water for hydrolysis. A similar outcome was obtained using LiAUfy to reduce the azide; the aziridine 14 was obtained directly within 24 hours. Spectroscopic data indicate clearly that reduction and ring closure have occurred. The i.r. spectrum no longer shows a band around 2100 cm"1 and NH stretching is observed at 3305 cm"1. The 1H-n.m.r. spectrum shows the two methine aziridine protons at 2.32 and 2.49 p.p.m. The coupling constant Ji,2 of 6 Hz and the absence of observable coupling between H-l and H-6 are typical of aziridines fused to six-membered rings.80 Furthermore, a 41 broad peak at 0.73 p.p.m. integrating for one proton disappears on D2O addition. Hydrogenation was then attempted on 14 with Pd/C 10% and ammonium formate as hydrogen donor in methanol. The starting material was consumed rapidly but once again, a complex mixture of benzylated products was obtained. 2.17 Alternative Approaches to 1 As was mentioned earlier, the dimesylate 12 could be hydrogenated in good yield and gave 1,2-di-O-methanesulfonyl-myo-inositol (17). Alternatively, compound 17 could be prepared by <9-deacylation of l,4,5,6-tetra-(9-acetyl-2,3-di-(9-methanesulfonyl-myo-inositol (16) with rt-butylamine. Compound 16 was obtained by mesylation of 15, the latter being prepared in two steps from 3 according to S.J. Angyal. 7 0 OAc O H 3 Therefore, we investigated the possibility of introducing the azide on this deprotected dimesylate followed by its reduction in some way other than by hydrogenation. Treatment of 17 with sodium azide in D M F gave three products in addition to starting material. In order to separate and purify them, the mixture was acetylated with acetic anhydride in pyridine followed by flash-chromatography. In order of decreasing polarity (light petroleum-ethyl acetate 2:1), the three products obtained, their percentage yields, and some relevant 42 spectroscopic data are listed in Table II. Table TL Products from azidolysis of 1,2-cU-O-methanesulfonyl-myo-inositol (17) followed by acetylation. Compound M+l Peak Yield Number of Number of Azido (Low Res.) (%) Acetyl Groups Mesyl Groups Groups (N.m.r.) (N.m.r.) (Lr.) 18 452 14.0 4 1 Present 20 399 11.7 4 0 Present 21 399 10.2 4 0 Present From Table II, it is clear that compounds 20 and 21 are in fact two isomers, each bearing two azido groups. Analysis of the JH-n.m.r. spectrum suggests that the structure of 20 is as shown in Scheme III. A number of such isomers have been reported in the literature and, as mentioned by T. Suami81, in general, "the signals of protons on carbon atoms bearing azido groups appeared at x 6.06-6.36 (axial) and x 5.70-5.90 (equatorial)" in the 1H-n.m.r. spectrum. Therefore, both 20 and 21 contain an axial and an equatorial azido group. The 1H-n.m.r. of 20 is well resolved and in good agreement with the structure proposed below (Scheme HI). Determination of structure 21 is not as straightforward as that of 20 but once again, observations by T. Suami81 suggest that the acetoxy methyl group at 2.04 p.p.m. is equatorial and adjacent to two acetoxy groups, the two acetoxy methyl groups at 2.10 p.p.m. are equatorial and adjacent to one azido group, and the acetoxy methyl group at 2.19 p.p.m. is either axial or flanked by two azido groups or both. On that basis and taking into account 43 the coupling constants of H-l and H-5, the structure is likely to be as shown in Scheme HI. The production of 21 presumably proceeds through epoxide formation followed by diaxial opening by an azide anion. 17 Scheme in Other possibilities for structure 21 would involve two adjacent azido groups. However, their formation could not simply be explained by epoxide intermediates. Among the three products, the compound of interest is 18 which can be converted to the desired 19 by mild deacylation with H-butylamine. Its 1H-n.m.r. spectrum shows that the azido (CHN3 : triplet, J 10.0 Hz) and the mesyl (CHOMs : triplet, J 10.0 Hz) groups are equatorial and adjacent to equatorial functional groups. The lack of resolution for the four protected carbinols is typical of fully equatorially substituted cyclitols. A decoupling experiment showed that the azido and mesyl groups are vicinal. Unfortunately, even though 18 has the proper stereochemistry and is a potential precursor to the desired aziridine, its yield, at best (1.3 equiv. of sodium azide) was no more 44 than 29.8%. Thus it was decided to investigate the azide formation reaction in the presence of acy! protecting groups. However, this proved no better since reaction of 16 with NaN 3 gave a mixture of five compounds as seen on tl.c. (mcluding starting material). Most products had similar polarities and could not be separated by chromatography on silica gel. Finally, the azidation reaction was attempted on l,4,5,6-tetra-0-benzoyl-2,3-di-<9-methanesulfonyl-myo-inositol (22) prepared by benzoylation of 17. This reaction was cleaner than the previous one and though some starting material was still present, a major product was formed together with a minor one. Unfortunately, the minor product happened to be the desired compound 23. This was confirmed by obtaining a sample of 23 from 19 by benzoylation. The major product which contain a mesyl group, was not characterized completely. On the basis of integration on iH-nmr. (OMs), the ratio for the major product to 23 (minor) to 22 (starting material), is 4.0 to 1.8 to 2.2. 2.18 Hydrogenation and Cyclisation A publication was then found 8 2 describing the successful hydrogenation of azides in the presence of strong acid. Hydrogenation of 13 was then attempted again in the presence of hydrochloric acid. The reaction proceeded smoothly at normal pressure over palladium in chloroform-ethanol. All four benzyloxy groups were removed (as deduced by n.m.r.) and the azido group was reduced to an amine as shown by i.r. Although a trace of free mesylate anion can be observed in the ^H-n.mx spectrum at 2.77 p.p.m., the presence of a covalently linked mesylate (3.31 p.p.m.) indicates that no cyclisation had occurred. Microanalysis OBz OBz 23 45 results indicated that the hydrogenated product 24 is isolated with one equiv. of HC1 and half an equiv. of H2O. The reason why 24 can be formed in the presence of hydrochloric acid, yet not with acetic acid, is unclear. The product 24 was relatively unstable as compared to other tetrahydroxycyclitols such as 17,19, and 31 prepared in this work. With this hydrogenation successfully achieved, attempts were made to hydrogenate the diazide 11 under similar conditions. Unfortunately, this was unsuccessful. Attempted cyclisation of 24 with sat hydrogen carbonate followed by heating (~70°) or with sodium hydroxide at 0° ended up in formation of several products as seen by t.l.c. (isopropanol-water 3:1). On the other hand, heating 24 at 55° with diethanolamine in water (pH 9) gave cleaner chromatograms with only two spots at Rf 0.63 and 0.02. A similar pattern was obtained with DMSO as solvent and triethylamine as base. The less polar compound (Rf 0.63) could be visualized on the plates with ninhydrin but, as for aziridine 14, required a pre-treatment with HC1. This product was isolated in low yield (7%) after purification by preparative layer chromatography on silica gel (isopropanohwater 3:1) Its 1 H -n.m.r. spectrum in D2O is in good agreement with the expected conduritol aziridine (see spectral index). Among the six proton signals, the two methine aziridine protons are upfield at 2.34 p.p.m.(doublet J 6.2 Hz) and 2.62 p.p.m. (doublet of doublets J 6.2, 3.3 Hz). Assignments for all 6 protons were obtained via a series of decoupling experiments. No coupling was observed between H - l and H-6 as is generally the case for aziridines fused to six-membered rings (including compound 14). Furthermore, no mesyl group nor mesylate anion was observed by either n.m.r. or i.r. O H H O - ^ V - ^ - — ^ > N H H O ^ ^ ^ ^ V O H 1 46 The 1H-n.m.r. of product 1 is therefore consistent with the structure of conduritol aziridine. However, a satisfactory microanalysis result could not be obtained. This may be due to some impurities eluted from silica gel by the aqueous solvent during the separation step. The mass spectrum (DCI) gives a base peak of 160 (M+-1). Another peak at mlz 196 indicates that 1 is present as its hydrochloride. Surprisingly, another intense peak is observed at mlz 321 suggesting the possibility of a dimer. Whether this dimer is an artifact of the mass spectrometer, or whether it is truly present in the sample is unclear. However, no evidence of such a dimer species was observed by t.l.c. or n.m.r. 47 2.2 Synthesis of a Potential Reversihle Inhibitor OH ^ n NR2 25 The different structural characteristics of a molecule such as the enamine 25, that make it a potential reversible inhibitor for glucosidases have been summarized in Section 1.5. This compound is similar to conduritol aziridine in several aspects. Therefore, myo-inositol was once again the starting material and much of the chemistry involved in the synthesis of the aziridine 1 could be applied, at least for the first steps, towards the preparation of the enamine 25. 2.21 The Reductive Amination Approach Among the various ways to form the required C-N bond, a very common method is to react a carbonyl with an amine followed by reduction of the resulting imine (or enarnine) by a hydride. The two transformations are often part of a one-pot reaction and called reductive amination.83 Having l,4,5,6-tetra-C^ benzyl-3-0-toluenesulfonyl-my0-inositol (6) in hand, our first plan was to oxidize its free hydroxyl group to a ketone followed by a reductive amination with a secondary amine. Removal of the four benzyl groups and elimination of the p-toluenesulfonyl group would lead to the desired product (Scheme IV). 48 OBzl 6 OXIDATION Bzl< 1 0 — ^ - ^ ^ v ^ O T s OBzT^o 2 6 H O • REDUCTIVE j AMINATION t OBzl N R 2 1 - D E P R O T E C T I O N Bzl< M ~2~EDMINAT10N~ " i 2 5 2 7 ShemelV Oxidation of the alcohol 6 by pyridinium dichromate in methylene chloride in the presence of acetic anhydride84 gave the ketone 2 6 in a rather low isolated yield (42%). The infrared spectrum of this product shows an absorption at 1750 cm - 1 for the carbonyl, and the iH-n.m.r. shows doublets at 4.21 and 5.18 p.p.m. for protons on each side of the carbonyl group. A coupling constant of 10.3 Hz for these two protons indicates that no epimerization occurred at these centers. Reductive amination of ketone 2 6 with dimethylamine hydrochloride in the presence of sodium cyanoborohydride was attempted. Compound 2 6 was consumed slowly and after 72 h, the product, after purification, proved to be the enone 2 7 (mlz 520), obtained by simple elirnination of p-toluenesulfonic acid. OBzl Bzl< 2 7 49 The same elimination product was obtained by simply reacting 26 with triethylamine over 5 h. The reductive arnination was then abandoned. 2.22 Preparation of 1 -amino-2-f l -benzovl- l -deoxv-scvZ/o- inosi to l f31) Hydrochloride The reductive amination was investigated no further and a second approach to introduce the amine was attempted, involving a nucleophilic substitution by azide anion. Hydrogenation of the azide and the four benzyl groups followed by selective amine alkylation (or imine formation) and elimination would lead to the desired enamine (Scheme V). Elimination of benzoic acid could give two isomers. One possible way to force the elimination towards the enamine formation is to replace the amine alkylation step by an imine condensation as shown below. In this case, elimination would likely proceed towards the thermodynamically more stable conjugated product The aminomesylate 24 could have been employed for the enamine synthesis but it was not known at the time (the synthesis of conduritol aziridine and the enamine being conducted in parallel) whether compound 24 would tend to form epoxide or aziridine rings during the course of those transformations. O H H O O H 24 50 1- PhCOCl,Pyr. » 2- MsCl, Pyr. OBzl B z l O - ^ - \ ^ - ^ O C O P h B z l O ^ ^ ^ ^ A BzlO I OR 28 R=H 29 R=S02CH3 1NaN3 DMF OH OBzl HO-^-V-A^^-OCOPh H2,Pd/C B z l C K ^ ^ ^ O C C H O - \ ^ ^ \ ^ N H 2 B z l O ^ ^ - - ^ \ ^ N OH BzlO 31 R2CO OCOPh 3 30 OH HO OCOPh HO N=CR2 ELIMINATION HO HO OH 32 OH OH M— N=CR0 Scheme V l-0-benzoyl-3,4,5,6-tetra-0-benzyl-myo-inositol (28) was prepared essentially according to the basic procedure described in the literature.85 Since both free hydroxyl groups are easily benzoylated, regiospecific benzoylation at the equatorial position requires greater care (-1.1 equiv. of PhCOCl, mixing the reagents at 0°) than the corresponding monotosylation (Section 2.13). Compound 28, like compound 6, was left unreacted on treatment with tosyl chloride under reflux for 3 h in pyridine. However, mesylation proceeded smoothly at room temperature. Analysis of the product 29 by ^H-n.ms. indicates that the signal (J 3.2 Hz) for the proton situated on the O-mesylated ring carbon is now at 51 5.38 p.p.m., almost one p.p.m. downfield from the corresponding signal (4.40 p.p.m.) of the starting free alcohol. Nucleophilic substitution of the mesylate by the azide anion in D M F posed no difficulty and a single product (30) was formed in 81% yield. The n.m.r. spectrum for 30 is not well resolved. Indeed, all the cyclitol ring protons but the one on the carbon bearing the benzoate ester (5.22 p.p.m.), absorb between 3.5 and 3.7 p.p.m..The presence of the azido and benzoate groups is confirmed by absorptions in the i.r. at 2105 and 1730 c m - 1 respectively. Moreover, as was the case for 13, hydrogenation of 30 in the absence of HC1 failed, the two products formed both showing benzylic hydrogens in the iH-n.m.r. between 4.5 and 5.0 p.p.m. An alternative approach was attempted involving an initial hydrogenation of 29 giving l-O-benzoyl-2-O-methanesulfonyl-myo-inositol (33) in high yield (88%). However, an attempt to introduce the azide directly by displacement of the mesylate in D M F produced a mixture of at least three compounds that could not be separated by chromatography. Reduction of 30 with triphenylphosphine in THF-water (no NH4OH) resulted in the reduction of the azido group but, as was indicated by the position of the C=0 band, 1640 cm - 1 in the infrared, the benzoyl group migrated from the oxygen to the neighboring nitrogen atom to give 35 instead of 34. 52 Hydrogenation of 30 in the presence of HC1 was then attempted and the expected compound 31 was indeed obtained in the form of its hydrochloride salt. The infrared spectrum, in which no azide absorption can be seen, shows an ester carbonyl peak at 1725 cm - 1 , a strong indication that no benzoyl migration occurred. This was confirmed by n.m.r. where the triplet at 5.23 p.p.m. and its relatively large coupling constant (J 9.4 Hz) prove that the equatorial benzoate ester is intact and adjacent to two functional groups which are also equatorially oriented. The remaining five carbocyclic protons have their signal between 3.4 and 3.8 p.p.m. as expected. 2.3 EnzvmplpgY Irreversible inhibition Conduritol aziridine 1 was tested for inhibitory activity against pABG5 pVglucosidase and yeast a-glucosidase. Since 1 might enter a glucosidase active site with two possible epimine ring orientations (Section 2.4), inhibitory activity is possible for both the glucosidases. For irreversible inhibition, the following process is assumed: 53 Where £ is free enzyme, I is free inhibitor, EI is non-covalent enzyme-inhibitor complex, EI' is covalent enzyme inhibitor complex, Ki is dissociation constant at equilibrium, and kj is inactivation rate constant The rate of inactivation vi at any concentration of I is vi-kitEI] Therefore, measuring inactivation rates at different EI concentrations will give the inactivation rate constant ki. The concentration of EI will depend on the analogue concentration according to the expression ir rem K i " [EI] Where the free enzyme concentration [ E ] is the difference between total enzyme concentration [EQ] and [EI]. Therefore, Kj [EI] = ([Eo] - [EIDm and l t l J Ki+rrj And the rate of enzyme inactivation can be expressed as follows: » " K i + [i] or \Zd _ K L _ + _L V i " ki[I] ki Therefore, irreversible inactivation of an enzyme can be detected and quantified by measuring loss of enzyme activity as a function of time after addition of inactivator (Fig. 1). 54 From the previous analysis, the loss of enzyme activity should be a pseudo first order process since the concentration of inactivator is essentially constant (mM concentrations of inactivator versus }iM concentrations of enzyme). 0.07-• • • 0.06-D D • 0.05- D 0.04- a a < 0.03-0.02-o D D o 0.01 -0.00- -* r 1.32 mM 2.63 mM 6.58 mM 9.87 mM 1 - i — i —f-10 15 20 25 Time (min.) Fig. 1. Loss of yeast a-glucosidase activity as a function of time at different aziridine concentrations. The rate of enzyme inactivation (vi) was measured at the aziridine concentrations listed in Table HI. Rates were obtained by removing aliquots at time intervals from the enzyme mixture and diluting into large volumes of substrate for assay (see Section 3.2 for details). 55 Tabic m Concentrations of conduritol aziridine 1 and corresponding [EO] /VJ values for irreversible inhibition of pABGS {S-glucosidase (0.011 mg/mL, SO - 200 uL) and yeast a-glucosidase (0.024 mg/mL, 100 - 200 uL).  a-Glucosidase P-Glucosidase [1] [Eol/Vi [1] [EoVvi (mM) (min) (mM) (min) 1.32 0.046 0.90 0.018 2.63 0.084 1.50 0.027 6.58 0.16 2.99 0.038 9.87 0.19 4.49 0.047 5.98 0.050 For both enzymes, an exponential loss of activity was observed indicating a pseudo-first order rate of inactivation (coefficient of correlation > 0.99). Complete inactivation of a-and P-glucosidases occurred within 20 and 60 min respectively. No reactivation was observed in the presence of a large excess of PNPG for either enzyme. A plot of [Eo]/vi versus 1/[TJ (Fig. 2 and 3) gives a straight line with an intercept on the [EQ] /VJ axis of 1/kj and an intercept of -1/Ki on the 1/JTJ axis. 56 [Eo]/vi 30" (min.) 0 H • 1 • 1 • 1 • 1 0.00 0.20 0.40 0.60 0.80 I/FT] (mM) Fig. 2. Inactivation of yeast a-glucosidase as a function of conduritol aziridine concentration. 10 H — ' — i — 1 — i — ' — i — 1 — i — ' — i — 1 — i -0.00 0.20 0.40 0.60 0.80 1.00 1.20 1/TJJ (mM) Fig. 3. Inactivation of pABG5 P-glucosidase as a function of conduritol aziridine concentration. The values of Kj and ki for the two enzymes were determined by computerized weighted linear regression86 and are listed in Table IV. 57 Table IV Dissociation constants (Kj) and inactivation rate constants (ki) at pH 6.8 for irreversible and reversible inhibition of pABG5 P-glucosidase at 37° and yeast a-glucosidase at 25° by analogues 1,24, and 31.  Compound cc-Glucosidase {3-Glucosidase Type of Ki kj Ki ki Inhibition (mM) (min"1) (mM) (min-1) Irreversible 1 9.5 0.39 3.0 0.077 Reversible 24 15 - 1.5 -31 8.5 - 0.55 -These data indicate that conduritol azMdine is an irreversible inhibitor for the a- and P-glucosidases tested. Further evidence of the conduritol aziridine entering the P-glucosidase active site was provided by a protection experiment. Addition of glucosyl benzene (a known reversible inhibitor of this enzyme) significantly lowered the rate of aziridine inhibition. The relatively low Kj value of 3.0 mM for the aziridine with P-glucosidase indicates an appreciable non-covalent binding and is about 38 times the pseudo-equilibrium constant (Km) value of 0.08 mM observed for PNPG. This difference in the binding may well be due to the absence of hydroxymethyl group in the structure of aziridine 1. Binding to the cc-glucosidase is equally good with a Kj value (9.5 mM) higher than the corresponding K m value (0.29 mM) by 33 times. This bmding to yeast a-glucosidase by an inositol analogue compares advantageously, for example, with the normal sugar methyl a-D-glucopyranoside which shows a Kj value of 30 mM for the same enzyme. Another case of glycosidase inactivation by an aziridine analogue was published earlier this year by B. Ganem et al.62 In this case, green coffee bean a-galactosidase was inactivated by the aziridinyltriol shown below: 58 HO OH The Kj and kj values for this compound were calculated to be 7.1 uM and 0.018 rnin"1 respectively. Evidence suggests that the enzyme was covalently inactivated through an ester linkage. freliminary testing of the aminomesylate 24 for irreversible inactivation indicated a slow covalent binding to p-glucosidase. The loss of enzyme activity was exponential and complete inactivation of the glucosidase was observed after 18 h. Even though the data accumulated here were not sufficient for accurate measurements of the Kj and ki values, they were evaluated to be in the 4 mM and 10"3 min-1 ranges respectively. The arninobenzoate 31 hydrochloride was also tested for irreversible inhibition but, as expected, showed no significant activity against P-glucosidase (incubation with 8.9 mM of analogue over 17 h). 24 and 31 were then tested as reversible (non-covalent) inhibitors. Reversible inhibition For competitive inhibition, the following process is assumed: Km E ES • E + P EI where S is substrate, ES is non-covalent enzyme-substrate complex, and P is product. The Michaelis-Menten equation is modified as follows: 59 v = Vm [S] [S] +Km(l+ Ifl) K; where v is initial velocity, V m is maximal initial velocity, and K m is the Michaelis constant; a pseudo-equilibrium constant for substrate binding. For competitive inhibition, the enzyme was incubated with a fixed concentration of substrate and varying concentrations of the analogues (Table V) and rates were (ktermined. Table V Concentrations of inositol analogues 24 and 31 for reversible inhibition of pABG5 P-glucosidase (0.00016 mg/mL, 37°) and yeast a-glucosidase (0.00034 mg/mL, 25°) at fixed concentrations of substrates p-PNPG (0.080 mM) and a-PNPG (0.20 a-Glucosidase pVGlucosidase [24] [31] [24] [31] (mM) (mM) (mM) (mM) 0 0 0 0 3.07 2.36 1.19 0.56 6.15 4.72 2.38 1.11 12.3 7.08 3.56 1.67 9.44 5.94 2.22 11.8 3.89 5.55 60 Initial reaction velocity was measured for each concentrations. Approximate values of i were obtained (Table IV) from Dixon plots (Fig 4,5,6 and 7), where [IJ = -Ki when 1/v 1/V m as shown by the arrows. -16 -11 - 6 - 1 4 9 14 P] (mM) Fig. 4. Dixon plot of the inhibition of a-glucosidase by 24. -10 -8 -6 -4 -2 0 2 4 6 8 10 12 [I] (mM) Fig. 5. Dixon plot of the inhibition of a-glucosidase by 31. 61 1/v 50" (min/mM) -2 -1 0 1 2 3 4 5 6 m (mM) Fig. 6. Dixon plot of the inhibition of P-glucosidase by 24. -1.5 -0.5 0.5 1.5 2.5 3.5 4.5 5.5 [TJ (mM) Fig. 7. Dixon plot of the inhibition of P-glucosidase by 31 62 The Ki values of 1.5 and 0.55 mM observed for analogues 24 and 31 respectively, indicates that their non-covalent interactions with pABG5 P-glucosidase are at least as important as in the conduritol aziridine case. The binding interactions with both analogues to yeast a-glucosidase are also significant. For comparison, the Ki of methyl a-D-glucopyranoside is 30 mM whereas myo-inositol showed no binding at all (at 30 mM) presumably at least partially due to the lack of a hydroxymethyl group. The azidobenzoate 31, in spite of its bulky O-benzoyl group, is not prevented from entering the active sites and binds as efficiently as 24 and 1. The nature of the slow covalent binding by aminomesylate 24 on the P-glucosidase was not investigated further due to limitations of time and material. There are two possible causes of such inactivation. Firsdy the mesyl group may be displaced by an enzyme nucleophile at the active site. A second possibility is the cyclisation of 24 at the active site resulting in the formation of conduritol aziridine, followed by enzyme inactivation as for compound 1. The mesylate anion, if liberated during incubation, has no inhibitory activity on the P-glucosidase (no inactivation observed by pure sodium mesylate at 23 mM). The affinity between the aminoinositols and the two glucosidases is likely to be due, in part, to the amino groups. Protonation of the amine by an enzyme carboxy lie acid at the active site would result in electrostatic interaction for the EI complex. To probe the importance of the amino group, the P-glucosidase was studied for inhibition by the azidomesylate 19. Unfortunately, the data obtained were not reliable, possibly due to some impurity. Three recrystallizations of the sample gave the same results. 63 On Conduritol Aziridine The concept, developed by E . Fischer, of complementary relationship applies not only to enzyme-substrate complexes but to enzyme-inhibitor complexes as well, thus, it is likely that only a single enantiomer of conduritol aziridine is capable of inhibition. For the corresponding epoxide, it was shown that the D-enantiomer* was responsible for 0-glucosidase inactivation (a-glucosidase inhibition was less efficient)10 whereas the L -enantiomer inhibited neither a- nor P-enzymes. Optical resolution of racemic conduritol aziridine would provide this kind of information and would also discard any possibility that the less potent enantiomer compete with the most potent one for the active site. Another improvement that could be made to conduritol aziridine is the substitution of the hydroxyl group at the 3-position (correspondingly, the 5-position in normal sugars) by a hydroxymethyl group, so as to mimic the substrate more efficiently and thereby improve the binding interactions in the non-covalent enzyme-inhibitor complex. This would enable syntheses of inhibitors with greater affinity and with greater specificity for anomeric configuration. *Structures of racemic conduritol epoxide and racemic conduritol aziridine (e.g. section 1.4 and 1.5) are represented in this work by the D-configuration. 64 OH NH OH OH On Enamine 25 The synthesis of enamine 25 was not completed due to a lack of time. Prior to its eventual reaction with a carbonyl compound, attempted deprotonation of the hydrochloride salt of amine 31 in mild aqueous conditions with the basic ion exchange resin AG 1 X8 (OH-form) apparently ended up in benzoate ester hydrolysis together with partial benzoyl transfer to the amino group ^H-n.m.r.). Such a problem might be solved by use of a less basic resin and/or non-aqueous conditions. The parent amine 24 hydrochloride which bears a mesylate group instead of the benzoate ester might also be a precursor to the enamine 25. In fact, 24 was deprotonated with potassium carbonate without epoxide or aziridine formation. 65 EXPERIMENTAL 66 3.1 Synthetic Methods Melting points were determined with a Fisher-Johns melting point apparatus and are uncorrected. Infrared spectra were recorded neat between NaCl plates or as KBr disks on either a Perkin-Elmer model 783 or a Nicolet 5DX FT-IR spectrophotometer. Absorption positions (D) are given in cm*1 units and are calibrated by means of the 1601 cm - 1 band of polystyrene. Proton magnetic resonance spectra were recorded either on a Varian XL-300 (300 MHz) or on a unit consisting of an Oxford instrument 63.4 K G superconducting magnet and a Nicolet 32 K computer (270 MHz), and shifts are reported in p.p.m. downfield from " tetramethylsilane used as an internal reference. Multiplicity, integrated area, coupling constants, and proton assignments (whenever possible) are indicated in parentheses. Mass spectra were recorded on either a modified Kratos MS 9 (low resolution), or a Kratos MS 50 (high resolution) or a Delsi-Nermag Rio-IOC Quadrupole (DQ) mass spectrometer. T.l.c. development: benzylated inositols were visualized under U V light and/or by spraying with a solution of sulfuric acid in methanol (10% v/v), followed by heating. Unprotected and acylated inositols were developed simply by exhaustive heating. Aminoinositol derivatives (including hydrochloride salts) were developed by spraying with a solution of ninhydrin (1 g/L in alcohol), followed by heating and showed pink or brown spots. Visualization of aziridines with ninhydrin required pre-treatment with hydrochloric acid. Elemental analyses were performed by Mr. P. Borda of the micToanalytical laboratory, U B C . Silica gel flash chromatography employed silica gel 60,230-400 mesh, as supplied by E . Merck Co. Thin-layer chromatography (t.l.c.) and preparative layer chromatography were carried out on aluminium and glass plates respectively, coated with silica gel 60 F-254 supplied by E.Merck Co. 67 Dry solvents and reagents where indicated were prepared as follows: acetonitrile and methylene chloride by distillation from phosphorus pentoxide; benzoyl chloride was distilled before use followed by storage over 3A molecular sieves; diethyl ether and tetrahydrofuran (THF) by refluxing over calcium hydride followed by distillation and storage over 3A molecular sieves; N^ V-dimethylformamide by distillation from 3A molecular sieves followed by storage over the same sieves; and pyridine by refluxing over barium oxide followed by distillation and storage over potassium hydroxide. Azides are potentially explosive compounds. They were therefore handled in small amounts and heat and light were avoided to prevent detonation. 68 1,4^,6-Tetra-O-benzyl-myo-inositol (5). OBzl Bzl< OH e 5 was prepared in three steps from myo-inositol according to the basic procedure of D. E. Kiely71: m.p. 113.5-114.5° (Lit. m.p. 114-115°); u K B r 3400 cnr* (OH); iH-n.m.r. data (270 MHz, CDC13): 8 730 (m, 20 H, Ar), 4.80 (m, 8 H, OCH2), 4.18 (br s, 1 H, H-2), 3.96 (t, 1 H, J 9.6 Hz, H-4), 3.81 (t, 1 H, J 9.6 Hz, H-6), 3.45 (m, 3 H, H-1,3,5), 2.52 (br s, 1 H, OH), 2.44 (d, 1 H, J 2.0 Hz, OH). 1,4^,6-Tetra-0-benzyl-3-0-toluemsulfonyl-myo-inositol (6). OBzl B z l C K ^ — ^ - ^ - O T s B z l c A ^ ^ - ^ A 6 was prepared according to the known method73: m.p. 116-117° (Lit. m.p. 115-117°); 1)^3520 (OH), 1360, and 1175 cnr* (SO2); ^-n.m.r. data (300 MHz, CDCI3): 8 7.77-7.02 (m, 24 H, Ar), 4.90-4.50 (m, 8 H, OCH2), 4.45 (m, 2 H, H-2,3), 4.04 (t, 1 H, J 8.8 Hz, H-4), 3.96 (t, 1 H, J 8.8 Hz, H-6), 3.47 (m, 2 H, H-1,5), 2.58 (s, 1 H, OH), 2.36 (s, 3 H, Tosyl CH3). 6 69 Acetylation of 1,4J,6-Tetra-0-benzyl-3-0-tosyl-myo-inositol (6). The alcohol 6 (0.50 g, 0.72 mmol) was treated with 4.0 mL of pyridine-acetic anhydride (1:1 v/v). After 24 hours, the mixture was poured into ice-water and extracted twice with chloroform. The combined organic extracts were washed twice with water, dried over magnesium sulfate and evaporated in vacuo. Recrystallization from hot methanol gave analytically pure acetylated 6 (0.43 g, 0.58 mmol, 81%); m.p. 117-119°; 1765 (OAc), 1375, and 1180 cnr* (SO2); iH-n.m.r. data (270 MHz, CDCI3): 8 7.78-7.15 (m, 24 H , Ar), 5.73 (t, 1 H , J 3.4 Hz, H-2), 4.88-4.38 (m, 8 H , O C H 2 ) , 4.51 (dd, 1 H , J 10.0, 3.4 Hz, H -3), 3.91 (t, 1 H , J 10.0 Hz, H-4), 3.79 (t, 1 H , J 10.0 Hz, H-6), 3.48 (m, 2 H , H-1,5), 2.37 (s, 3 H , Tosyl C H 3 ) , 2.10 (s, 3 H , OAc). Anal. Calc. for C43H44O9S: C, 70.09; H , 6.02. Found: C, 70.20; H , 6.07. 1 -azido-2,3,4£-tetra-0-benzyl-l -deoxy-chko-inositol (8). Method A: A mixture of 6 (0.50 g, 0.72 mmol) and sodium azide (0.20 g, 3.1 mmol) was refluxed in 5 mL of dry dimethylformamide for 5 h. After cooling, 20 mL each of diethyl ether and water were added. The aqueous phase was extracted with two additional 20 mL portions of ether and the combined extracts were washed with water, dried over magnesium sulfate, and evaporated. The products were separated by flash chromatography eluting with light petroleum ether-ethyl acetate (3:1) to give the oil 8 (20 mg, 0.035 mmol, 4.9%) and crystalline 2,4-dibenzyloxyphenol 9 (71 mg, 0.23 mmol, 32.2%, m.p. 94-96° L i t 93-9473). BzlO BzlO-8 70 Compound 8: Dneat 3430 (OH), and 2105 cm-1 (N3); lH-n.m.r. data (300 MHz, CDCI3): 8 7.32 (m, 20 H, Ar), 4.95-4.60 (m, 8 H, OCH2), 4.03 (m, 2 H), 3.93 (t, 1 H, J 2.9 Hz), 3.84 (t, 1 H, J 8.8 Hz), 3.75 (t, 1H, J 8.8 Hz), 3.67 (dd, 1 H, J 9.1, 2.9 Hz), 2.58 (s, 1 H, OH). Exact mass calc. for C34H35NO5 m/z 537.2516. Found: 537.2510. Method B: A solution of 6 (0.30 g, 0.43 mmol) and sodium azide (31 mg, 0.48 mmol) in 15 mL of dry dimethylformamide was stirred for 24 h at 135°. Extraction and purification as described in method A afforded 8 (66 mg, 0.12 mmol, 27%) and crystalline 2,4-dibenzyloxyphenol 9 (14 mg, 0.046 mmol, 10.6%) with n.m.r. and t.l.c. characteristics for 8 and 9 identical with those obtained by method A. 1A $ ,6-tetra-0-benzyl-2-0-methamsulfonyl-3-Q^ (10). Compound 6 (0.30 g, 0.43 mmol) was dissolved in anhydrous pyridine (5.0 mL) and methanesulfonyl chloride (0.25 g, 5 equiv.) was added. The solution was stirred for three days at room temperature then poured into ice-water, and extracted into ethyl acetate. The ethyl acetate layer was washed twice with water and dried over magnesium sulfate. Evaporation of the solvent and recrystallization from ether-light petroleum ether gave 10 as a white crystalline solid (0.22 g, 0.28 mmol, 66%); m.p. 135-136°; xpBr 1355 and 1180 cm"1 (SO2); 1H-n.m.r. data (300 MHz, CDCI3): 8 7.85-7.10 (m, 24 H, Ar), 5.45 (t, 1 H, J 3.0 Hz, H-2), 4.92-4.53 (m, 9 H, H-3, OCH2), 3.89 (t, 1 H, J 12.0 Hz, H-4), 3.85 (t, 1 H, J OBzl OMs 10 71 10.8 Hz, H-6), 3.55 (dd, 1 H, J 10.8, 3.0 Hz, H-l), 3.50 (t, 1 H, J 10.2 Hz, H-5), 3.08 (s, 3 H, Mesyl CH3), 2.38 (s, 3 H, Tosyl CH3). Anal. Calc. for C42H44O10S2: C, 65.27; H, 5.74. Found: C, 65.43; H, 5.75. 12-diazido-3,4J5,6-tetra-O-benzyl-l,2-dideoxy-myo-inositol (11). Compound 10 (10 mg, 0.013 mmol) and an excess of sodium azide (10 mg, 0.15 mmol) were stirred in 3.0 mL of dry dimethylformamide for 18 h at 85°, after which time no progress was shown by t.l.c. analysis. Thus, the temperature was raised to 100° and the solution stirred for 24 h. After cooling, the mixture was partitioned between diethyl ether and water, and the aqueous phase was extracted three times with ether. The combined organic layers were washed with water and dried over anhydrous magnesium sulfate and the solvent was removed in vacuo to give the white solid 11 (6 mg, 0.010 mmol, 79%); m.p. 117.5-118.5°; \)K B r 2100 cm-1 (N3); ^ -n.m.r. data (270 MHz, CDC13): 8 7.30 (m, 20 H, Ar), 4.70-4.90 (m, 8 H, OCH2), 3.95 (t, 1 H, J 3.2 Hz, H-2), 3.89 (t, 1 H, J 9.6 Hz, H-4), 3.79 (t, 1 H, J 9.6 Hz, H-6), 3.53 (dd, 1 H, J 9.6, 3.2 Hz, H-3), 3.45 (t, 1 H, J 9.6 Hz, H-5), 3.34 (dd, 1 H, J 10.3, 3.2 Hz, H-l). Anal. Calc. for C34H34N604: C, 69.14; H, 5.80; N, 14.23. Found: C, 69.35; H, 5.73; N, 14.30. Bzl< 11 72 1,4,5,6-tetra-Q-benzyl-2,3 -di-O-mettonesulfonyl-myo-inositol (12). OBzl Bzli OMs iMs 12 Methanesulfonyl chloride (6.36 g, 55 mmol) was added dropwise to a solution of 5 (3.0 g, 5.5 mmol) in 25 mL of dry pyridine. The reaction mixture was stirred at room temperature for three days, then poured onto ice-water and extracted with chloroform. The combined extracts were washed with water and dried over magnesium sulfate. Elution from silica gel with ethyl acetate and recrystallization in methanol yielded the white crystalline solid 12 (3.4 g, 4.8 mmol, 87%); m.p. 151-152°; xF*r 1360, and 1180 cm"1 (SO2); ^-n.m.r. data (270 MHz, CDCI3): 6 7.30 (m, 20 H, Ar), 5.37 (t, 1 H, J 2.7 Hz, H-2), 4.90-4.61 (m, 8 H, OCH2), 4.55 (dd, 1 H, J 9.9, 2,7 Hz, H-3), 3.94 (t, 1 H, J 9.9 Hz, H-4), 3.87 (t, 1 H, J 9.9 Hz, H-6), 3.54 (m, 2 H, H-1,5), 3.05 (s, 3 H, Mesyl CH3), 2.90 (s, 3 H, Mesyl CH3). Anal. Calc. for C36H40O10S2: C, 62.05; H, 5.79. Found: C, 62.12; H, 5.64. l-azido-2,3,4^-tetra-0-benzyl-l-deoxy-6-0-methanesulfonyl^ (13). A mixture of 12 (0.50 g, 0.72 mmol), sodium azide (61 mg, 0.94 mmol), and dry dimemylformaniide (25 mL) was heated to 85° for 24 h. After the solution had been cooled, OBzl BzlO 13 73 20 mL of ether was added, followed by 20 mL of water. The aqueous layer was separated and extracted twice with ether. The organic extracts were combined and washed with water, dried over anhydrous magnesium sulfate and then evaporated in vacuo to provide a mixture of three components, which was chromatographed on silica gel. Elution with light petroleum ether-ethyl acetate (5:1) afforded 13 as a white crystalline solid (0.28 g, 0.43 mmol, 61%); starting material (50 mg, 0.07 mmol, 10%); and diazido derivative 11 (80 mg, 0.14 mmol, 19%). Compound 13: m.p. 90-92°; \)KB r 2115 (N3), 1360 and 1180 cm"1 (S02); ^ -n.m.r. data (300 MHz, CDCI3): 8 7.32 (m, 20 H, Ar), 4.88 (m, 8 H, OCH2), 4.43 (t, 1 H, J 6.6 Hz, H-6), 3.55 (m, 5 H, H-1,2,3,4,5), 3.02 (s, 3 H, Mesyl CH3). Anal. Calc. for C35H37N3O7S: C, 65.30; H, 5.79; N, 6.53. Found: C, 65.45; H, 5.75; N, 6.61. 12-dideoxy-3,4£,6-tetra-0-benzyl-l,2-epimino-vayo-inositol (14). Method A: A solution of 13 (0.10 g, 0.16 mmol) in ether (5 mL) was added dropwise to a stirred suspension of LiAlH4 (0.10 g, 2.63 mmol) in anhydrous ether at -78°. Stirring was continued for 1 h at -78°, and then for 24 h at room temperature. The excess hydride was decomposed by addition of water and the solution filtered and concentrated. Elution of the crude product from silica gel with ethyl acetate afforded colorless crystals of 14 (50 mg, 0.096 mmol, 62%); an analytical sample was prepared by recrystallization from methanol; m.p. 105-106°; 3305 cm-1 (NH); lH-n.nu. data (300 MHz, CDCI3): 8 7.32 (m, 20 H, Ar), 4.78 (m, 8 H, OCH2), 3.85 (m, 2 H), 3.62 (t, 1 H, J 9.8 Hz), 3.45 (t, 1 H, J 9.0 Hz), OBzl OBzl 14 74 2.49 (dd, 1 H, H-2), 2.32 (d, 1 H, H-l), 0.73 (br s, 1 H, NH); (CDC13 + D20): 8 2.49 (dd, 1 H, J 6.0, 3.4 Hz, H-2), 2.32 (d, 1 H, J"i,2 6.0 Hz, H-l). Anal. Calc. for C34H35NO4: C, 78.28; H, 6.76; N, 2.69. Found: C, 77.72; H, 6.84; N, 2.49. Exact mass calc. for C34H35NO4 mlz 521.2557. Found: 521.2567. Method B: A mixture of 13 (50 mg, 0.078 mmol) and triphenylphosphine (28 mg, 0.11 mmol) in 1.0 mL of dry tetrahydrofuran was stirred at room temperature for 24 h. Cone, ammonium hydroxide was added, the mixture was stirred for 5 days, then concentrated and eluted from silica gel with light petroleum ether-ethyl acetate (1:1) to afford crystalline 14 (28 mg, 0.054 mmol, 69%); with m.p., n.m.r., and t.l.c. characteristics identical with those of a sample of 14 obtained by method A. 1,4^,6-tetra-O-acetyl-myo-inositol (15). 15 was prepared in three steps from myo-inositol according to the known method70: m.p. 122-1230 (Lit. m.p. 118°); D K B r 3465 (OH), and 1750 cm.-* (OAc); lH-n.m.r. data (270 MHz, CDCI3): 8 5.56 (t, 1 H, J 10.0 Hz, H-6), 5.35 (t, 1 H, J 10.0 Hz, H-4), 5.11 (t, 1 H, J 10.0 Hz, H-5), 4.93 (dd, 1 H, J 10.0, 3.0 Hz, H-l), 4.26 (br s, 1 H, H-2), 3.74 (br d, 1 H, J 10.0 Hz, H-3), 3.47 (br s, 1 H,OH), 3.25 (br s, 1 H, OH), 2.08, 2.06, and 1.99 (3 s, 3,3„ and 6 H, 4 OAc). OAc 15 OH 75 l,4^,6-tetra-0-acetyl-2,3-di-0-methanesulfonyl-myo-inositol (16). OAc OMs 16 OMs A solution of 15 (2.0 g, 5.7 mmol) and methanesulfonyl chloride (3.3 g, 28.8 mmol) was stirred at room temperature for 18 h. The mixture was then treated in the manner described for 12 and recrystallized in absolute ethanol to yield 16 (2.1 g, 4.2 mmol, 73%); m.p. 215-2170 (Lit.8? 211-213°); x>™ 1755 (OAc), 1360 and 1180 cm-1 (S02); iH-n.m.r. data (270 MHz, CDCI3): 8 5.50 (t, 1 H, J 10.2 Hz, H-4), 5.49 (t, 1 H, J 10.7 Hz, H-6), 5.34 (t, 1 H, J 2.9 Hz, H-2), 5.22 (t, 1 H, J 9.9 Hz, H-5), 5.12 (dd, 1 H, J 10.7, 2.9 Hz, H-3), 4.97 (dd, 1 H, J 10.2, 2.9 Hz, H-l), 3.21 (s, 3 H, Mesyl CH3), 3.12 (s, 3 H, Mesyl CH3), 2.10, 2.08, 2.02, and 2.01 (4 s, 3,3,3, and 3 H, 4 OAc). 12-di-O-methanesulfonyl-myo-inositol (17). Method A: From hydrogenation of 12. To a solution of 12 (0.33 g, 0.47 mmol) in 60 mL of methanol-ethyl acetate (2:1) was added 100 mg of Pd/C 10%. The mixture was hydrogenated for 24 h at atmospheric pressure. The catalyst was filtered off and the filtrate was evaporated to dryness. The solid residue was recrystallized from hot methanol to yield white crystalline 17 (0.14 g, 0.42 mmol, 88%) ; m.p. 207-209° (dec.); u^r 346O (OH), 1335 and 1170 cm-1 (SO2); 1H-n.m.r. data (270 MHz, D20): 8 5.22 (t, 1 H, J 3.0 Hz, H-2), OH 17 OMs 76 4.74 (dd, 1 H, J 10.0, 3.0 Hz, H-l), 3.81 (dd, 1 H, J 10.0, 3.0 Hz, H-3), 3.79 (t, 1 H, J 9.6 Hz, H-6), 3.61 (t, 1 H, J 9.6 Hz, H-4), 3.19 (t, 1 H, J 9.6 Hz, H-5), 3.30 (s, 3 H, Mesyl CH3), 3.25 (s, 3 H, Mesyl CH3). Anal. Calc. for C8Hi6OioS2: C, 28.57; H, 4.80. Found: C, 28.79; H, 4.95. Method B: From deacylation of 16. Compound 16 (2.4 g, 4.76 mmol) was dissolved in 50 mL of n-butylamine and the solution was stirred for 18 h at room temperature. The reaction mixture was evaporated and the residue was stirred one more hour in 30 mL of fresh amine. The solution was concentrated and the product was recrystallized from hot methanol to afford crystalline 17 (1.31 g, 3.89 mmol, 82%) with n.m.r. and t.l.c. characteristics identical with those obtained by method A. Azidolysis of 1,2-di-O-methanesulfonyl-myo-inositol (17). The dimesylate 17 (1.0 g, 3.0 mmol) was stirred with sodium azide (0.31 g, 4.7 mmol) in 25 mL of dry cUmethylformamide at 95°. After 48 h, the solution was concentrated in vacuo and 20 mL of acetic anhydride-pyridine (1:1) was added. The mixture was stirred 24 h, and poured onto ice-water. The aqueous solution was extracted with chloroform, and the organics were washed with water and dried over magnesium sulfate. The solution was evaporated, the residue redissolved in toluene and evaporated again. Separation by flash chromatography on silica gel (light petroleum ether-ethyl acetate 2:1) afforded crystalline 18 and 20, and the oil 21, with the following m.p. and spectroscopic data: 77 2,3,4£-tetra-0-acetyl-l-azido-l-deoxy-6-0-methanesrf (18). OAc A c C K ^ A ^ - ^ ' O M s A c O A ^ - ^ ^ \ ^ N 3 OAc 18 (0.19 g, 0.42 mmol, 14%); m.p.l74-176°; t^Br 2120 (N3), 1760 (OAc), 1370 and 1230 cnr 1 (S02); ^-luxur. data (300 Mhz, CDC13): 8 5.22 (m, 4 H, H-2,3,4,5), 4.67 (t, 1 H, J 10.8 Hz, H-6), 3.80 (t, 1 H, J 10.2 Hz, H-l), 3.17 (s, 3 H, Mesyl CH3), 2.13, 2.11, 2.02, and 2.01 (4 s, 3,3,3, and 3 H, 4 OAc). Exact mass calc. for C15H22N3O1 i S mlz 452.0976. Found: 452.0970. 3,4,5,6-Tetra-O-acetyl-l,2-diazido-l2-dideoxy-myo-inositol (20). (0.14 g, 0.35 mmol, 12%); m.p. 133-134°; vKBr 2120 (N3), and 1750 cm-1 (OAc); *H-n.m.r. data (300 MHz, CDCI3): 8 5.48 (t, 1 H, J 10.4 Hz, H-4), 5.38 (t, 1 H, J 10.4 Hz, H-6), 5.06 (t, 1 H, J 10.4 Hz, H-5), 4.96 (dd, 1 H, J 10.4, 4.1 Hz, H-3), 4.20 (t, 1 H, J 4.1 Hz, H-2), 3.69 (dd, 1 H, J 10.4, 4.1 Hz, H-l), 2.11, 2.07, and 1.99 (3 s, 3,3, and 6 H, 4 OAc). Exact mass calc. for C14H19N6O8 mlz 399.1266. Found: 399.1257. 78 2,3,4,6-tetra-O-acetyl-lJ5-diazido-l^-dideoxy-chiro-inositol (21). OAc A c O - " ^ - ^ \ AcO^^Nf-^-A^ OAc 21 (0.12 g, 0.30 mmol, 10%); u n e a t 2115 (N3), and 1755 cm"1 (OAc); ^-n.m.r. data (300 MHz, CDC13): 8 5.48-5.23 (m, 4 H, H-2,3,4,6), 4.13 (t, 1 H, J 4.2 Hz, H-l), 3.82 (dd, 1 H, J 9.6, 4.2 Hz, H-5), 2.19, 2.10, and 2.03 (3 s, 3,6, and 3 H, 4 OAc). Exact mass calc. for C14H19N6O8 m/z 399.1266. Found: 399.1253. 1 -Azido-l-deoxy-2-O-methanesulfonyl-scyllo-inositol (19). Compound 18 (0.41 g, 0.91 mmol) was dissolved in 20 mL of n-butylamine and stirred overnight at room temperature. The solvent was evaporated in vacuo, 10 mL of fresh n-butylamine was added and the solution evaporated again. Chloroform (10 mL) was added to the oil and the resulting solid was filtered. Recrystallization in chloroform-methanol 20:1 gave 19 (0.18 g, 0.64 mmol, 70%); m.p. 156-157°; xP&x 3380 (OH), 2120 (N3), 1175, and 1340 cm-l ( S O 2 ) ; ^-n.mx data (300 MHz, D20): 8 4.44 (t, 1 H, J 10.2 Hz, H-2), 3.69 (t, 1 H, J 10.2 Hz), 3.63 (t, 1 H, J 10.2 Hz), 3.42 (m, 3 H), 3.31 (s, 3 H, Mesyl CH3). OH OH 19 79 Anal. Calc. for C7H13N3O7S: C, 29.68; H, 4.63; N, 14.83. Found: C, 29.60; H, 4.80; N, 15.00. l,4£,6-Tetra-0-benzoyl-2,3- a^-O-methanesulfonyl-myo-inositol (22). Compound 17 (0.25 g, 0.74 mmol) was dissolved in 5 mL of dry pyridine and benzoyl chloride (0.83 g, 5.9 mmol) was added over a period of 5 min. The mixture was heated to 70° for 0.5 h and, after cooling, poured onto ice-water and the product extracted into chloroform. The organic extracts were washed successively with hydrochloric acid 5M, aqueous sodium bicarbonate, and water. The solution was dried over magnesium sulfate and the solvent evaporated. Recrystallization from methanol gave 22 (0.45 g, 0.60 mmol, 81%); m.p. 243-245°; \)K B r 1740 (OCOPh), 1360, and 1180 cm"1 (S02); ^-n.m.r. data (300 MHz, CDCI3): 8 8.03-7.25 (m, 20 H, Ar), 6.20 (t, 1 H, J 9.0 Hz), 6.11 (t, 1 H, J 9.0 Hz), 5.92 (t, 1 H, 9.0 Hz), 5.43 (m, 2 H), 5.30 (dd, 1 H, J 9.0, 2.3 Hz), 3.22 (s, 3 H, Mesyl CH3), 2.98 (s, 3 H, Mesyl CH3). Anal. Calc. for C36H32O14S2: C, 57.44; H, 4.28. Found: C, 57.63; H, 4.49. OBz 22 80 23,4J-Tetra-O-benzoyl-l-azido-l-deoxy-6-0-metnanesulfonyl-scy\\o-inositol (23). OBz BzO-*V—V.—-~V-OMs BzO-X^^Y-A-^Nj OBz 23 Compound 19 (10 mg, 0.035 mmol) was dissolved in 2 mL of dry pyridine-benzoyl chloride (1:1) and stirred at room temperature for 24 h. The product was worked up as described for 22 and recrystallization in light petroleum ether-chloroform (10:1) yielded 23 (18 mg, 0.026 mmol, 73%); m.p. 230-235° (dec); u™' 2120 (N3), 1730 (OCOPh), 1365, and 1180 (S02); iH-n.mx data (300 MHz, CHCI3): 8 8.23-7.25 (m, 20 H, Ar), 5.87 (m, 3 H), 5.71 (t, 1 H, J 10.1 Hz), 5.02 (t, 1 H, J 10.1 Hz), 4.14 (t, 1 H, J 10.1 Hz). J -amino-1-deoxy-2-O-methanesulfonyl-scyllo-inositol (24)hydrochloride . OH HO—^-\^-*V -OMS HO~\^---^\^NH2 • HC1 OH 24 A mixture of 13 (1.0 g, 1.55 mmol) and Pd/C 10% (0.30 g) in ethanol (50 mL), chloroform (25 mL) and 5M hydrochloric acid (3.5 mL) was hydrogenated for 48 h at atmospheric pressure. The catalyst was filtered off and washed with ethanol. The solution was concentrated in vacuo and the resulting oil was crystallized from methanol-chloroform (1:1) to give white hygroscopic 24 (0.30 g, 0.99 mmol, 64%); m.p. 143-145°; u*811345 and 1180 cm"1 (SO2); ^-n.m.r. data (300 MHz, D2O): 8 4.68 (t, 1 H, J 10.2 Hz, H-2), 3.69 (m, 1 H), 3.55 (m, 1 H), 3.42 (m, 3 H), 3.31 (s, 3 H Mesyl CH3). 81 Anal. Calc. for C7Hi6ClN07S 0.5 H20: C, 27.77; H, 5.66; N, 4.63. Found : C, 27.89; H, 5.68; N, 4.66. 1,2 -Dideoxy-12 -epimino-myo-inositol (1). Compound 24 (50 mg, 0.17 mmol) was dissolved in 2 mL of water and triethanolamine 1M was added drop wise until the mixture reached pH 9 as determined using a pH meter. The solution was stirred at 55-60° for 24 h. The solvent was then evaporated in vacuo and the less polar compound (Rf 0.63) was separated by preparative chromatography on a silica gel plate (isopropanol-water 3:1). The product was then filtered over a short column of silica gel. Elution with methanol yielded 1 as a gum (2 mg, 0.019 mmol, 7%); D K B r 3480 cm-1 (OH); ^-n.m.r. data (300 MHz, D20): 8 3.88 (m, 1 H, H-3), 3.68 (m, 1 H, H-5), 3.20 (m, 2 H, H-4,6), 2.61 (dd, 1 H, J 6.2, 3.3 Hz, H-2), 2.34 (d, 1 H, J 6.2 Hz, mlz (rel. intensity) 160 (M+-1,100), 196 (32), 198 (24), 321 (66). 1,4£,6-tetra-0-benzyl-3-0-toluenesulfonyl-myo-inosose-2 (26). OBzl BzlO—^A ^ -OTs BzlO^\^-—-^\ OBzl^ o 26 To a stirred suspension of pyridinium dichromate (0.35 g, 0.93 mmol) and acetic OH OH 1 H-l). 82 anhydride (0.28 g, 2.74 mmol) in 10 mL of dry dichloromethane, a solution of the alcohol 6 (0.55 g, 0.79 mmol) in 5 mL of dichloromethane was added. The mixture was refluxed for 24 h and after cooling, 10 mL of ethyl acetate was added. The precipitate was filtered on a short column of silica gel and the dissolved product eluted with ethyl acetate. Further purification by flash chromatography (light petroleum-ethyl acetate 5:1) gave white crystals of 26 (0.22 g, 0.32 mmol, 40%); m.p. 135-136°; u K B r 1750 (CO), 1360, and 1180 cm"1 (S02); iH-n.m.r. data (300 MHz, CDC13): 8 7.88-7.08 (m, 24 H, Ar), 5.18 (dd, 1 H, J 10.3, 1.0 Hz, H-3), 4.92-4.45 (m, 8 H, OCH2), 4.21 (dd, 1 H, J 10.3, 1.0 Hz, H-l), 3.85 (t, 1 H, J 9.0 Hz), 3.61 (t, 1 H, J 9.8 Hz), 3.60 (t, 1 H, J 9.8 Hz). Anal. Calc. for QiILjoOgS: C, 71.08; H, 5.82. Found: C, 71.09; H, 5.92. 3,4£,6-tetra-Q-benzyl-2 -cyclohexene-1 -one (27). OBzl Method A: To a solution of 26 (20 mg, 0.029 mmol) and dimethylamine hydrochloride (14 mg, 0.17 mmol) in 3 mL of methanol-ether 3:1 was added sodium cyanoborohydride (2 mg, 0.032 mmol). The solution was stirred for 72 h at room temperature. Aqueous 5N hydrochloric acid was added to drop the pH below a value of 2 (pH paper) and the solvent was removed in vacuo. Water (5 mL) was added and the solution was extracted with ether. The aqueous phase was brought to pH 10 or higher with solid potassium hydroxide and extracted again with ether. The organic extracts were dried over magnesium sulfate and evaporated in vacuo. Chromatography on silica gel (light petroleum ether- ethyl acetate 3:1) gave the oil 27 (6 mg, 0.012 mmol, 40%); u n e a t 1675 (CO), and BzlO-BzlO-27 83 1640 cm-l (C=C); *H-n.m.r. data (270 MHz, CDCI3): 8 7.35 (m, 20 H), 5.98 (s, 1 H), 5.50 (d, 1 H, J 12.0 Hz), 5.37 (d, 1 H, J 11.6 Hz), 4.70-4.45 (m, 4 H), 4.24 (d, 1 H, J 3.6 Hz), 3.90 (dd, 1 H, J 4.4, 4.4 Hz), 2.91 (dd, 1 H, J 17.2, 4.0 Hz), 2.65 (dd, 1 H, J 16.8, 4.0 Hz). Exact mass calc. for C34H32O5: mlz 520.2251. Found: 520.2254. Method B: The ketone 26 (50 mg, 0.071 mmol) and triethylamine (20 mg, 0.20 mmol) were stirred in dry acetonitrile until the ketone was completely consumed (t.l.c). The pink solution was concentrated and chromatographed on silica gel flight petroleum ether-ethyl acetate 3:1) to give the oil 27 (17 mg, 0.033 mmol, 47%) with t.l.c. and n.m.r. characteristics as in Method A. 1 -0-benzoyl-3,4J,6-tetra-0-benzyl-myo-inositol (28). OBzl BzlO-^V-^^^^OCOPh B z l O . \ ^ ^ ^ \ BzlO I OH 28 28 was prepared by benzoylation of 5 according to the known method84 : m.p. 147-148° (Lit. m.p. 144-145°); 3520 (OH), and 1718 cm"1 (OCOPh); ^ -n.m.r. data (270 MHz, CDCI3): 8 8.08-7.08 (m, 25 H, Ar), 5.12 (dd, 1 H, J 10.0, 2.0 Hz, H-l), 4.90-4.66 (m, 8 H, OCH2), 4.40 (br s, 1 H, H-2), 4.24 (t, 1 H, J 10.0 Hz, H-6), 4.01 (t, 1 H, J 10.0 Hz, H-4), 3.61 (dd, 1 H, J 10.0, 2.0 Hz, H-3), 3.59 (t, 1 H, J 10.0 Hz, H-5), 2.52 (br s, 1 H, OH). 84 /-0-benzoyl-3,4J,6-tetra-0-benzyl-2 -O-methanesulfonyl-myo-inositol (29). OBzl BzlC)---**V-A----**^OCOPh BzlO 1 OMs 29 Method A: From 5. To a solution of 5 (3.54 g, 6.55 mmol) in 10 mL of dry pyridine at 0° was added benzoyl chloride (0.93 g, 6.61 mmol). The mixture was allowed to warm to room temperature and stirred for 24 h, a further 0.05 g of benzoyl chloride was then added and the solution stirred for another 24 h. Methanesulfonyl chloride (1.50 g, 13.1 mmol) was added directly to this mixture and, after 48 h, the mixture was poured into ice-water and extracted twice with chloroform. The combined organic extracts were washed with water, dried over magnesium sulfate and concentrated to dryness, taken up in toluene and again concentrated to dryness. Recrystallization from hot methanol afforded 29 (3.3 g, 4.57 mmol, 69%); m.p. 131-132°; u^r 1725 (OCOPh), 1360 and 1180cm-l ( S O 2 ) ; ^-n.m.r. data (270 MHz, CDCI3): 5 8.09-7.08 (m, 25 H, Ar), 5.38 (t, 1 H, J 3.2 Hz, H-2), 5.18 (dd, 1 H, J 9.6, 3.2 Hz, H-l), 4.90-4.68 (m, 8 H, OCH2), 4.12 (t, 1 H, J 9.6 Hz, H-6), 3.93 (t, 1 H, J 9.6 Hz, H-4), 3.69 (dd, 1 H, J 9.6, 3.2 Hz, H-3), 3.63 (t, 1 H, J 9.6 Hz, H-5), 2.95 (s, 3 H, Mesyl CH3). Anal. Calc. for C42H42O9S: C, 69.79; H, 5.86. Found: C, 69.68; H, 5.90. Method B: From 28. A solution containing 28 (100 mg, 0.16 mmol) and methanesulfonyl chloride (50 mg, 0.44 mmol) was stirred at room temperature for 48 h. Extraction and recrystallization as in method A yielded 29 (85 mg, 0.12 mmol, 76%) with m.p., n.m.r. and t.l.c. identical with those of method A. 85 1 -azido-2-0-benzoyl-3,4£,6-tetra-O-benzyl-l-deoxy-scyUo-inositol (30). OBzl Bzl< OCOPh 30 A mixture of the ester 29 (1.20 g, 1.66 mmol) and sodium azide (0.22 g, 3.38 mmol) in dry dimemylformamide was stirred at 90° for 24 h. After cooling, 20 mL each of water and chloroform were added and the two phases were separated. The aqueous phase was extracted twice more with chloroform and the combined organic extracts were washed twice with water, dried over magnesium sulfate and the solvent was evaporated. Recrystallization from ethanol afforded 30 (0.91 g, 1.35 mmol, 81%); m.p. 144.5-145.5°; u K B r 2105 (N3), and 1730 cm-1 (OCOPh); 1H-n.m.r. data (270 MHz, CDCI3): 8 8.04-7.00 (m, 25 H, Ar), 5.22 (t, 1 H, J 10.4 Hz, H-2), 5.00-4.56 (m, 8 H, OCH2), 3.67-3.49 (m, 5 H, H-1,3,4,5,6). Anal. Calc. for C41H39N3O6: C, 73.52; H, 5.87; N, 6.27. Found: C, 73.50; H, 5.78; N, 6.43. l-Amino-2-O-benzoyl-l-deoxy-scyWo-inositol (31) Hydrochloride. OH HO OH 31 86 Compound 30 (0.10 g, 0.15 mmol) was dissolved in a mixture of 3 mL of chloroform, 4 mL of ethanol, and 0.3 mL of 5M hydrochloric acid. It was then hydrogenated at atmospheric pressure over Pd/C 10% (40 mg) for 48 h. After filtration, the solution was concentrated in vacuo to provide crystals of 31 (30 mg, 0.091 mmol, 61%); m.p. 233-235° (dec.); i)KB' 3380 (OH), 3280 (NH), and 1725 cm"1 (OCOPh); ^-n.m.r. data (300 MHz, D20): 5 8.12-7.50 (m, 5 H, Ar), 5.23 (t, 1 H, J 9.4 Hz, H-2), 3.72 (m, 1 H), 3.62 (m, 1 H), 3.46 (m, 3 H). Anal. Calc. for Ci3Hi7NO6.HC1.0.5 H2Or C, 47.50; H, 5.83; N, 4.26. Found: C, 47.74; H, 5.80; N, 4.12. l-O-benzoyl-2-O-methanesulfonyl-myo-inositol (33). OH OHI OMs 33 The ester 29 (0.20 g, 0.28 mmol) was dissolved in 50 mL of methanol-ethyl acetate (2:1), Pd/C 10% (0.10 g) added, and the mixture hydrogenated at atmospheric pressure for 3 days. The catalyst was filtered off and washed with methanol (5 mL), the filtrate evaporated and the residue recrystallized from chloroform-methanol (10:1) to give white crystalline 33 (88 mg, 0.24 mmol, 88%); m.p. 201-203° (dec.); v^r 1710 (OCOPh), 1345 and 1180 cnr* (SO2); ^-n.m.r. data (300 MHz, DMSO-6D): 8 8.03-7.48 (m, 5 H, Ar), 5.61 (d, 1 H, J 4.5 Hz), 5.36 (d, 1 H, J 5.5 Hz), 5.11 (m, 2 H), 4.92 (m, 2 H), 3.14 (s, 3 H, Mesyl CH3). Anal. Calc. for C14H18O9S: C, 46.41; H, 5.01. Found: C, 45.74; H, 5.13. 67 / -Benzamido-2£,4£-tetra-0-benzyl-l -deoxy-scyWo-inositol (35). OBzl BzlO-^-X^-^Y^OH ° BzlO^^---Y-\^NfHCC 6H 5 OBzl 35 Compound 30 (0.10 g, 0.15 mmol) and triphenylphosphine (50 mg, 0.19 mmol) were dissolved in 10 mL of THF-H2O (7:3) and the mixture was refluxed for 48 h. After cooling, the solution was evaporated in vacuo and chromatographed on silica gel (ethyl acetate-light petroleum ether 1:1) to give crystalline 35 (41 mg, 0.064 mmol, 43%); m.p. 226-227°; uKBr 3420 (OH), 3300 (NH), and 1640 cnr* (CON); lH-n.m.r. data (300 MHz, CDCI3): 8 7.52-7.18 (m, 25 H, Ar), 6.14 (d, 1 H, J 6.7 Hz, CONH), 4.92-4.65 (m, 8 H, OCH2), 3.97 (m, 1 H, H-l), 3.82-3.52 (m, 5 H, H-2,3,4,5,6). Anal. Calc. for C41H41NO6: C, 76.49; H, 6.42; N, 2.18. Found: C,76.23; H, 6.49; N, 2.10. 88 3.2 Enzvmological Methods Chemicals Inositol derivatives 19,24, and 31 were recrystallized prior to enzyme assays and their purity was confirmed by microanalysis. The substrates p-nitrophenyl a - D -glucopyranoside (a-PNPG) and p-nitrophenyl p*-D-glucopyranoside (P-PNPG) were purchased from Sigma Chemical Co. Phosphate buffer (50 mM, pH 6.8) was prepared by mixing the correct ratio of monobasic and dibasic sodium phosphates. Enzymes a-Glucosidase Type HI from yeast was purchased from Sigma Chemical Co. as a suspension in 3.2 M ammonium sulfate solution containing approximately 25% BSA. The mixture was dialysed in 50 mM phosphate buffer before use. The P-glucosidase used is an enzyme originally isolated from Alcaligenes faecalis and since cloned into E. Coli. This enzyme, referred to as pABG5 p-glucosidase, was isolated from this organism in this laboratory essentially as described previously.88 Enzyme assays Al l of the kinetic experiments were conducted in collaboration with Ms Karen Rupitz. Absorbance measurements were carried out on a Pye Unicam PU-8800 UV/Visible spectrophotometer thermostated with a Julabo F-10 circulating bath. Measurements of pH were carried out on a Radiometer PHM pH meter. Assays of enzyme activity were performed by measuring the rate of release of p-89 nitrophenol from the nitrophenyl glucopyranoside substrate and by monitoring change in absorbance at 400 nm of reaction mixtures (coefficient of absorption 7280 M^cnr 1 ). Reactions with a-glucosidase were performed at 25° with a-PNPG and those with P-glucosidase at 37° with P-PNPG. All reactions were carried out in 50 mM phosphate buffer at pH 6.8. The inositol analogues were tested for both irreversible and reversible inhibition of pABG5 P-glucosidase and yeast a-glucosidase as follows: Irreversible inhibition The enzyme (50-200 uL, 0.011 mg/mL for p-glucosidase, and 100-200 uL, 0.024 mg/mL for a-glucosidase) was incubated at the appropriate temperature in the presence of the analogue at the concentration indicated in Table in. Aliquots (10 \iL) of the enzyme mixture were removed at time intervals and assayed for activity by dilution into a large volume (1.0 mL) of substrate PNPG (1.1 mM for P-glucosidase, and 2.8 mM for a-glucosidase) and residual activity thereby determined directly. Decreases in enzyme activity were analyzed by computer fitting the residual activities to a single exponential curve giving values for the pseudo-first order rate constants for inactivation at each concentration. A replot of reciprocal inactivation rate constant versus reciprocal inhibitor concentration yielded values for the dissociation constant Ki and the inactivation rate constant ki. Reversible inhibition Those compounds showing no covalent inhibition were tested as reversible (non-covalent) inhibitors as follows. 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Day, and S. G. Withers, Biochem. Cell. Biol, 64 (1986) 914. 97 SPECTRAL INDEX 58 Conduritol Aziridine (l^-dideoxy-l^-epimino-m>o-inositol, 1) 99 100 101 l-Airanc^ 2-0-bcnzoyl-l-o^ xy-5cy//o-jnositol (31) Hydrochloride 

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