9.2 Carbasugars: Synthesis and Functions

Yoshiyuki Kobayashi Daiichi Sankyo Research Institute, 4250 Executive Square, La Jolla, California 92037, USA [email protected]

1 Introduction ...... 1915 2Overview...... 1915 3 Synthesis of Carbasugars: New Generations of Glycosidase Inhibitors . . . . . 1916 3.1 New Generations of Glycosidase Inhibitors Bearing 6-Membered Cyclitol . . . . . 1916 3.1.1 Cyclophelitol: A β-Glucosidase Inhibitor...... 1916 3.1.2 Tamiflu: A Neuraminidase Inhibitor ...... 1933 3.2 New Generations of Glycosidase Inhibitors Bearing 5-Membered Cyclitols . . . . 1951 3.2.1 Allosamizoline: The Aglycone of the Chitinase-Specific Glycosidase Inhibitor, Allosamidin...... 1952 3.2.2 Trehazolin, Trehalamine and Its Aminocyclitol Moiety, Trehazolamine: A Trehalase-Specific Glycosidase Inhibitor ...... 1964 4 New Methods for Conversion of Sugars to Carbasugars ...... 1982 4.1 Carbasugar Formation via SnCl4-Promoted Intramolecular Aldol Condensation 1982 4.2 SmI2-Mediated Carbasugar Formation ...... 1983 4.2.1 Cyclization between Carbonyl Compounds and α, β-Unsaturated Esters ...... 1985 4.2.2 Cyclization between Carbonyl Compounds and Simple Olefins ...... 1986 4.2.3 Cyclization between Carbonyl Compounds and Oximes ...... 1987 4.3 SmI2-Mediated Pinacol Coupling ...... 1988 4.4 SmI2- or Zirconium-Mediated Ring Contraction of Hexapyranoside Derivatives to 5-Membered Carbosugar...... 1988 5Conclusion...... 1992

Abstract It is well recognized that glycosidase inhibitors are not only tools to elucidate the mechanism of a living system manipulated by glycoconjugates but also potential clinical drugs and insecticides by inducing the failure of glycoconjugates to perform their function. In this chapter, the syntheses and functions of natural glycosidase inhibitors (cyclophelitol, allosamidine, and tre- hazoilin), which possess highly oxygenated and functionalized cyclohexanes or cyclopentanes in their structures and are deﬁned as carbasugars, and the structure and activity relationships (SAR) of their derivatives are described. Also, recently much attention has been focused on

In: Glycoscience. Fraser-Reid B, Tatsuta K, Thiem J (eds) Chapter-DOI 10-1007/978-3-540-30429-6_49: © Springer-Verlag Berlin Heidelberg 2008 1914 9 Glycomimetics neuraminidase inhibitors as anti-influenza drugs since relenza, which was derived from sialic acid, and also, tamiflu, which is the artificial carbasugar designed as a transition state analogue in the hydrolysis pathway of substrates by neuraminidase, were launched in the market. Here- in, the medicinal chemistry efforts to discover tamiflu and some efficient syntheses applicable to process chemistry are described. Finally, useful synthetic methodologies for carbasugar formation from sugars are also introduced in this chapter.

Keywords Carbasugars; Cyclitols; Glycosidase inhibitors; Cyclophelitol; Allosamidin; Trehazolin; Neuraminidase inhibitor; Tamiﬂu; Intramolecular [3+2] cycloaddition; The Ferrier reaction

Abbreviations CDMA catalytic desymmetrization of meso-aziridine Chx cyclohexyl DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DDMPO diisobutylaluminum 2,6-di-tert-butyl-4-methoxyphenoxide DEIPS diethylisopropylsilyl DIBALH diisobutylaluminum hydride DIPEA diisopropylethylamine DMAP N, N-dimethylaminopyridine DMF N, N-dimethylformamide 2,2-DMP 2,2-dimethoxypropane DMSO dimethylsulfoxide HA hemagglutinin HPLC high pressure liquid chromatography KHMDS potassium (1,1,1,3,3,3)-hexamethyldisilazide mCPBA m-chloroperbenzoic acid MOM methoxymethyl NA neuraminidase NBA N-bromoacetamide NBS N-bromosucciimide NIS N-iodosucciimide NMO 4-methylmorpholine N-oxide PPTS pyridinium p-toluenesulfonate RCM ring-closing metathesis SEMCl 2-(trimethylsilyl)ethoxymethyl chloride TBAI tetrabutylammonium iodide TBAF tetrabutylammonium fluoride TBDMS t-butyldimethylsilyl TBDPSCl t-butyldiphenylsilyl chloride TFA trifluoroacetic acid TFAA trifluoroacetic anhydride THAB tetrahexylammonium bromide TMSCN trimethylsilyl cyanide Carbasugars: Synthesis and Functions 9.2 1915 1 Introduction

Recently, a great deal of attention has been focused on the glycosidase inhibitors under the premise that glycoconjugates such as oligosaccharides, glycolipids, and glycoproteins play pivotal roles in living systems, and recently a scientific field called “glycogenomics” has been developed as a part of genomics science. Glycosidase inhibitors possess interesting enzyme- specific inhibitory activities, therefore, they are expected not only to be tools to elucidate the mechanisms of a living system manipulated by the glycoconjugates but also to be potential clinical drugs and insecticides by inducing the failure of glycoconjugates to perform their function: anti-obesity drugs, anti-diabetics, anti-fungal, and anti-viral including substances active towards the human immunodeficiency virus (HIV) [1,2,3,4,5,6,7]. Most of the glycosidase inhibitors are isolated from natural sources, and they possess interesting structures in their molecules. Some of them possess the highly functionalized and oxygenated cyclohexane or cyclopentane moieties. In general, these moieties are called cyclitols, and also in this chapter, glycosidase inhibitors possessing cyclitols in their molecular structures are defined as carbasugars. Biochemically, carbasugars and cylitols themselves are recognized as the pseudo-sugars in a living system, and they show interesting biological activities based on the structure similarity to sugars. In the meantime, chemically, such highly functionalized molecules are quite challenging targets for synthetic studies. The most interesting and significant points for the synthesis of glycosidase inhibitors possessing cyclitols are how we can form the frameworks of the cyclitols and how we can introduce the functional groups essential to generate their specific and interesting biological activities. In the last few decades new generations of glycosidase inhibitors, e. g., cyclophelitol, allosamidin, mannnostatin, and trehazolin, have been isolated from natural sources, and the appearance of these natural products has contributed to the development of the new methodologies needed to perform the aforementioned synthetic tasks. Also, due to tech- nical developments in the field of structure biology, the interaction between enzymes and inhibitors can now be visualized. This is helpful in the design of new scaffolds and elucidation of the detailed mechanisms of action involved. The creation of tamiflu, which is an artificially designed carbasugar showing neuraminidase (NA) inhibitory activity, is a good example favored by structure biology, and it has already been approved by US Food and Drug Administration (FDA) as an orally available anti-influenza drug. Herein, I will describe a variety of synthetic strategies directed to total syntheses and chemical modifications of these glycosidase inhibitors and the structure and activity relationships of their derivatives. Also, the methodologies of carbasugar formation from sugars will be described.

2Overview

One of the key points in the synthesis of carbasugars is the formation of the scaffolds of the cyclitol moieties, and three types of synthetic strategy can be considered:

(a) Transformation of sugars to cyclitols; (b) Synthesis from nonsugar substrates by using asymmetric synthetic methodologies; (c) Utility of the framework of intact cyclitols. 1916 9 Glycomimetics Also, in addition, much attention should be focused on the structure-activity relationships (SAR) on enzyme inhibitory activities inﬂuenced by their stereochemistry and their functional groups. Therefore, the synthetic strategies should be applicable to the synthesis of their stereoisomers and congeners, and, in practice, some of the synthetic strategies have also been designed in consideration of this point. In this chapter, case studies on total synthesis of cyclophelitol, tamiﬂu, allosamidin, and trehazolin, including the synthesis of their stereoisomers and congeners, will be described, and SAR of these compounds will be discussed. Finally, SnCl4-promoted cyclitol formation and SmI2-mediated cyclitol formation will be described as useful methodologies for the conversion of sugars to cyclitols as well as SmI2-mediated carbasugar formation.

3 Synthesis of Carbasugars: New Generations of Glycosidase Inhibitors

3.1 New Generations of Glycosidase Inhibitors Bearing 6-Membered Cyclitol

3.1.1 Cyclophelitol: A β-Glucosidase Inhibitor Cyclophelitol (1)isaβ-glucosidase inhibitor isolated from a culture ﬁltrate of the mushroom strain, Phellinus sp., and shows inhibitory activity towards almond β-glucosidase. In general, a series of β-glucosidase inhibitors such as castanospermine and 1-deoxynojirimycin have been reported to inhibit syncytium formation and infection with human immunodeﬁciency virus (HIV), possibly by perturbing the gp 120-linked glycan structure [8]. The structural feature of cyclophelitol is the fully oxygenated cyclohexane, which possesses the stereochemistry of the hydroxyl groups corresponding to that of D-glucose and the β-epoxy group at the C1, C6 positions. This β-epoxy group is recognized to be the equivalent of a β-glycoside moiety by enzyme, and it is considered that this is possibly the reason for the inhibitory activities exhibited towards β-glucosidases [9,10].

This interesting structure encouraged many organic chemists to undertake synthetic projects to develop appropriate synthetic methodologies, and several elegant syntheses of this compound have been reported. The ﬁrst total synthesis of cyclophelitol was achieved by Tatsuta’s group in 1990 [11]. The key point of their synthetic strategy is the construction of this highly oxygenated cyclohexane via intramolecular [3+2] cycloaddition of a nitrile oxide to an alkene. Oxime 3, which was derived from L-glucose via stereoselective hydroboration of the exo- oleﬁn of L-xylo-hex-5-enopyranoside 2 [12] with dicyclohexyl borane, Swern oxidation of the Carbasugars: Synthesis and Functions 9.2 1917

⊡ Scheme 1

corresponding alcohol, subsequent Wittig alkenylation of the resulting unstable aldehyde with Ph3P=CH2, and acid hydrolysis, was treated with NaOCl to obtain isoxazoline 4 as a single product via the corresponding nitrile oxide [13,14]. The ring opening of isoxazoline 4 was achieved with H2 and Raney nickel W-4 in aqueous dioxane in the presence of AcOH to afford the corresponding keto-diol 5. After diethylisopropylsilylation [15], which can be easily removed under hydrogenolysis conditions using Pd(OH)2 on carbon, the resulting ketone was reduced with BH3·SMe2 stereoselectively, to furnish the desired α-alcohol. Mesylation of this α-alcohol provided the labile mesylate 6, which was subjected to hydrogenolysis with Pd(OH)2 on carbon to give the de-O- benzylated compound. Epoxidation of this debenzylated compound with NaOMe and subsequent removal of the silyl groups of the corresponding epoxide with TBAF afforded cyclophelitol (1)(> Scheme 1). 1918 9 Glycomimetics For further studies regarding structure-activity relationships on the enzyme inhibitory activities inﬂuenced by the stereochemistry of cyclophelitols, Tatsuta’s group also synthesized a series of cyclophelitol-related compounds.

The syntheses of 1,6-epi-cyclophelitol (7) and the 1,2,6--cyclophelitol α-manno-type analogue (8) are outlined in > Scheme 2 and > Scheme 3 [16]. These compounds were synthesized from

⊡ Scheme 2 Carbasugars: Synthesis and Functions 9.2 1919

⊡ Scheme 3

a common intermediate, isoxazoline 14a, which was derived from methyl 2,3,4-tri-O-benzyl- α-D-galactopyranoside via the above-mentioned intramolecular [3+2] cycloaddition. Interest- ingly, in the case of the synthesis of 1,6-epi-cyclophelitol (7), isoxazoline 14a although sub- stantially undesired to produce compound 7, which is derived from oxime 13, was subjected to acidic hydrogenolysis with Raney nickel W-4 to afford the desired keto-alcohol 15 with epimerization at the C1 position. In contrast, in the case of the synthesis of the α-manno-type analogue, hydrogenolysis of isoxazoline 14a which is desired to obtain compound 8 was conducted using Raney nickel and B(OH)3 to furnish the desired keto-alcohol 16 in a quantitative yield (> Scheme 2). These keto-alcohols 15 and 16 were converted into 1,6-epi-cyclophelitol (7) and the 1,2,6-epi- cyclophelitol α-manno-type analogue (8), respectively, according to the synthetic route shown in > Scheme 3. Furthermore, on the basis of these syntheses, this group also synthesized the β-azirizine analogue (9) from 1,6-epi-cyclophelitol (7)andtheα-azirizine analogue (10) from cyclophelitol (1), respectively [17,18]. In addition, the thiirane analogues (11)and(12) were also synthe- 1920 9 Glycomimetics

⊡ Scheme 4

sized from 1,6-epi-cyclophelitol (7) and cyclophelitol (1), respectively, by treatment of their O-PMB derivatives with Ph3P=S and triﬂuoroacetic acid, followed by de-O-p-methoxybenzy- lation with DDQ (> Scheme 4)[18]. As shown in > Table 1, glycosidase inhibitory activities of these cyclophelitol-related compounds were evaluated [19]. In contrast to cyclophelitol (1) with inhibitory activity toward only β-D-glucosidase at an IC50 of 0.8 µg/ml, 1,6-epi-cyclophelitol (7) exhibited inhibitory activity toward only α-D- glucosidase at an IC50 of 10 µg/ml and α-manno-type analogue (8) showed inhibitory activity toward α-mannosidase at an IC50 of 19 µg/ml. Also, the β-azirizine analogue (9) exhibited high inhibitory activity toward β-glucosidase at an IC50 of 0.22 µg/ml, while the α-azirizine analogue (10) showed little α-glucosidase inhibitory activity. Interestingly, both thiirane ana- Carbasugars: Synthesis and Functions 9.2 1921 ⊡ Table 1 Inhibitory activity of cyclophelitol (1) and its related compounds 7–9 towards glycosidases

logues (11)and(12) showed no significant activities. With respect to this SAR on glycosidase inhibitory activities of these compounds, Tatsuta’s group concluded the following; Structurally, cyclophelitol (1) and its β-azirizine analogue (9)havequasi-equatorially ori- ented C1–O and C1–N bonds, which correspond to the equatorial C1–O bond β-glucopy- ranosides, whereas 1,6-epi-cyclophelitol (7)andα-manno-type analogue (8)havequasi-axial C1–O bonds corresponding to the axial C1–O bond of α-D-glycopyranosides. Their glycosidase inhibitory activities emphasized that the α-andβ-glycosidase recognized especially the C-1 positions and the residual portions as corresponding to those of α-andβ-glycopyranosides. Consequently, these glycosidase inhibitors (1), (7), (8), and (9) serve as antagonists of the corresponding α-andβ-D-glycopyranosides. Nakata et al. reported the straightforward and practical synthesis of cyclophelitol (1)and 1,6-epi-cyclophelitol (7) from D-glucose via SmI2-mediated reductive coupling [20]. Hydrob- oration of olefin 21, which was derived from methyl α-D-glucopyranoside (19) in seven steps, with dicyclohexylborane afforded the mixture of alcohols 22 (22a:22b = 4:1). After silylation with TBDPSCl, followed by selective acid hydrolysis of the Tr group, which furnished both the desired diol 23a and the undesired one 23b in 47 and 12% yield, respectively, subsequent Swern oxidation of the desired alcohol 23a afforded dialdehyde 24. This dialdehyde 24 was treated with SmI2 [21,22,23,24,25], to give separable cis-diols 25a and 25b (> Scheme 5). Each diol was converted to the same olefin 26 by mesylation, reduction, and subsequent desilylation. Compound 26 was transformed to cyclophelitol (1) via stereoselective epoxidation, and on the other hand, debenzylation of 26 followed by epoxidation provided 1,6-epi-cyclophelitol (7)(> Scheme 6). 1922 9 Glycomimetics

⊡ Scheme 5

Fraser-Reid’s group reported the synthesis of Tatsuta’s penultimate intermediate, the mesy- lates for cyclophelitol (1) and 1,6-epi-cyclophelitol (7)viaa6-exo-dig radical cyclization of 2-deoxy-2-iodo-6-alkenyl glycoside and completed the formal total synthesis of cyclophelitol (1) and 1,6-epi-cyclophelitol (7)[26]. Swern oxidation of glucal 27, alkynylation of the corresponding aldehyde and subsequent acetylation of the resulting alcohols afforded an epimer- ic mixture 28. Thiem’s iodoalkylation of 28 [27,28] provided the 2-deoxy-2-iodo-6-alkynyl glycoside 29, and the radical cyclization of 29 with Bu3SnH and AIBN [29,30,31,32]gave a quantitative yield of material which, after deacetylation, could be fractionated into two sets of isomers comprised of diastereomeric [2.2.2]oxabicycloglycoside 30 and 2-deoxy epimer 31. Finally, compound 30 was converted into mono alcohol 35 in four steps (> Scheme 7). On the basis of the conﬁguration at C1 (cyclophelitol numbering), the components of this diastereomeric mixture were divided into 35a and 35b, which were correlated to Tatsu- ta’s intermediates. Compound 35a was transformed to Tatsuta’s mesylate 39 for 1,6-epi- Carbasugars: Synthesis and Functions 9.2 1923

⊡ Scheme 6 cyclophelitol (7) in seven steps including C2–OH inversion via oxidation/reduction with the Dess–Martin reagent [33]/NaBH4, ozonolysis followed by BH3·SMe2 reduction, and mesylation of the corresponding alcohol. On the other hand, 35b was also converted into Tat- suta’s mesylate 47 for cyclophelitol (1) in ten steps involving ozonolysis followed by stereo- controlled reduction with NaBH(OAc)3 [34,35] via complex 40 and C2–OH inversion via oxidation/reduction with the Dess–Martin reagent/BH3·SMe2 (> Scheme 8). Ferrier reaction [36,37] is well-known as a representative of the synthetic methodologies for the conversion of intact sugars into cyclitols. It is not exceptional that Ferrier reaction was also used for total synthesis of cyclophelitol as a key step for cyclitol formation. The synthesis reported by Sato et al. [38]isshownin> Scheme 9. The features of this synthesis are the stereoselective introduction of a dichloromethyl function to C2-ketone 48 [39], which was derived from D-glucose, and the conversion of 49 with NaBH4 in DMSO into the 2-deoxy-2-C-hydroxymethyl α-D-glucopyranoside derivative 50, and furthermore, Ferrier reaction of exo-oleﬁn 51, which was derived from 50 in two steps, with HgCl2 in acetone/H2O, and subsequent treatment of the product with MsCl and Et3N provided the desired α, β-unsaturated ketone 52. Stereoselective 1,2-reduction of 52 with NaBH4 and CeCl3·7H2O, protection of the corresponding alcohol with a TBDMS group, and subsequent cleavage of acyl groups under the basic conditions afforded triol 53. The epoxidation to the oleﬁn part of triol 53 was proceeded stereoselectively favored by the bulky TBDMS group, to afford epoxide 54 which 1924 9 Glycomimetics

⊡ Scheme 7

possesses the desired stereochemistry, and subsequent acid desilylation furnished cyclophelitol (1)(> Scheme 9). Also, Jung et al. reported the total synthesis of cyclophelitol (1) and 1,6-epi-cyclophelitol (7) using Ferrier reaction as its key steps for cyclitol formation [40]. The known methyl ben- zylidenemannoside 55 [41], which was derived from D-mannose in two steps, was converted into alkene 56 via regioselective benzylation of the equatorial hydroxyl group of 55 [42,43], Swern oxidation of the remaining hydroxyl group, and subsequent Wittig reaction. Hydrob- oration of 56 was expected to occur preferentially from the axial direction, anti to the sterically hindering axial methoxy group. However, unexpectedly, the reaction furnished a 1:1 mixture of two isomeric hydroxymethyl compounds 57a and 57b. Even with a sterically bulky borane such as 9-BBN the reaction afforded a 1:2 mixture in favor of 57b. The problem of the production of exclusively the equatorial isomer was solved by Swern oxidation of the mixture, subsequent quantitative equilibration to the β-aldehyde in a mild base, and reduction of the corresponding α-aldehyde to give only the desired equatorial hydroxymethyl compound 57a. Benzylation, reductive opening of the benzylidene acetal, iodination, and elimination furnished exo-oleﬁn 58. Ferrier reaction of 58 and subsequent elimination gave the desired enone 59. 1,2-reduction of 59 to the corresponding allylic alcohol and subsequent benzoyla- tion gave compound 60, and epoxidation of 60 with mCPBA provided a mixture of the epoxides. Cleavage of all protective groups produced cyclophelitol (1) and 1,6-epi-cyclophelitol (7) (> Scheme 10). Recently, ring-closing metathesis (RCM) using Grubbs catalyst [44] became popular in the ﬁeld of the synthesis of natural products. This methodology was also used in the synthesis of cyclophelitols. The synthesis using RCM was reported by Ziegler et al. and started from D-xylose as shown in > Scheme 11 [45]. Didithioacetal 61, which was derived from D-xylose, Carbasugars: Synthesis and Functions 9.2 1925

⊡ Scheme 8 was converted into aldehyde 63 in three steps that were silylation of the primary alcohol, benzylation of all the remaining secondary hydroxyl groups, and subsequent removal of dithioacetal with HgO and HgCl2. Methylenation to aldehyde 63 was performed with Tebbe’s reagent in place of the Wittig reaction because the basic methylenation with Wittig reaction gave sev- 1926 9 Glycomimetics

⊡ Scheme 9

eral by-products such as the unsaturated aldehyde and its diene derived from β-elimination of benzyl alcohol from aldehyde 63 as well as the desired olefin 64. After Swern oxidation of the remaining primary hydroxyl group, Wittig reaction of the corresponding aldehyde with PPh3=CHCO2Me afforded α, β-unsaturated ester 65. One of the problems to be solved here was the stereoselective 1,4-addition to α, β-unsaturated ester 65. In this case, the best method for this objective is the magnesium-based vinyl cuprate, which was employed under the protocol reported by Hanessian [46]togive66 with both high yield and high stereoselectivity. Afterwards, RCM to 66 using Grubbs catalyst provided cyclohexene 67 efficiently, and subsequent iodolactonization to 67 afforded iodide 68. Iodide 68 was converted into the β-epoxide 70 according to the Saurez procedure [47,48]. After reduction of 68 with DIBALH, the resulting lactol efficiently afforded diiodideformate 69 upon irradiation in the presence of PhI(OAc)2/I2 [49]. Subsequently, base treatment of 69 gave iodoepoxide 70.Thetrans- formation of iodine to a hydroxyl group was accomplished by radical oxygenation to afford a mixture of epoxyalcohol 71a and epoxydiol 71b. Finally, hydrogenolysis of a mixture of the epoxides 71a and 71b removed the benzyl groups to afford cyclophelitol (1)(> Scheme 11). Carbasugars: Synthesis and Functions 9.2 1927

⊡ Scheme 10

Kornienko’s group reported the formal synthesis of cyclophelitol (1) via RCM to the diene derived from D-xylose [50]. Their synthetic strategy was interestingly focused on the latent plane of chirality present in D-xylose as shown in > Fig. 1, and the enantiodivergent synthesis of (+)- and (–)-cyclophelitol from D-xylose was achieved (> Fig. 1). Their enantiodivergent strategy relies on the synthesis of both enantiomeric forms of Ziegler’s enoate 72 (=65). They have reported a straightforward preparation of enoate 72 from 2,3,4- tri-O-benzyl-D-xylopyranose by way of Wittig methylenation, Swern oxidation, and reaction of the resulting aldehyde with PPh3=CHCO2Me [51]. On the other hand, the synthesis of ent-72 by directly reversing the order of the two oleﬁnation steps was not always successful because of an enoate reactivity problem. To circumvent this problem, the anomeric position of 2,3,4-tri-O-benzyl-D-xylopyranose was protected to form ethyl thioacetal, and subsequent Swern oxidation of the remaining primary hydroxyl group, followed by the intermediate treatment of the corresponding aldehyde with PPh3=CH2, deprotection of ethyl thioacetal, and oleﬁnation of the corresponding aldehyde with PPh3=CHCO2Me cleanly provided the desired 1928 9 Glycomimetics

⊡ Scheme 11

enantiomeric enoate ent-72 in excellent yield. With the synthetic route to 1 and ent-1 available, the feasibility of accessing each cyclophelitol enantiomer was demonstrated by completing the synthesis of the (+)-antipode. Addition of a vinylcopper reagent to enoate 72 provided ester 73 in excellent yield and exclusive anti selectivity. Treatment of potassium enolate of ester Carbasugars: Synthesis and Functions 9.2 1929

⊡ Figure 1 “Latent symmetry” approach for the synthesis of cyclitol enantiomers

73 with Davis oxaziridine gave a 1:1 mixture of α-hydroxylated derivatives 74,whichwas reduced to diols 75. Cleavage of the vicinal diol functionality with NaIO4 followedbytreat- ment of the corresponding aldehyde with NaBH4 gave alcohol 76. Finally, RCM of alcohol 76 with the ﬁrst generation Grubbs’ ruthenium catalyst resulted in conduritol analogue 77. Since the transformation of 77 to (+)-cyclophelitol (1) by way of directed epoxidation and hydrogenolytic deprotection was reported by Trost’s group [52,53], this pathway constituted a formal synthesis of the natural product (> Scheme 12). Madsen et al. also reported the synthesis of cyclophelitol (1) from D-xylose via RCM [54]. The key transformations involve a zinc-mediated fragmentation of benzyl protected methyl 5-deoxy-5-iodo-xylofuranoside (78) followed by a highly diastereoselective indium-mediated coupling with ethyl 4-bromocrotonate as shown in > Scheme 13. Compound 78,which was derived from D-xylose in three steps including acidic methyl glycosylation, iodination to primary alcohol, and subsequent benzylation to the remaining hydroxyl groups with benzyl trichloroacetimidate under acidic conditions, was sonicated with zinc to afford unsaturated aldehyde 79. The indium-mediated addition reaction to 79 with ethyl 4-bromocrotonate and 60 mesh indium powder (99.999% pure) in the presence of La(OTf)3 [55,56,57] provided the desired product 81 as only one diastereomer. The generation of the only desired diastereomer 81 can be explained by invoking a chelated intermediate such as 80.Diene81 was converted into the corresponding cyclohexene 82 by RCM with Grubbs’ second generation catalyst [58]. The ester functionality was reduced with DIBALH to afford diol 83. This reduction was quite sluggish and afforded minor amounts of the intermediate aldehyde as a byproduct. Using a larger excess of DIBALH or a higher temperature did not solve the problem. It is interesting that water and NaBH4 instead were added in the workup to drive the reaction to completion. Epoxidation of 83 with mCPBA was completely stereoselective to afford the desired epoxide without another isomer, and ﬁnally the corresponding epoxide was deprotected by hydrogenolysis to furnish cyclophelitol (1)(> Scheme 13). An interesting approach to cyclophelitol (1) is its synthesis via the nonracemic Diels–Alder reaction to form the scaffold of cyclophelitol as performed by Schlessinger’s group, shown 1930 9 Glycomimetics

⊡ Scheme 12

in > Scheme 14 [59]. To the nonracemic furan which was prepared by deprotonation of the vinylogous lactone 84, followed by treatment of the resulting potassium enolate with TBDP- SCl, dimethyl 2,3-pentadienedioate 85 was added [60], and the resulting compound 87,which contained the requisite elements of structure and functionality for conversion to cyclophelitol (1), was obtained as a crude product via the proposed transition state 86. Acid hydrolysis of 87, and subsequent reduction of the corresponding bromoketone with NaBH4 gave the syn- Carbasugars: Synthesis and Functions 9.2 1931

⊡ Scheme 13 bromohydrin 88, which possesses the correct functionality and stereochemistry at C1 and C2, respectively, as required by the structure (1). To reform C4 and C5 of 88 for the structure (1), ﬁrstly the stereochemistry of the axial ester at C5 was adjusted by treatment with DMAP via intermediate 89, to afford the epimerized C5 equatorial ester 90. Ozonolysis of 90, reduction of the resulting ketone of 91 with DIBALH, and subsequent benzylation of the corresponding alcohol under acidic conditions afforded compound 92 possessing the correct stereochemistry at C4 for cyclophelitol synthesis. The oxabicycloketal moiety of tribenzyl ether 92 was cleaved smoothly with BF3·Et2O to afford the tractable cyclohexanone. Diisobutylalu- minum 2,6-di-t-butyl-4-methylphenoxide (DDMPO) [61,62] treatment of this cyclohexanone gave 93 stereoselectively. After the transformation of bromohydrin 93 to the epoxide with KHMDS, hydrogenolysis of the corresponding, fully protected epoxide furnished cyclophelitol (1)(> Scheme 14). Finally for this section, as another asymmetric synthesis of cycliphelitol (1), the synthetic effort of Trost’s group will be introduced [52]. Their synthesis was conducted based on the palladium-catalyzed kinetic resolution of racemic conduritol B tetraacetate (±)-94. Since racemic 1932 9 Glycomimetics

⊡ Scheme 14

conduritol B is easily accessed from benzoquinone, two issues needed to be addressed: (1) how could racemic conduritol B easily be resolved and (2) how could the hydroxyl groups be readily differentiated. Trost’s group considered that asymmetric palladium catalysts offer a simple solution to both of these issues. On the basis of their preliminary study on a palladium- catalyzed kinetic resolution of the racemic C2-symmetric tetraacetate (±)-94, they expected that it should be possible using the chiral ligand (R, R)-95, since, with respect to the ligand, one enantiomer of (±)-94 would provide a “matched” substrate for ionization while the oth- Carbasugars: Synthesis and Functions 9.2 1933 er would be “mismatched” [63]. In the meantime, a pivalate was chosen as the carboxylate nucleophile for the resolution because the resultant allyl pivalate was anticipated to ionize much more slowly than the starting material and the pivalate should provide a means for easy differentiation of the alcohol protecting groups later on. The kinetic resolution of (±)-94, which was synthesized from in three steps from benzoquinone, was carried out using 0.65 equiv. of sodium pivalate (formed in situ from 0.80 equiv. of PvOH and 0.65 equiv. of NaOH) with 1 mol% of 96 and 3 mol% of 95 at 0.5 M in a two-phase methylene chloride/water system with tetrahexylammonium bromide (THAB) as a phase transfer catalyst. The reaction stopped cleanly at 50% conversion providing a quantitative yield (based on 50% theoretical yield) of recovered tetraacetate (+)-94 and an 88% yield (based upon a 50% theoretical yield) of monopivalate (–)-97 with only 1% of dipivalate isolated. Chiral HPLC analysis of (–)-97 showed it to have 97% ee. Cleavage of Ac groups of (–)-97 in the presence of the Pv group was straightforward with NH4OH in MeOH to provide triol 98. With access to enantiomerically pure triol 98, the conversion of 98 into cyclophelitol (1) was carried out as summarized in > Scheme 15. They made the synthetic strategy using 2,3-sigmatropic rearrangement of an alkoxymethyl anion [64] as the key step, and use of this rearrangement required protection of the free hydroxyl groups as the benzyl ethers. After pivalate cleavage of the corresponding benzyl ethers, the tin derivative 99 was readily formed. Tin-lithium exchange promoted the 2,3-sigmatropic rearrangement to put the hydroxymethyl group in place with correct regio- and diastereochemistry with production of 101 (=77) via transition state 100. Epoxidation with mCPBA, directed by the homoallylic alcohol, gave a 78% yield of the desired epoxide, with a 7% yield of the diastereomeric epoxide. Completion of the synthesis by the hydrogenation procedure published by Ziegler et al., gave (+)-cyclophelitol (1)(> Scheme 15). In this section, a variety of the synthetic efforts on cycliphelitol (1) were introduced. From these efforts, useful synthetic methodologies to synthesize highly functionalized cyclitols were developed, and, in practice, they favored SAR analysis on glycosidase inhibitory activities of cyclophelitol (1) and its stereoisomers. This means that the appearance of cyclophelitol (1) paved the way for a new wave of drug discovery based on the science of glycosidase inhibitors and this will contribute to the further development of glycoscience and the development of new synthetic methodologies for total synthesis.

3.1.2 Tamiflu: A Neuraminidase Inhibitor Despite considerable progress in elucidating the molecular mechanism and cellular biology of influenza virus, influenza infection continues to be the most serious respiratory disease both in terms of morbidity and mortality [65]. Amantidine and its analogue rimantidine represent the compounds licensed for the treatment and prophylaxis of influenza A infection. However, these compounds are not effective against influenza B viruses, and their clinical use has been limited by side effects and the rapid emergence of resistant viral strains [66]. On the other hand, vaccine development has been partially successful in the control of influenza epidemics due to the highly variable mutation of the influenza virus [67,68]. This means that promising anti-influenza drugs don’t always exist in medical practice. Recently, a great deal of attention in this field has been focused on the unique replication mechanism of the influenza virus, and this has allowed the relevant scientists to identify a number of potential molecular targets for drug design. Those targets include hemagglutinin (HA) [69,70], neuraminidase [71,72], M2 1934 9 Glycomimetics

⊡ Scheme 15 Carbasugars: Synthesis and Functions 9.2 1935 protein [73], and endonuclease [74]. Hemagglutinin and neuraminidase are two major surface glycoproteins expressed by both influenza A and B viruses. HA is known to mediate binding of viruses to target cells via terminal sialic acid residue in glycoconjugates. This binding is the first step of viral infection. In contrast to HA activity, NA catalyzes removal of terminal sialic acids linked to glycoproteins and glycolipids. Although the biological consequences of this activity are not completely understood, it has been postulated that NA activity is necessary in the elution of newly formed viruses from infected cells by digesting sialic acid in the HA receptor [75,76]. NA may also promote viral movement through respiratory tract mucus, thus enhancing viral infectivity [74. 75]. Therefore, NA has been considered to be a suitable target for designing new types of anti-influenza drugs. In earlier studies, 2,3-didehydro-2-deoxy-N-acetylmeuraminic acid (Neu5Ac2en, 102)was found to be an influenza NA inhibitor with a Ki of 4 µM [78,79]. Biochemical studies [80] indicate 102 is considered a transition state-like analogue binding to the active site of NA [81,82]. Afterward, on the basis of structural information generated from X-ray crystallographic study of 102 complexed with NA, the following rationally designed NA inhibitors were prepared: 2,3-didehydro-2, 4-dideoxy-4-amino-N-acetylneuraminic acid (4-amino-Neu5Ac2en, 103) and its guanidine analogue (4-guanidino-Neu5Ac2en, 104)[81,83,84]. In comparison to 102, both 103 and 104 are more potent NA inhibitors with Ki values of 10−8 M and 10−10 M, respectively. Both the amino group in 103 and the guanidino group in 104 are suggested to form salt bridges with Glu119 in the NA active site, while the latter adds a strong charge- charge interaction with Glu227 [84]. In addition, 104 also exhibited potent antiviral activity against a variety of influenza A and B strains in the cell culture assay [85]. Eventually, 104 was approved by the FDA, and it has been launched as an anti-influenza drug called relenza [86,87, 88,89]. However, poor oral bioavailability and rapid excretion precluded 104 as a potential oral agent against influenza infection and 104 has to be administered by either intranasal or inhaled routes. In the case of an influenza epidemic, oral administration may be a more convenient and economical method for treatment and prophylaxis. Therefore, it would be desirable to have a new class of orally active NA inhibitors as potential agents against influenza infection. The research group of Gilead Science tried to discover a new class of compounds by using a carbocyclic template in place of the dihydropyran ring of the Neu5Ac2en system, and it was expected that the carbocyclic ring would be chemically more stable than the dihydropyran ring and easier to modify for optimization of antiviral and pharmacological properties. Conse- quently, they succeeded in identifying GS-4071 (105) and its prodrug GS-4104 (106), which is called tamiflu and has been launched as an orally available neuraminidase inhibitor for the treatment of influenza [90]. In this section, I will describe the story of the discovery of tamiflu. This represents a success story of an artificial carbasugar launched as an actual drug. 1936 9 Glycomimetics Design NA has been classified into nine subtypes for type A influenza virus strains according to their serological properties. However, there are no subtypes in the B-type virus. Although amino acid sequence homology among NA from both type A and type B virus strains has been found to be only 30% [91,92], the enzyme activity of NA among the different strains is the same, indicating the highly conserved nature of the active site of the enzyme [93]. The X-ray crystallographic structures of NA have been determined from three influenza subtypes: A/Tokyo [94], A/Tern [95], and B/Beijing [96]. The structures displayed a symmetrical folding pattern of six four-stranded antiparallel β-sheets arranged like blades of a propeller. NA exists as a mushroom-shaped spike with a boxlike head on top of a long stalk containing a hydrophobic region by which it is embedded in the viral membrane. Crystallographic studies of NA reveal that, in the active site, the amino acids which line and surround the walls of the binding pocket are highly conserved among all influenza strains examined so far. The high-resolution crystallographic structure of sialic acid (107a) complexed with NA revealed that sialic acid binds the enzyme in a considerably deformed conformation due to the strong ionic interactions between the carboxylate of the substrate and Arg118, -292, and -371 in the active site of the enzyme [97]. In solution the carboxylate of sialic acid is axial, but the deformation of the ring on binding places the carboxylate into a pseudoequatorial position. This binding mode is similar to that found in the X-ray crystal structure of Neu5Ac2en (102) complexed with NA [95]. In this case, the double bond of Neu5Ac2en constrains the pyranose ring of the sugar into a planar structure around the ring oxygen. On the basis of this structural information, it has been proposed that the catalytic mechanism for the cleavage of sialic acid from glycoconjugates (107b) implicates the formation of the C2 carbonium cation (108)whichis stabilized by the neighboring oxygen atom as shown in > Fig. 2 [81,82]. Furthermore, kinetic isotope studies have provided convincing evidence for the C2 carbonium cation formation as an intermediate structurally similar to the transition state in the sialic acid cleavage by NA [80]. In general, transition-state mimics frequently are potent inhibitors for the catalyzing enzyme. The concept of structural similarity to the transition state has found wide application in drug design over the years. The multitude of enzyme-inhibitor interactions are governed by steric as well as electronic factors. In theory, compounds that closely resemble the transition-state structure should give high binding affinity toward the target enzyme [98,99], and the cases of neuraminidase inhibitors are not exceptional. Using intermediate 108 as a key transition-state mimic is a reasonable approximation in view of the X-ray crystallographic studies described above. Their earlier attempts to mimic the intermediate 108 with a completely flat benzene ring did not lead to potent NA inhibitors [100,101], suggesting the importance of the stereochemistry of substituents around the ring in the design of inhibitors with high affinity for NA. Therefore, considering the flat oxonium cation in 108 as an isoster of the double bond and the fact that the carbocyclic system would be expected to be chemically versatile for the manipu- lation of side chains attached to the ring, Gilead’s group selected the cyclohexene scaffold as a replacement for the oxonium ring of 108 which would keep the conformational changes to a minimum (> Fig. 2).

Syntheses of the Prototypes In their initial stage of designing carbocyclic NA inhibitors, the oleﬁnic isomers that are shown in structures 109 (Type [I]) and 110 (Type [II]) were considered as two possible prototypes of transition-state analogues. The isomer 109 is structurally closer to transition-state 108 than isomer 110. However, Gilead’s group expected that it would be Carbasugars: Synthesis and Functions 9.2 1937

⊡ Figure 2 Rational design of carbocyclic transition-state analogues difﬁcult to assess a priori which isomer would be a preferred NA inhibitor, especially, in light of the potent NA inhibitory activity displayed by Neu5Ac2en (102) and its analogues 103 and 104, in which the double bond was located in the position corresponding to isomer 110 and the fact that Chandler et al. reported carbocyclic analogues have some interesting NA inhibitory activity [102]. In the meantime, the molecular modeling of isomers 109 and 110 indicated that these two molecules overlay well. Therefore, Gilead’s group synthesized the simple compounds 109a and 110a, and evaluated which would be a more appropriate prototype for further extensive structure-activity relationships studies (> Fig. 2). 1938 9 Glycomimetics

⊡ Scheme 16

A comparison of structural similarities revealed that (–)-shikimic acid (111)and109a share common structural features. However, the conversion of 111 to 109a requires the effective stereochemical control for transforming the trans C4 and C5 hydroxyls of 111 to the trans C4 and C5 amino groups of 109a. The approach selected for this transformation, as outlined in > Scheme 16, relied on the conversion of 111 to aziridine 116 followed by azide ion attack in a regio- and stereospecific manner as a key reaction. The synthesis began with the preparation of epoxide 112 from 111 as described in the literature [103]. Nucleophilic ring opening of MOM-protected epoxide 113 with NaN3 in the presence of NH4Cl generated azido alcohol 114. The ring-opening of the epoxide was both region- and stereospecific as depicted in 114, and this could be attributed to the steric and electronegative inductive influence of the MOM group in 113. Conversion of azide 114 to aziridine 116 was efficiently accomplished via a two-step sequence: (1) mesylation of the hydroxyl group in 114 and (2) reduction of the azide functionality in 115 with PPh3 in the presence of Et3N and H2O. The aziridine ring opening of 116 with NaN3 gave 117 exclusively. This selective ring opening was again a consequence of the favored azide ion attack at the C5 position due to the steric and electronegative inductive effects of the MOM group. Finally, acetylation of 117 followed by reduction of the azide group in 118 and saponification of the methyl ester and deprotection of the MOM group in 119 with TFA provided 109a (> Scheme 16). Carbasugars: Synthesis and Functions 9.2 1939

⊡ Scheme 17

On the other hand, 110a was synthesized from (–)-quinic acid (120). The cyclohexene intermediate 122, readily available from 121 by literature methods [104], possesses considerable structural similarity to 110a.TheC5 hydroxyl of 121 was protected as a pivaloyl ester, and mild acid hydrolysis of 122 furnished the corresponding diol. Exposure of the diol to SOCl2 in the presence of Et3N generated cyclic sulfite 123. The sulfite ring opening of 123 with NaN3 gave azide 124 as a single product. This complete regiospecific ring opening is a consequence of the favored azide ion attack at allylic C3 position of 123.Theβ-hydroxy azide moiety in 124 was then converted in a three-step sequence to aziridine 126, analogous to the conversion of 114 to 116. Ring opening of acetylated aziridine 126 with NaN3 was completely regiospecific, giving azide 127 followed by reduction of azide functionality which provided 110a (> Scheme 17). 109a and 110a showed interesting inhibitory activities in a NA enzymatic assay (> Fig. 3). While 109a proved to be a potent NA inhibitor with an IC50 of 6.3 µM, 110b did not exhibit inhibitory activity at concentrations up to 200 µM. 1940 9 Glycomimetics

⊡ Figure 3 Inﬂuenza neuraminidase inhibition by prototypes 109a and 110a

It was concluded that this result demonstrated that the double bond position in the design of carbocyclic NA inhibitors plays an important role in NA activity; however, further structural investigation is required to illustrate the binding differences of 109a and 110a in the NA active site. Eventually, based on this result, the structure of 109 was selected as the prototype for further drug discovery attempts.

SAR Analysis Crystallography studies of Neu5Ac and its analogues bound to NA appear to indicate that the C7 hydroxyl of the glycerol side chain does not interact with any amino acids of the NA active site [72]. This suggested that the C7 hydroxyl could be eliminated from the carbocyclic analogues without losing binding affinity to NA. Furthermore, it was also realized that, in the transition-state intermediate 108, the oxonium double bond is highly polarized and electron deficient. Considering these features, in the carbocyclic structure, the CHOH group at the C7 position of the glycerol side chain in the Neu5Ac system was replaced with an oxygen atom as shown in structure 109.ThisC3 oxygen atom would reduce the electron density of the double bond via the σ bond electronegative inductive effect. In addition to these rationales, Gilead’s decision to have the C3 oxygen atom was based on the synthetic practicality of modifying R2 groups to optimize NA inhibitory activity and pharmacological properties (> Fig. 2). X-ray crystallographic structures of Neu5Ac and its analogues complexed with NA show that two terminal hydroxyls of the glycerol side chain form a bidentate interaction with Glu276 [94,96]. However, it is also noted that C8 of the glycerol chain makes hydrophobic contacts with the hydrocarbon chain of Arg224 [72]. Therefore, it was expected that the optimization of this hydrophobic interaction would lead to new NA inhibitors with increased lipophilicity while maintaining potent NA inhibitory activity. This consideration was especially important for designing orally bioavailable drugs since balancing lipophilicity and water solubility could be as critical as the size of the molecule for its absorption from the intestinal tract. For optimization of hydrophobic interactions, the dimensions of the spacer would be expected to play an important role in binding affinity to the enzyme, as factors such as length, geometry, and conformational mobility. On the basis of this premise, a systematic modification of the R2 portion in 109 with various aliphatic side chains was conducted. Previous studies [72] suggested that the carboxylate of Neu5Ac2en (102) and its analogues form strong ionic interactions with three guanidino groups of Arg118, -292, and -371. The acetamido Carbasugars: Synthesis and Functions 9.2 1941 moiety at C5 of Neu5Ac2en and its analogues interacts with Arg152 and Glu227, and the methyl group fits nicely into a hydrophobic pocket formed by Trp180, Ile224, and Arg226. The acetamido group was also demonstrated to be optimal for antiviral activity [105]. The amino and guanidino groups at the C4 position of the Neu5Ac2en system were optimal for NA activity as described above [79,84]. Therefore, in the carbocyclic structure 109,theC1 carboxylate, C4 acetamido, and C5 amino groups were kept constant, while the C3 aliphatic group was optimized for antiviral activity. For structure-activity relationship studies of carbocyclic analogues 109, a general and efficient route to introduce various alkyl ethers at the C3 position was required. Also, although shikimic acid as a chiral starting material is convenient because of easy conversion to the key epoxy intermediate 112, its high cost and low availability in large quantities made it impractical for scale-up of 112. This meant that this would become a formidable challenge when multi-kilograms of material were needed for evaluation of clinical candidates for further drug development as well as frequent consumption of common synthetic intermediate 112 for SAR studies. In this stage, the development of the synthetic route, including the solutions of these issues, was achieved. With respect to the improvement of the synthetic route of 112, (–)-quinic acid (120), which was used as a starting material for the synthesis of 110a as shown in > Scheme 17, appeared to be an ideal starting material because of its low cost and commercial availability. The focus on the synthesis is the selective dehydration of the C2 hydroxyl in 129. Tosylate 129 was reacted with sulfuryl chloride in pyridine, followed by acetonide cleavage in refluxing methanol in the presence of p-TsOH. Interestingly, the desired diol 130 was directly crystallized out of the reaction mixture in 54% overall yield, and in the dehydration and acetonide removal sequence for the conversion of 129 to 130, the other olefinic regioisomer of 130 was aromatized under the reaction condition and easily separated by crystallization. Gilead’s group reported that this procedure would be amenable to a several hundred gram scale. Finally, epoxide 112 was produced in quantitative yield by treatment of 130 with DBU in THF, and the synthesis of epoxide 112 from quinic acid has been achieved in good overall yield without column chromatography. Also, regarding a general and efficient route to introduce various alkyl ethers at the C3 position, the approach, as outlined in > Scheme 18, relied on the aziridine opening of 132 with alcohols, which should in principle be highly regioselective due to the preferred nucleophilic attack at the C3 allylic position. The requisite aziridine 132 was derived from the trans amino alcohol 131 by the two-step, one-pot process: (1) selective protection of the amino functionality with TrCl and (2) mesylation of the hydroxyl in the presence of Et3N. Under these conditions, the mesylate intermediate was converted into aziridine 132. Treatment of 132 with various alcohols in the presence of BF·Et2O followed by acetylation of the crude product provided the ether 133 in 55–88% yield. This aziridine ring opening proceeded regio- and stereospecifically, and no other regio- and stereoisomer were observed. Finally, reduction of the azide functionality and saponification of the ester group in 133 gave 109. Thus, the convergent and efficient synthesis of various ether analogues of 109 has been achieved from intermediate 112 which is readily available from quinic acid as well as shikimic acid (> Scheme 18). The NA inhibitory activities of the carbocyclic analogues were evaluated in two assay systems (> Table 2). The intrinsic activity of each compound was assessed by measuring the inhibition of enzymatic activity, and compounds that exhibited potent NA inhibitory activity were further evaluated in cell culture by a plaque reduction assay using an influenza A (H1N1) strain. 1942 9 Glycomimetics

⊡ Scheme 18

As shown in > Table 2, the length, size of branching, and geometry of the alkyl groups in 109 profoundly influence the NA inhibitory activity. In a series of linear alkyl analogues (109b–e), steady increases in the enzyme inhibitory activity were observed up to the n-propyl analogue 109d. The over 20-fold increase in the NA inhibitory activity for 109d compared to the methyl counterpart 109b implicated a significant hydrophobic interaction of the n-propyl group with amino acids in the active site. Branching at the β-carbon of the n-propyl group (compound 109f) resulted in no enhancement of NA inhibitory activity compared to that of 109d. In contrast, branching at the α-carbon of the n-propyl group resulted in almost 20-fold enhance- Carbasugars: Synthesis and Functions 9.2 1943 ⊡ Table 2 Influenza neuraminidase inhibition and plaque reduction by carbocyclic analogues

ment of NA inhibitory activity. This improved potency undoubtedly arose from additional hydrophobic interactions in the NA active site generated by the α-methyl group. Remarkably, the absolute stereochemistry of the α-methyl group (109g and 109h) did not influence the NA inhibitory activity as they exhibited almost equal potency. Gilead’s group concluded that this result means it can be speculated that the methyl and ethyl groups in the sec-butyl analogues 109g and 109h contribute almost the same binding energy by an equal degree of interaction with amino acids in the active site. This hypothesis was further supported by NA activity of 3-pentyl analogue 109i (=105), which possesses two identical branched ethyl groups and shows the NA inhibitory activity increase of almost 10-fold that of either isomers 109g and 109h. Further extension of the two ethyl groups to the 4-heptyl analogue 109j resulted in a decrease of NA inhibitory activity, suggesting that the 3-pentyl group in 109i (=105) might provide the optimum hydrophobic contact with the NA active site. Interestingly, the enzymatic and plaque reduction activities of 109i (=105) were comparable to those of 104,which exhibited over 100-fold increase in activity compared to that of amino analogue 103. Replace- 1944 9 Glycomimetics ment of the amino group in 109i (=105) with the guanidino moiety resulted in a significant increase in its enzymatic and cell culture activity compared to 109i (=105). Thus, carbocyclic NA inhibitors represented by 109i (=105) and its guanidino analogue are more potent NA inhibitors than any inhibitors including compounds 103 and 104. Finally, ethyl ester of 109i (=105) (designated as GS-4104, 106) exhibited good oral bioavailability in several animals (mice, rats, and dogs) and demonstrated oral efficacy in the mouse and ferret influenza mod- el. On the basis of potent in vitro/in vivo activity and favorable pharmacological properties, GS-4104 (106) has been selected as a clinical candidate for the oral treatment and prophylaxis of influenza infection.

Synthetic Method for Process Chemistry After GS-4104 (106) was identified as a clinical candidate, a practical kilogram-scale preparation of 106 was necessary to supply an acceler- ated program of clinical and toxicological studies. The key structural feature in 106,interms of both pharmacological activity and synthetic challenge, was the 3-pentyl ether group. The aforementioned discovery synthesis of 106 constructed this 3-pentyl ether by acid-catalyzed opening of the tritylaziridine 132 with 3-pentanol (Route [A], > Fig. 4). The tritylaziridine 132 was prepared stereospecifically from either (–)-quinic acid (120) or (–)-shikimic acid (111), but both sequences required double inversion and repeated protection/deprotection of the (R)-3-hydroxyl group. The generality of the tritylaziridine opening for a wide variety of alcohols and other nucleophiles facilitated the rapid creation of structural diversity in the discovery program. However, identification of the lead compound 106 allowed for the design of a more efficient approach. Rohloff et al. developed a new 12-step synthesis of 105 utilizing a novel and efficient reductive ketal opening of 134 to construct the 3-pentyl ether (Route [B], > Fig. 4)[106]. This new process is highly applicable to kilogram-scale synthesis in that it fea-

⊡ Figure 4 Synthetic strategies of 105 and 106 Carbasugars: Synthesis and Functions 9.2 1945

⊡ Scheme 19

tures only three isolated crystalline intermediates and requires no chromatography (> Fig. 4). Compound 134 was synthesized in 80% yield from natural shikimic acid (111)(> Scheme 19). However, due to the limited commercial availability of (–)-shikimic acid (111), an efficient preparation of ketal 134 from (–)-quinic acid (120) was developed as shown in > Scheme 20. The quinic lactone acetonide 135a was prepared in 90% yield from 120 by modification of the method of Shing [107], and this was converted to a 1:5 equilibrium mixture of lactones : hydroxyl ester 135a:136a in anhydrous EtOH (1 M) containing catalytic NaOEt. Separation of the 135a:136a mixture by fractional crystallization was found to be inefficient on the kilogram scale, and instead, the crude 135a:136a mixture was treated with MsCl (1.1 equivalents) in CH2Cl2 in the presence of Et3N to chemoselectively afford the monomesylates 135b:136b in an unchanged ratio of 1:5. Undesired, but highly crystalline, lactone mesylate 135b was readily removed by filtration of an EtOAc slurry of the 135b:136b mixture. Upon evapora- tion of the filtrate, crude oily 136b was isolated in 69% overall yield from (–)-quinic acid (120). Dehydration of 136b to form the unsaturated “shikimic” ring system was accomplished ◦ with SO2Cl2 and pyridine in CH2Cl2 at –20 . A mixture of 1,2- and 1,6-olefin regioisomers 137:138 (ratio 4:1) was obtained in 60% yield along with 10–15% of oily α-chloro compound 139. Because of the high crystallinity of both olefin isomers, a high-throughput fractional crystallization could not be accomplished. Instead, it was found that treatment of the crude 137:138:139 mixtures with pyrrolidine and catalytic (Ph3P)4Pd in EtOAc led to selective conversion of the undesired 1,6-olefin (allylic mesylate) 138 into pyrrolidine substitution product 140 [108,109]. This 1,6-olefin adduct was readily removed by aqueous H2SO4 extraction. The pure 1,2-olefin isomer 137 was then isolable by crystallization from EtOAc/hexane in 30% overall yield from 120. In order to complete the link to the (–)-shikimic acid route, the quinic acid-derived acetonide 137 was transketalized using catalytic perchloric acid in 3-pentanone at ambient temperature with continuous vacuum distillation of acetone. The 3,4-pentyliden ketal 134 was isolated in nearly quantitative yield and was identical in all respects to the ketal 134 prepared from (–)-shikimic acid (111). A more concise route based on initial formation of the 3,4-pentylidene ketal analogues of 135a, 136a,and136b from (–)-quinic acid (120)was found to be impractical for scale-up due to the lack of crystallinity in the 3,4-pentylidene series (> Scheme 20). Treatment of 134 with TMSOTf and BH3·Me2SinCH2Cl2 under modified Hunter conditions [110], gratifyingly afforded a 10:1:1 mixture of isomeric pentyl ethers 141a:141b and diol 141c in 75% yield. Separation of the 141a:141b:141c mixture was not possible through fractional crystallization or chromatography, but heating the crude mixture in aqueous EtOH in the presence of KHCO3 selectively converted 141a into the alkane-soluble epoxide 142. Heptane extraction gave crystalline epoxide 142 in 60% overall yield from ketal 134. Epoxide 1946 9 Glycomimetics

⊡ Scheme 20

◦ 142 was heated at 70 C with NaN3 and NH4Cl in aqueous EtOH to afford an oily 10:1 mixture of isomeric azido alcohols 143a:143b. Intramolecular reductive cyclization of the crude ◦ 143a, b mixture with Me3P in anhydrous MeCN at 35 C cleanly afforded a single aziridine ◦ 144 of ca. 70% purity. Aziridine opening proceeded smoothly at 80 C with NaN3 and NH4Cl in DMF affording the corresponding azidoamine, which was directly acylated with Ac2O. The resulting azidoacetamide 145 (m.p 137–8◦C) was isolated, after recrystallization in 37% overall yield from epoxide 142. Because of its stability and high crystallinity, azidoacetamide 145 has proven to be an excellent ﬁnal intermediate for drug substance synthesis. To complete Carbasugars: Synthesis and Functions 9.2 1947

⊡ Scheme 21

the synthesis, azide reduction of substrate 145 was accomplished using catalytic hydrogena- ◦ tion with Raney Ni in EtOH (1 atm H2)at35 C. After removal of catalyst by ﬁltration, 85% phosphoric acid was added. The salt 106·H3PO4 crystallized as feathery needles, and it was isolated in 71% yield from 145. Consequently, overall, 106 waspreparedin12stepsand4.4% yield from (–)-quinic acid (120) (Average 77.2% yield/step), and (formally) in ten steps and 21% yield from (–)-shikimic acid (111), and kilogram quantities of drug substance for clinical and toxicological studies have been prepared by this route (> Scheme 21).

Novel Synthetic Routes The recent emergence of the avian virus H5N1 raises the possibility of a pandemic wave of life-threatening influenza that requires prompt action. Currently, a four-pronged effort to avert widespread disease is now underway, and the effort consists of the following components: (1) worldwide surveillance of both wild and domesticated birds with quick culling of the latter, (2) development of recombinant vaccines against the H5N1 1948 9 Glycomimetics virus and its mutated forms whose production can be scaled up rapidly, (3) procedure for quar- antine, and (4) ramped up production of the orally effective, synthetic neuraminidase inhibitor tamiflu (106). Especially, the stock of tamiflu (106) is becoming a serious social problem, and it is true that many nations have planned to stock a significant amount of 106 to protect against a possible influenza outbreak. The current commercial synthetic route of 106 uses naturally occurring (–)-shikimic acid (111) or quinic acid (120) as a starting material. However, as mentioned in previous sessions, these starting materials are complex, relatively expensive, and of

⊡ Scheme 22 Carbasugars: Synthesis and Functions 9.2 1949 limited availability, therefore, a more reliable source is desired for constant and large-scale supply of 106. In 2006, two groups independently reported the novel asymmetric synthesis of tamiflu (106). Corey et al. reported a short enantioselective pathway for the synthesis of 106 from 1,3-butadiene and acrylic acid shown in > Scheme 22 [111]. The key steps of the synthesis are (1) Diels– Alder reaction of 1,3-butadiene (146) and trifluoroethyl acrylate (147) in the presence of chiral ligand 148 developed in the laboratory [112], (2) the introduction of two amino groups in tamiflu (106) without using potentially hazardous and explosive azide reagents, and (3) a novel SnBr4-catalyzed bromoacetamidation. The initial Diels–Alder step easily provided adduct 149 at room temperature on multigram scale in 97% yield and with > 97% ee; recovery of the chiral ligand corresponding to 148 is simple and efficient. Ammonolysis of 149 produced amide 150 quantitatively, and subsequent iodolactamization of 150 using the Knapp protocol [113,114] generated lactam 151, which was transformed by N-acylation with (Boc)2O into Boc derivative 152 in very high yield. Dehydroiodination of 152 occurred cleanly with DBU to give 153, which was allylical- ly brominated using NBS to generate 154 very efficiently. Treatment of 154 with CsCO3 in EtOH afforded the diene ethyl ester 155 quantitatively. The next step in the synthetic sequence was a novel SnBr4-catalyzed bromoacetamidation reaction which was completely region- and stereoselective using N-bromoacetamide (NBA) in MeCN at −40◦C that converted the diene 155 to the bromodiamide 156. It is surmised that this bromoacetamidation process involves + the transfer of Br from a SnBr4-NBAcomplexedtotheγ , δ-bond of the diene ester 155 followed by nucleophilic attack on the intermediate bromonium ion. Cyclization of 156 to the N-acetylaziridine was rapid and efficient using in situ generated tetra-n-butylammonium hexamethyldisilazane and provided the bicyclic product 157. Reaction of 157 in 3-pentanol solution containing a catalytic amount of cupric triflate at 0◦C occurred regioselectively to generate the ether 158. Finally, removal of the Boc group and salt formation with H3PO4 in EtOH afforded 106·H3PO4 (tamiflu) (> Scheme 22). On the other hand, Shibasaki et al. reported the synthesis of 106 utilizing a general catalytic enantioselective ring-opening of meso-aziridines with TMSN3 (> Fig. 5, > Scheme 23)[115].

⊡ Figure 5 Key step of asymmetric synthesis of tamiﬂu (106) utilizing a general catalytic enantioselective ring-opening of meso-aziridines with TMSN3 1950 9 Glycomimetics

⊡ Scheme 23

The basic concept of this synthesis is on the extension of catalytic desymmetrization of meso- azridines (CDMA) with TMSCN using a poly Gd complex derived from ligand 160 [116], and this synthesis also includes the investigation of the possibility of extending this catalysis to using TMSN3 as the nucleophile. As the results of optimization of the reaction conditions, N-3, 5-dinitrobenzoyl aziridines were selected as the substrates and the screening of rare earth i alkoxides as the catalyst metal source indicated that Y(O Pr)3 was optimum. On the basis of i these results, meso-azirizine 159 was treated with 2 mol% of Y(O Pr)3, 4 mol% of ligand 160, Carbasugars: Synthesis and Functions 9.2 1951 and 1.5 equivalents of TMSN3 in EtCN to afford the product 161 in 96% yield with > 91% ee (> Fig. 5). It can be predicted that the reaction should proceed through a mechanism similar to the CDMA with TMSCN [116]: generation of a reactive yttrium azide from TMSN3 through transmetalation [117] and intramolecular transfer of the azide to an activated acylaziridine by a Lewis acidic yttrium in the same poly Y catalyst [118]. Finally, products were converted to optically active C2 symmetric 1,2-diamines in excellent yield. The main tasks required for the synthesis of 106 from 161 were the introduction of an oxygen functionality at the allylic position and an ethoxycarbonyl group at the olefin. After obtaining enantiomerically enriched 161 by recrystallization, C2 symmetric diamide 163 was synthesized in four steps. Allylic oxidation of 163 with SeO2 in the presence of Dess–Martin periodinane produced a mixture of enone 164 and allylic alcohol 165 (ca. 2:3), which was treated without purification by Dess–Martin periodinane, giving 164 in 68% yield. Enantiomer- ically pure (> 99% ee) 164 was obtained at this stage by recrystallization. A 1,4-addition of TMSCN in the presence of 10 mol% of Ni(COD)2 followed by treatment with NBS and Et3N produced γ -keto nitrile 166, which was selectively reduced with bulky aluminum reagent, t LiAlH(O- Bu)3 to give alcohol 167. Aziridine formation of 167 under Mitsunobu conditions furnished 168 and, subsequently, BF3·Et2O-mediated aziridine opening of 168 with 3-pentanol afforded 169. Treatment of 169 with TFA, followed by protection of the sterically less hindered amine with a Boc group and acetylation afforded 170, and conversion of nitrile 170 to ethoxycarbonyl in acidic EtOH concomitant with removal of the Boc group, and H3PO4 salt formation afforded tamiflu (106)(> Scheme 23). Recent advances in structural biology have led to new drug discovery processes with the neuraminidase inhibitors such as tamiflu (106) and relenza (104) being notable achievements from this scientific area. It should be emphasized that tamiflu (106) does not originate from a natural substrate but is an artificially designed glycosidase inhibitor based on structural biological data, which has already been prescribed for influenza patients in practice. A variety of drugs designed using structural biology, in addition to tamiflu, will appear in the clinical market in the near future.

3.2 New Generations of Glycosidase Inhibitors Bearing 5-Membered Cyclitols

Among the glycosidase inhibitors, there is a type containing 5-membered cyclitol, which also exhibits interesting enzyme-specific inhibitory activities. Interestingly, most of the 5-membered cyclitols are aminocyclitols. For instance, mannostatine, which is a 5-membered aminocyclitol derivative, shows β-mannosidase-specific inhibitory activity, and allosamidin possessingthe [3.3.0] bicyclic aglycone called allosamizoline, which consists of a 5-membered aminocyclitol, exhibits inhibition towards chitinase specifically. Furthermore, trehazolin possessing the [3.3.0] bicyclic aglycone called trehalamine, which also consists of a 5-membered aminocyclitol, shows the trehalase-specific inhibitory activity. One of the scientific focuses on the researches of allosamidin and trehazolin is the elucidation of structure and activity relationships on the stereochemistry of their aglycons and their enzyme-specific inhibitory activities. . Therefore, while a variety of synthetic methodologies to form their scaffolds and to introduce the functional groups focused on total synthesis of the aforementioned glycosidase 1952 9 Glycomimetics inhibitors, have been developed, the general application of the methodologies for the synthesis of their related compounds to investigate the SAR have also been reported. Herein, the new generations of glycosidase inhibitors, allosamidin and trehazolin, which possess the 5-membered aminocyclitol unit and exhibit interesting tri- or disaccharide hydrase-specific inhibitory activities, will be introduced and a variety of syntheses of the aglycones of allosamidin, allosamizoline, and trehazolins will be described.

3.2.1 Allosamizoline: The Aglycone of the Chitinase-Specific Glycosidase Inhibitor, Allosamidin The importance of chitin as one of the main structural components of insect cuticles [119,120] and fungal cell walls [121,122] generates interest in discovering agents that may interact with its biosynthesis. With this in mind, allosamidin was discovered as expected. Allosamidin (171) is a novel pseudotrisaccharide as shown in Structure 4; it was isolated from the mycelial extract of Streptomyces sp. no. 1713, and shows the chitinase-specific inhibitory activity [123]. Its structure was deduced from hydrolysis experiments which produce 2 equivalents of D-allosamine and 1 equivalent of the aglycone moiety, allosamizoline (172). In particular, its aglycone moiety, allosamizoline has an interesting structure; it is a [3.3.0] bicyclic compound possessing aminocyclitol and dimethyl aminooxazoline parts. At first, the structure of allosamizoline was suggested to be the cis-diol (allo-isomer) 173 [124] and later revised to the trans-diol 172 [123. 124]. In the course of its correct structural assignment, the necessity for the synthesis of a series of allosamizolines (172–175) appeared and many chemists directed their efforts to the construction of these fascinating structures.

Trost’s group and Danishefsky’s group reported the total syntheses of allosamizoline independently and almost simultaneously. Both groups chose the racemic cyclopetenediol deriva- Carbasugars: Synthesis and Functions 9.2 1953

⊡ Figure 6 Diversity of the syntheses of allosamizoline (172) from cyclopentendiols 176 and 177

tive 176, which was derived from cyclopentadienylthallium [127], as the starting material, and also Ganem’s group also reported the synthesis of allosamizoline from the relevant cyclopentenediol derivative 177 later (> Fig. 6). First, Trost’s allosamizoline synthesis will be discussed [128,129]. The basic concept of their synthesis is shown in Fig. > Fig. 7. They have studied the use of Pd(0)-catalyzed reactions to provide the aminocyclitols with varying regio- and stereoselectivity. Also, they envisioned that the cis-vicinal aminocyclitols might be derived either from epoxides in a single step as in > Fig. 7 (Eq. (1), path a) [130,131] or from their synthetic equivalents such as 2-alkene-1,4- diols as illustrated in the one-pot sequence of > Fig. 7 (Eq. (1), path b) [132,133,134], with the bisurethane being generated in situ, and, in both cases, the regio- and diastereoselectivity might be assured by covalent tethering of the nitrogen nucleophile to the substrate. Moreover, they have found that signiﬁcant levels of enantioselectivity may be achieved in the ionization process of the Pd(0)-catalyzed alkylation illustrated in > Fig. 7 (Eq. (2)) and have developed a class of easily prepared chiral phosphine ligands which allow the prediction of the absolute stereochemistry of the oxazolidinone product based on the stereochemistry of the ligand precursors [133,134,135,136,137,138,139,140,141]. On the basis of their preliminary synthetic concept, the allosamizolines 172–175 were synthesized as shown in > Scheme 24, 25, and 26. The ﬁrst synthesized allosamizolines were racemates and were separated as optically pure components after glycosidation with a sugar moiety, to give di-D-allosamine derivatives. Furthermore, they are continuing development of the asymmetric version by using chiral phosphine ligands. The cyclopentenediol 176, which was derived from cyclopentadienylthallium, was treated with TsNCO and a Pd(0) catalyst to afford the oxazolidinone 178. In a further study of this step, the use of 3 mol% of (dba)3Pd2CHCl3 and 6 mol% of (–)-BINAPO at ambient temperature gave a 91% yield of oxazolidinone 178 with 59–65% ee [134]. After reductive de-N-tosylation of 178, subsequent O-methylation of oxazolidinone 179 with MeOTf and exposure of the resultant imino ether to dimethylamine furnished the aminooxazoline 180. Treatment of aminooxazoline 180 with 1954 9 Glycomimetics

⊡ Figure 7 Basic concept of Pd(0)-mediated optical resolution for the synthesis of allosamizoline (172) reported by Trost’s group

◦ 5.4 M CF3CO3H, exposure of the corresponding epoxide to 10% aqueous TFA at 40 C, and subsequent hydrogenolysis afforded allosamizoline 172 (> Scheme 24). In the meantime, the gulo-isomer 174 was also derived from the intermediate 180, via minor product 182 after the treatment with 1.2 M CF3CO3H and subsequent exposure of the corre- ◦ sponding epoxide to 10% aqueous TFA at 65 C(> Scheme 25). On the other hand, the initially proposed structure, the cis-diol (allo-isomer) 173 and its galac- to-type isomer 175 were synthesized from the same aminooxazoline 180 via dihydroxylation with OsO4 as shown in > Scheme 26. Also, the concept and outcomes for this synthetic study were applied to the syntheses of mannostatine A [129] and valienamine [142]. Carbasugars: Synthesis and Functions 9.2 1955

⊡ Scheme 24

⊡ Scheme 25 1956 9 Glycomimetics

⊡ Scheme 26

Next, Danishefsky’s allosamizoline synthesis will be described [143,144]. The key point of their synthetic strategy is the utilization of enzymatic optical resolution to the racemic substrate. As illustrated in > Fig. 8, there are two approaches for the enzymatic optical resolution. One is the enzymatic hydrolysis of a diester [145,146,147], and the other is the enzymatic transacylation of the meso-diol [148,149,150](> Fig. 8). In Danishefsky’s group, the former route was chosen as the key step. Treatment of diacetate 186 with electric eel acetylcholinesterase provided the monoacetate 187, which was reported by Deardorrf et al. [147]. This work was also applied to the synthesis of PG F2a in Danishefsky’s laboratory [151]. On the basis of the success of their synthesis of PG F2a, diacetate 188, which was derived from the 2-alkene-1,4-diol derivative 176, was treated with electric eel acetylcholinesterase as well. Interestingly, this treatment provided the unexpected monoacetate 189 in 95% yield, > 95% ee (> Fig. 8). Carbasugars: Synthesis and Functions 9.2 1957

⊡ Figure 8 Basic concept of enzymatic optical resolution for the synthesis of allosamizoline (172) reported by Danishefsky’s group

This result initially discouraged Danishefky’s group to complete the synthesis of the intact allosamizoline 172. However, eventually, Danishefsky et al. succeeded in the conversion of this undesired monoacetate ent-189 to the intact allosamizoline (172)shownin> Scheme 27. After silylation of the corresponding hydroxyl group of ent-189, the remaining Ac group was removed with NH3 in MeOH to give the alcohol 190. After introduction of a carbamate group by reaction with ClCO2Ph and NH3, the TBDMS group was cleaved to provide the alcohol 191. Treatment of 191 with TFAA and Et3N furnished the optically active oxazolidinone 179, which was the intermediate in Trost’s synthesis. O-Methylation of the optical active oxazolidinone 179 with MeOTf and exposure of the resultant imino ether to dimethylamine furnished the corresponding optical active aminooxazoline. This aminooxazoline was treated with CF3CO3H and the corresponding epoxide was exposed to 10% aqueous TFA; subsequent hydrogenolysis under acidic conditions afforded the optical active allosamizoline (172). In the meantime, the resulting dibenzyl ether 192, which was regioselectively benzylated after treatment of 181 with Bu2SnO, was converted into allosamidine (171) via the KHMDS-promoted coupling with bromosulfonamide-disaccharide, which is by way of the interesting glycosidation developed by this group (> Scheme 27). As mentioned above, Ganem’s group also synthesized allosamizoline (172) from the 2-alkene- 1,4-diol derivative 177 [152]. They derived the 2-alkene-1,4-diol derivative 177 from cyclopentadienylthallium [127] via alkylation with SEMCl, cycloaddition of the corresponding cyclopentadiene 193 with singlet oxygen, and subsequent reduction in situ of the transient endoperoxide. This 2-alkene-1,4-diol derivative 177 was treated with NBS in DMSO to afford 1958 9 Glycomimetics

⊡ Scheme 27 Carbasugars: Synthesis and Functions 9.2 1959

⊡ Scheme 28

a single bromohydrin 194. The structure was conﬁrmed by cyclization with Na2CO3-MeOH to furnish exclusively the epoxide 195 possessing the desired stereochemistry. Ring-opening with NaN3 furnished the racemic azidotriol 196, and subsequently compound 196 was reduced to corresponding aminotriol. Cyclization with thiocarbonyldiimidazole produced the thioox- azolidinone 107. After a one-step construction of the dimethylaminooxazoline ring had been achieved by heating with Me2NH-MeOH, deprotection of the silyl group gave the racemic allosamizoline (172)(> Scheme 28). Allosamizoline (172) was also synthesized from natural sugar. Tatsuta et al. reported the synthesis of allosamizoline (172) from D-glucosamine using an intramolecular [3+2] cycloaddi- tionasakeystep[153]. On the other hand, Kitahara’s group also independently achieved the synthesis of allosamizoline using the same key step [154]. First, Tatsuta’s allosamizoline synthesis will be described. Iodination of methyl 2-deoxy-2-phthalimide-D-glucopyranoside 199, which was readily derived from D-glucosamine 198, and subsequent reductive β-elimination of the corresponding iodide gave the 5-enofuranose 200 concomitant with the reductively dehalogenated C6 product. Treatment of this mixture with EtSH and concentrated HCl, and subsequent silylation, furnished dithioacetal 201. Dedithioacetalization of 201 with HgCl2 and CaCO3 provided the corresponding aldehyde, and, subsequently, the aldehyde was treated with NH2OH·HCl to afford oxime 202. Intramolecular [3+2] cycloaddition of oxime 202 with aqueous NaOCl furnished the isoxazoline 203 via nitrile oxide [13,14]. Isoxazoline opening 1960 9 Glycomimetics

⊡ Scheme 29

of 203 was a troublesome step, however, and as a result of screening the reaction conditions, it was completed by hydrogenolysis with Raney-Ni W4 in the presence of B(OH)3 and AcOH or ozonolysis [155]togivetheβ-hydroxy ketone 204. The reduction of 204 with Zn(BH4)2 proceeded stereoselectively and afforded a single diol 205.Afterde-N-phthaloylation of 205, benzyloxycarbonylation followed by base treatment gave the oxazolidinone 206. Acid desilylation and subsequent acetylation afforded the triacetate 207.ThenO-methylation of 207 with MeOTf, followed by the treatment with Me2NH·HCl and Et3N, as well as further acidic deacetylation furnished allosamizoline (172)(> Scheme 29). Carbasugars: Synthesis and Functions 9.2 1961

⊡ Scheme 30

As shown in > Scheme 30, Kitahara et al. also derived allosamizoline (172) from D-glucosamine 198 via Tatsuta’s intermediate, isoxazoline 203,aswell[154]. As described in the session on cyclophelitol, the Ferrier reaction is a very useful method to transform sugars into carbasugars, and Kuzuhara’s group used this reaction in the synthesis of allosamizoline (172)shownin> Scheme 31 [156]. A further interesting point in their synthesis is the transformation of the [4.3.0] bicyclic structure, which was derived from D-glucosamine 198 via the Ferrier reaction, to the [3.3.0] bicyclic scaffold of allosamizoline (172). It was performed by reductive ring contraction to the α-hydroxy tosylate under the action of L-selectride. Selective iodination of the N, N-dimethylurea-diol derivative 211 with NIS and PPh3 [157], whereby 211 was derived from D-glucosamine 198 via the 4, 6-O-benzylidene compound 210, silylation of the secondary hydroxyl group, and subsequent dehydroiodination of the corresponding iodide with t-BuOK gave the enol ether 212. For conversion of the ring system from pyranose to cyclohexane, the modiﬁed Ferrier reaction with HgSO4 [158]was employed and, furthermore, β-elimination of the resulting ketol with MsCl-pyridine provided the enone 213. 1,2-Reduction of this enone 213 with NaBH4 and CeCl3·7H2O proceeded stereoselectively and subsequent treatment of the corresponding allyl alcohol with Ms2Oand Et3N furnished the dimethylaminooxazoline ring 214 simultaneously [159]. Dihydroxylation of 214 with OsO4 proceeded exclusively from the convex face and, fortunately, the subsequent sluggish monotosylation gave the desired monotosylate 215. Ring contraction of 215 in basic media was regarded as the key step in this synthesis. As the expected rearrangement product 216 bearing an aldehyde group was assumed to lack stability in basic media, 1962 9 Glycomimetics

⊡ Scheme 31

an appropriate basic reducing agent [160] was screened for the ring contraction followed by immediate reduction of aldehyde. As the result, L-selectride was selected to give the desired cyclopentanemethanol 217. Finally, compound 217 was converted into allosamizoline (172) by deprotection of the TBDMS and Bn groups (> Scheme 31). Carbasugars: Synthesis and Functions 9.2 1963

⊡ Scheme 32

Another synthesis of allosamizoline (172) from sugar was reported by Simpkins’ group, and the key step of their synthesis is a radical cyclization to construct the 5-membered aminocyclitol [161,162]. So far, radical cyclization for the conversion of carbohydrates to carbasugars has been problematic because of the suitability of carbon-centered radicals for the preparation of the highly functionalized 5-membered ring [163]. However, significant contributions in this field, most notably by Rajanbabu [164], the possibility of using an aldehyde as the radical acceptor in the key cyclization reported by Fraser-Reid [165,166], and the cyclizations of radicals onto oxime ether reported by Bartlett [167] have paved the way to the formation of the highly functionalized 5-membered rings. Finally, Simpkins et al., focusing their attention 1964 9 Glycomimetics on a report by Bartlett [167], accomplished the synthesis of allosamizoline (172). In a way, Simpkins’ group has verified the usefulness of the concept of radical cyclization for the formation of the highly functionalized cyclitols through the synthesis of allosamizoline (172) shown in > Scheme 32. Treatment of the N-Cbz-tri-O-acetyl sugar 218, which was derived from D-glucosamine 198 in three steps, with the O-benzyl ether of hydroxylamine, followed by derivatization of the secondary alcohol with thiocarbonylimdazole gave the compound 219. This oxime derivative was treated with Bu3SnH and AIBN according to Bartlett’s protocol, to afford a mixture of diastereomeric products 220. Judging from the configuration of the major product 220a, obtained as a mixture of C1 epimers, it was expected to be convertible into allosamizoline (172). Oxidation of the mixture of benzyloxyamines with mCPBA afforded the corresponding oxime [168], and ozonolysis of the resulting oxime, followed by direct reductive work up gave the alcohol 221. Treatment of 221 with SOCl2 provided the oxazolidinone 207, and the reaction proceeded with inversion at C1. O-Methylation of 207, followed by the exposure to Me2NH, and deacetylation gave allosamizoline (172)(> Scheme 32). Interestingly, in each case of the synthesis of allosamizoline (172) from sugar, D-glucosamine 198 was chosen as the starting material, and, in fact, there is the notion that D-glucosamine 198 is considered to be a biosynthetic precursor of allosamizoline (172). In a way, these syntheses from D-glucosamine might pave the way to elucidate the biosynthetic route of allosamizoline (172).

3.2.2 Trehazolin, Trehalamine and Its Aminocyclitol Moiety, Trehazolamine: A Trehalase-Speciﬁc Glycosidase Inhibitor

Trehalose (222) is a structurally interesting disaccharide in which two D-glucoses are linked by an α(1–1)α glycosyl bond.

Biologically, it is considered that D-glucose degraded from trehalose (222) with trehalase, which is a kind of glycosidase and is possibly used as an energy source by insects, and a series of glycosidase inhibitors exhibiting inhibitory activity towards this enzyme, would be expected to be a new type of potential insecticide. In 1991, Ando et al. reported the isolation of trehazolin (223) from the culture broth of Micromonospora sp. strain SANK 62390 [169,170]. This compound is a unique natural pseudodisaccharide showing a strong trehalase-speciﬁc inhibitory activity. Nearly concomitantly, the isolation of a compound named trehalostatin (224) from the culture broth of Amicoratopsis trehalostatica was also reported [171]. Carbasugars: Synthesis and Functions 9.2 1965

Trehalostatin (224) was reported to be the C5 epimer of the aglycone of trehazolin (223). According to the reported physical and biological data, trehalostatin (224) seems to be identical to trehazolin (223). Because both compounds are noncrystalline, X-ray crystallography could not be used to determine the correct stereochemistry; hence, the stereochemistry at the C5 position of the aglycones remains unknown. Therefore, a series of degradation studies of trehazolin (223) was attempted to try to obtain degradation products from which the correct stereochemistry could be deduced. Hydrochloric acid degradation provided degradation products: the aglycone moiety trehalamine (225), D-glucose, and the aminocyclitol moiety (226), from which we next obtained the acetylated product 227. However, all products were also noncrystalline. Consequently, they could not be used to determine the correct stereochemistry. In the meantime, further NMR analysis of trehazolin (223) and a series of biochemical studies to elucidate its trehalase-specific inhibitory activity were undertaken, and on the basis of 1966 9 Glycomimetics these studies, it was surmised that the actual structural resemblance between trehazolin (223) and trehalose (222) may bear on the generation of trehazolin’s activity towards various trehalases, and the absolute configuration of its aminocyclitol moiety (226) was hypothesized as [1R-(1α,2β,3α,4β,5β)]. Then, in order to verify the proposed correct structure of trehazolin (223), a series of synthetic studies were performed. The first enantioselective synthesis of trehazolin (223) and its components was accomplished by Kobayashi and Shiozaki et al. [172,173,174]. It was performed from D-glucose, and intramolecular [3 + 2] cycloaddition was used as the key step to form the 5-membered scaffold of the aminocyclitol moiety. As the first stage of the synthesis, the hexaacetate of the aminocyclitol moiety was synthesized to determine the absolute configuration of the aminocyclitol unit shown in > Scheme 33 and > Scheme 34 [173,174]. Intramolecular [3+2] cycloaddition of the oxime 229, derived from D-glucose according to the method developed by Bernet and Vasella [175], furnished the corresponding isoxazoline 230. In general, hydrogenolysis of isoxazolines 230 with Raney Ni in the presence of boric acid [176] results in conversion to a hydroxymethyl ketone. However, in this case, by virtue of the electron-withdrawing effect of the benzoyl group, β-elimination of the ben- zoyloxy group by the generated ketone was induced and the corresponding hydroxymethyl

⊡ Scheme 33 Carbasugars: Synthesis and Functions 9.2 1967

⊡ Scheme 34

enone 231 was obtained. Silylation of the enone 231 and subsequent 1,2-reduction of the corresponding ketone afforded a separable 1:2.5 mixture of alcohols 232. The configurations at the C1 positions of these alcohols 232 were determined by analysis of the 1H-NMR data of the acetates 233; that is, NOEs were observed between C5-H and C4-H of compound 233α and between C5-H and C3-H of compound 233β (> Scheme 33). Benzylation of the alcohol 232β, possessing the desired configuration, and subsequent removal of the TBDMS group of compound 234 afforded the corresponding allyl alcohol 235.Sever- al types of epoxidation towards the allyl alcohol 235 were attempted; Sharpless’ epoxidation using diisopropyl L-tartrate [177] furnished the desired epoxide 236 as a single isomer. Finally, the synthesis of hexaacetate of the aminocyclitol moiety (227) was accomplished by regiospecific azide opening towards the chiral epoxide 236, deprotection, and subsequent complete acetylation. This synthetic aminocyclitol hexaacetate was identical in all respects to the aminocyclitol hexaacetate obtained from the degradation product of natural trehazolin, and the absolute configuration was found to be [1R-(1α,2β,3α,4β,5β)] as expected (> Scheme 34). Next, the synthesis of trehazolin aglycone, trehalamine (225) was conducted as shown in > Scheme 35. The key point of this synthesis was the method by which the aminooxazoline ring was formed. Initially, the aminooxazoline formation, which was used for allosamizoline synthesis [128,129,143,144,153,161,162], was attempted via O-methylation of 240 or 241 derived from epoxide 236.TheO-alkylated compounds derived from 240 and 241 were exposed to benzylamine derivatives, but this method did not yield the desired product 242. Finally, the cyclization of thiourea alcohol via the carbodiimide alcohol was attempted. The aminooxazoline ring can be considered as an equivalent of the cis-carbodiimide alcohol, 1968 9 Glycomimetics

⊡ Scheme 35

and a number of methods to generate carbodiimides were investigated with the method of Mukaiyama [178,179] being selected. This synthetic method to derive carbodiimides from thiourea alcohol uses 2-chloro-3-ethylbenzoxazolium tetraﬂuoroborate 245 and related reagents, and was also applied to the synthesis of indolemycine [180]. The thiourea alcohol 244 derived from the azide alcohol 239 was treated with 2-chloro-3-ethylbenzoxazolium tetraﬂuoroborate 245, furnishing the aminooxazoline derivative 247 via transformation of Carbasugars: Synthesis and Functions 9.2 1969

⊡ Scheme 36 the cis-thiourea alcohol 244 to the corresponding carbodiimide alcohol 246 and subsequent cyclization of 246. Finally, the aminooxazoline 247 was hydrogenolyzed to cleave three benzyl groups and to give trehalamine (225). This synthetic trehalamine was identical in all respects to natural trehalamine (> Scheme 35). On the basis of the synthesis of trehalamine (225), the synthesis of trehazolin (223)was undertaken. Coupling between the amino alcohol derived from the azide compound 239, and the α-D-glucopyranosyl isothiocyanate derivative 248, as synthesized by Camarasa [181], afforded the α-D-glucopyranosylthiourea derivative 249. Treatment of this thiourea 249 with 2-chloro-3-ethylbenzoxazolium tetraﬂuoroborate 245 furnished the aminooxazoline derivative 251 via carbodiimide alcohol 250 as the intermediate. Finally, this aminooxazoline 251 was hydrogenolyzed to cleave the benzyl groups and to generate trehazolin (223). This synthetic trehazolin was identical to the natural trehazolin in all respects, including biological activities (> Scheme 36). Also, Ogawa’s group reported the synthesis of racemic trehazolin aminocyclitol derivatives, as well as trehazolin and its diastereoisomers from myo-inositol, and thus contributed independently to the determination of the correct stereochemistry of trehazolin [182,183,184,185,186]. The successful total synthesis of trehazolin (223) encouraged Kobayashi and Shiozaki et al. to investigate structure-activity relationships regarding the inhibitory activities towards vari- 1970 9 Glycomimetics

⊡ Figure 9 Synthesis of the trehazolin β-anomer

ous α-glucosidases resulting from the stereochemistry of trehazolin. They then designed the trehazolin stereoisomers.

To investigate the inﬂuence of the stereochemistry of the anomeric position on the inhibitory activities, the β-anomer of trehazolin (252) was synthesized from the azide 239 and the β-D-glucopyranosyl isothiocyanate derivative 254 [187]. To avoid anomerization and con- tamination of the α-anomer, 2-chloro-1-methylpyridinium iodide 255 was used in place of 2-chloro-3-ethylbenzoxazolium tetraﬂuoroborate 245 for the ring formation (> Fig. 9). At the step of the 1,2-reduction of the hydroxymethylenone 231, the allyl alcohol 232α was also obtained. Trehalostatin (the C5-epimer of trehazolin) (224) was then synthesized from this allyl alcohol 232α according to the synthetic route to trehazolin (223)[188]. The physical data, including the 1H-NMR spectrum, of trehalostatin (224), were quite close to those of trehazolin (223) itself, but the inhibitory activities of this compound towards trehalases were much weaker than those of trehazolin (223). As a result, the argued stereochemistries of the aminocyclitol moiety of trehazolin (223) and trehalostatin (224) were determined to be [1R- (1α,2β,3α,4β,5β)] and [1R-(1α,2α,3α,4β,5β)] by these synthetic studies, respectively (> Scheme 37). Carbasugars: Synthesis and Functions 9.2 1971

⊡ Scheme 37

Next, in order to investigate further the structure-activity relationships regarding the inhibitory activities towards various α-glucosidases resulting from the stereochemistry of the aminocyclitol moiety, the C-6 epimer of trehazolin (253) was synthesized [189,190]. In this synthesis, the challenging tandem aldol-Wittig type reaction was performed to construct the enone [II]. As shown in > Fig. 10, it was expected that treatment of the silylenol lactone [III],whichwas derived from D-ribonolactone, with the α-lithiated phosphorane [191] would give the enone [II] via the aldol reaction and subsequent intramolecular Wittig reaction in one pot (> Fig. 10). > Scheme 38 shows the practical synthesis of the C-6 epimer of trehazolin (253). Treatment of the silylenol lactone 259, which was derived from D-ribonolactone 258 in three steps, with the lithiated phosphorane furnished the cyclopentenone 260 in moderate yield. This reaction should thus have synthetic utility for the one-step synthesis of cyclic α, β-unsaturated ketones from cyclic enol ester-type derivatives. Afterwards, the synthesis proceeded basically according to the trehazolin synthesis, including 1,2-reduction of the enone 260, stereoselective epoxidation, regiospeciﬁc azide-opening of the epoxide 262, coupling between the amine derived from compound 263 and the isothiocyanate 248, and cyclization to form the aminooxazoline ring 265. Finally, compound 265 was hydrogenolyzed to cleave the benzyl groups and to furnish trehazolin C-6 epimer (253) (> Scheme 38). 1972 9 Glycomimetics

⊡ Figure 10 Synthetic strategy of trehazolin C-6 epimer (253) via tandem aldol-Wittig reaction

Ganem et al. also reported the synthesis of the trehazolin C-6 epimer (253) and the formal total synthesis of trehazolin (223)[192,193]. The synthetic strategy used was related to the synthesis of allosamizoline, and involved the cyclopentadiene derivative 266,which was also used for the synthesis of allosamizoline, as the starting material. Treatment of the cyclopentadiene 266 [194] with (S)-mandelohydroxamic acid 267 in the presence of Bu4NIO4 led to a mixture of the desired cycloadduct 268 and its diastereomers. The inseparable mixture was reduced using Na(Hg) for separation to afford the pure cyclopentene 269a and the minor cycloadduct 269b. Compound 269a was converted to acetate 270 for characterization of the structure. Dihydroxylation of 270 with OsO4 favored syn addition, and subsequent acetylation gave compound 271a selectively and quantitatively. The next key point in Carbasugars: Synthesis and Functions 9.2 1973

⊡ Scheme 38 the synthesis was the introduction of a quaternary stereocenter. Hydrogenolysis of both ben- zylic ether and Ac groups of 271a using Pd(OH)2 on carbon afforded the alcohol 272,and nitrophenylselenation followed by in situ oxidative elimination according to the method of Grieco et al. [195] cleanly converted 272 into alkene 273. Flanked by two allylic substituents shielding the top face of the 5-membered ring, the exocyclic alkene increment in 273 underwent vicinal hydroxylation exclusively from the opposite face and subsequent acetylation furnished acetate 274. Exhaustive acid hydrolysis of 274 provided the aminocyclitol 275 required for the synthesis of the C6-epimer of trehazolin (253). This aminocyclitol hydrochloride 275 was converted into the C6 epimer of trehazolin (253) in analogy to the syntheses of trehazolin (223) and its stereoisomers 224, 252,and253 reported by Kobayashi and Shiozaki et al. [172,173,174,187,188,189,190] and Ogawa’s group [182,183,184,185,186](> Scheme 39). 1974 9 Glycomimetics

⊡ Scheme 39

Moreover, these authors prepared the aminocyclitol 226 needed for the synthesis of trehazolin (223) from the allyl alcohol 269a. Epoxidation of 269a with mCPBA gave the desired epoxide 276 exclusively. The epoxide 276 was exposed to 2:1 H2O-TFA to open the epoxy ring, and afforded the alcohols 277. They were transformed to peracetates 278a and 278b, Carbasugars: Synthesis and Functions 9.2 1975

⊡ Scheme 40 1976 9 Glycomimetics

⊡ Scheme 41 Carbasugars: Synthesis and Functions 9.2 1977

⊡ Scheme 42

respectively, for further characterization of the structure of the alcohols 277. The desired per- acetate 278a was converted into the aminocyclitol 226 necessary for the synthesis of trehazolin (223), according to the synthetic procedure for the C-6 epimer of trehazolin (253), and thus completed the formal total synthesis of trehazolin (223)(> Scheme 40). On the other hand, Carreira et al. also reported the total synthesis of trehazolin from the optically active spirocycloheptadiene [196], which was prepared from the (R)-epichlorohydrin 282 and lithium cyclopentadienide, shown in > Scheme 41. Treatment of lithium cyclopentadienide (CpH+BuLi) with (R)-epichlorohydrin 282 afforded the optically active spirocycloheptadiene 283 in 91% ee. Compound 283 was converted into trichloroacetimidate 284 by treatment of NaH and Cl3CCN [197], and subsequent treatment of 284 with I(syn-collidine)2ClO4 gave the alcohol 287 via the unstable intermediates 285 and 286. After silylation of the secondary hydroxy group of 287, the imidate underwent nucleophilic opening upon treatment of the corresponding silyl-protected imidate with Li2NiBr4 to yield the cyclopropylcarbinyl bro- 1978 9 Glycomimetics mide 288 [198]. The alkene moiety in 288 was converted into the epoxide 289 with dimethoxy- dioxorane [199], and subsequent treatment of the resulting epoxide 289 with BF3·Et2O afforded the alcohol 290.Conversionof290 to 291 was performed under free radical conditions, which also resulted in partial reduction of the trichloromethyl moiety. Oxazoline 291 was directly converted into 292 by treatment with aqueous PPTS, followed by acetylation. Hydrob- oration of the terminal olefin unit in 292, followed by Swern oxidation of the resulting primary alcohol, provided aldehyde 293 [200,201]. Conversion of 293 to the corresponding phenyl ketone 294 was achieved by treatment of 293 with PhMgBr and oxidation of the resulting secondary alcohol. The aryl ketone 294 underwent Norrish type II cleavage upon irradiation through a Pyrex filter in degassed PhH, giving the corresponding alkene, which, without purification, was reacted with catalytic OsO4 to yield diol 295 as a single diastereomer. Exhaustive acid hydrolysis of 295 provided the aminocyclitol hydrochloride 226 necessary for the synthesis of trehazolin (223), and this corresponding aminocyclitol 226 was transformed to trehazolin (223) according to the preceding synthesis (> Scheme 41). After the total synthesis of trehazolin (223) and its stereoisomers, the further focus of synthetic studies on trehazolins was directed to the design and exploration of the other glucosidase- specific inhibitors possessing the trehalamine moiety, because trehalamine is considered to be a kind of pseudo-D-glucose. Compound 296 was designed by Knapp et al. as a pseudo- maltose and it was expected to have a potential for maltase inhibition [202]. On the other hand, compound 296 and its related compounds 297, 298 and 299 were independently designed by Kobayashi and Shiozaki et al. [203]. Carbasugars: Synthesis and Functions 9.2 1979

⊡ Scheme 43

Knapp et al. achieved the synthesis of the trehazolin aminocyclitol moiety (trehazolamine) as the hexaacetate 227 in the process of the synthesis of a maltose-type of trehazolin derivative 296. The allylic alcohol 300, available from D-ribonolactone 258 in several steps according to the method of Marquez [204], was converted to its thiocarbamate derivative by condensation with p-methoxybenzyl isothiocyanate, which in turn was cyclized with I2 to afford the iodoox- azolidinone 301. This is a rare example of formal anti-Markovnikov iodocyclization and may result from the kinetic preference for 5-membered (rather than 6-membered) and fused (rather than bridged) ring formation. One-pot treatment of 301 with Ac2OandH2SO4 followed by activated Zn provided allylic acetate 302, whose conﬁguration was inverted at the C3 position (trehazolin aminocyclitol numbering), by the Mitsunobu procedure [205], and the allyl alcohol 303 was obtained. Epoxidation of the resulting alcohol 303 gave the desired epoxide 304 as a single stereoisomer detected, and then hydrolysis of 304 at the less substituted position (C2 position, trehazolin aminocyclitol numbering) led to the triol 305. The stereochemistry of the cyclitol chemistry was proven by the conversion of 305 to the aminocyclitol hexaacetate 227 (> Scheme 42). Moreover, the triol 305 was converted into the O-benzylated compound 308, which was transformed to compound 296 via coupling with D-glucopyranosyl isothiocyanate 309 [206], formation of aminooxazoline ring, and complete removal of protecting groups (> Scheme 43). Kobayashi and Shiozaki et al. synthesized the trehazolin-related compounds 296–299 from aminocyclitol 226 degraded from natural intact trehazolin (223)[203]. The purpose of the synthesis was to discover the pseudosaccharides with interesting α-glucosidase inhibitory activities, and especially, compounds 296 and 297 were designed as intestinal maltase inhibitors and 298 was designed as an intestinal isomaltase inhibitor, and these inhibitors were directed towards potent therapies for noninsulin-dependent diabetes. The general synthetic route is summarized in > Fig. 11, and this route was established, based on the experience that cycliza- 1980 9 Glycomimetics

⊡ Figure 11 General synthetic route of trehazolin-related derivatives 296–299 Carbasugars: Synthesis and Functions 9.2 1981 ⊡ Table 3 Inhibitory activity of trehazolin-related compounds 296–299 toward glucosidases

tion of compound [IV] proceeded through path A to form thermodynamically stable [3, 3, 0] bicyclic structure in their synthesis of trehazolin C-6 epimer (253)[189,190]. Compounds 296–299 were synthesized according to this general synthetic route successfully and inhibitory activities of these compounds are shown in > Table 3. Interestingly, the structure-activity relationships of these compounds based on structural similarities couldn’t be found. Compound 298, which was designed as a pseudo-isomaltose, inhibited maltase and sucrase more potently than trehazolin (223); whereas compounds 296, 297, and 299 did not exhibit inhibitory activities toward maltase, isomaltase, and sucrase, compared to trehazolin (223). On the other hand, while none of the derivatives inhibited silkworm trehalase at a concentration of 100 µg/ml, only compound 297 possessed inhibitory activity toward porcine trehalase, with an IC50 value of 0.245 µg/ml. These results suggest that the interaction between an α-glucosidase and a glucose unit would be rather diverse among various glucosidases even though they can catalyze common substrates, and particularly, in the case of α-glucosidase reacting to disaccharide speciﬁcally, it was concluded that accurate structural analyses of the complexes consisting of the inhibitors and target enzymes are necessary for the design of the inhibitors with speciﬁc α-glucosidase inhibitory activity. 1982 9 Glycomimetics 4 New Methods for Conversion of Sugars to Carbasugars

The definition of carbasugars can be described as highly oxygenated and functionalized carbocycles. Also, these types of carbocycle components are included in the biologically significant molecules, such as a variety of enzyme inhibitors represented by glycosidase inhibitors, nucle- oside mimetics, and natural products. Therefore, the methodologies of carbocycle formation should be designed to be applied to a wide range of the synthesis. In general, as mentioned earlier, the Ferrie reaction, intramolecular [3 + 2] cycloaddition of nitrile oxide and cyclization of the diene with Grubbs’ catalysts are powerful methods as the key reactions for conversion of sugars to carbasugars. In addition to these, regarding the synthesis of 6-membered carbasugars, transformation of furanolactones to carbasugars via C1 unit prolongation and Lewis acid-mediated aldol-like cyclization, is also a useful synthetic method [207,208,209]. Also, regarding the transformation of a 6-membered to a 5-memberd ring, the development of methodologies has received much attention. Most of the methodologies involve a free-radical approach in which Bu3SnH mediated a well-known 5-hexenyl-type radical cyclization from bromide, iodide, or thiocarbonyl imidazole precursors [210,211,212]. In the meantime, the chemical characters of Sm and Zr have been a topic of interest and their utility in synthetic chemistry continues to be examined. In particular, synthetic exploitation of the reagent SmI2 has rapidly become one of the most significant fields in organic chemistry [213], and a great number of important synthetic transformations with SmI2 involve the one-electron reduction of ketones and aldehydes to a samariumketyl radical anion which can promote cyclizations, deoxygenations, and reductions. In fact, during this decade, some new methodologies for the transformation of pyranose derivatives to 5-memberd carbasugars using Sm and Zr reagents have been reported. In this section, novel synthetic methodologies for conversion of sugars to carbasugars will be introduced.

4.1 Carbasugar Formation via SnCl4-Promoted Intramolecular Aldol Condensation

Tatsuta et al. developed the method of carbasugar formation using SnCl4-promoted intramolecular aldol condensation as a key step. The methodology is shown in > Fig. 12,andakey precursor of carbasugar, α-hydroxymethyl enone [XI] was achieved according to the following steps: (1) 1,2-addition of lithiated MeSO2Ph to furanolactones containing a dimethyl acetal [VII], (2) transformation of compound [VIII] to linear silyl enol ether [IX],(3)SnCl4- promoted aldol-like cyclization of [IX] to α-phenylsulfonyl cyclohexenone [X] and (4) conversion of the corresponding α-phenylsulfonyl cyclohexenone [X] to α-hydroxymethyl cyclohexenone [XI] through the Michael-type addition of tributylstannyl-lithium followed by trapping with formaldehyde and desulfonylation. This transformation is ideally suited to the synthesis of carbocycle-containing natural products and carbasugars, since the core skeleton arises after appropriate replacement of the phenylsulfonyl group, and, in practice, this method was applied to total synthesis of progesterone receptor ligands, (–)-PF1092A, B, and C [207], and (–)-glyoxalase inhibitor and its precursor (–)-KD16-U1 (> Fig. 12)[208]. As shown in > Scheme 44, this method was also applied to total synthesis of (+)-valienamine (320) and (+)-validamine (324)[209]. Silyl enol ether 314 derived from D-xylose in nine steps Carbasugars: Synthesis and Functions 9.2 1983

⊡ Figure 12 Key steps of the synthesis of the 6-membered carbasugar using SnCl4-promoted intramolecular aldol condensation

was converted to α-hydrixymethyl cyclohexenone 316 via SnCl4-promoted intramolecular aldol condensation and Michael-aldol reaction with tributylstannyl-lithium and formaldehyde in good yield. Cyclohexenone 316 was converted to (+)-valienamine (320) and (+)-validamine (324) via the common intermediate, azide 318 in good yield (> Scheme 44). It can be expected that this transformation would be one of the methods useful for the synthesis of 6-membered carbasugars.

4.2 SmI2-Mediated Carbasugar Formation

First, the intramolecular cyclizations between carbonyl compounds and olefins will be described. This field can be classified into three types as illustrated in > Fig. 13.Oneis a type of cyclization between carbonyl compounds and α, β-unsaturated esters (type [A]), the next is a type of cyclization between carbonyl compounds and simple olefins (type [B]), and the last is a type of cyclization between carbonyl compounds and oximes (type [C]) (> Fig. 13). The general feature of these reactions is as follows: When treated with SmI2, a reductive cyclization between the carbonyl compound and the β-carbon of the olefin of [XIII] derived from sugars [XII] gives the desired polyhydroxylated cyclopentane [XIV],inwhichanew C1/C5 bond (carbohydrate numbering) is formed between sp2 centers. In the overall sequence, the sp3 alcohol stereocenter at C5 is destroyed when oxidation occurs to form the carbonyl moieties and subsequently reinstated, upon treatment with SmI2, to form a new hydroxy- 1984 9 Glycomimetics

⊡ Scheme 44 Carbasugars: Synthesis and Functions 9.2 1985 bearing stereocenter. In contrast, the existing 5-hexenyl free radical methods for the cyclization of carbohydrates do not allow for the incorporation of this additional sp3 alcohol center which provides further functionality for subsequent synthetic manipulations. This is a general difference between the traditional radical cyclization and the SmI2-mediated radical cyclization [214].

4.2.1 Cyclization between Carbonyl Compounds and α, β-Unsaturated Esters

Enholm et al. reported the SmI2-mediated construction of carbocycles from carbohydrate templates in 1989 [214]. This is the example of the type [A] reaction in > Fig. 13.Thisstrat- egy, which permits the conversion of pyranose sugars into highly oxygenated cyclopetanes, is illustrated in > Fig. 14 (Eq. (1)). Treatment of carbohydrate templates [XVI], which are derived from sugars [XV] via Wittig reaction with stabilized ylides and subsequent PDC oxidation, with the one-electron reducing reagent SmI2 prompts the intramolecular coupling of two sp2-hybridized carbon centers and ﬁnally constructs a highly oxygenated cyclopentane

⊡ Figure 13 SmI2-mediated carbasugar formation

⊡ Figure 14 Cyclization between carbonyl compounds and α, β-unsaturated esters 1986 9 Glycomimetics ring [XVII]. Interestingly, the geometry of the olefin unit of the substrates influences the stereochemistry at C1 and C5 (carbohydrate numbering) of the products, and there is the tendency that Z-olefins give the anti-alcohols and E-olefins provide the syn-alcohols exclusively. Also, this method was applied to the synthesis of the natural product, anguidine [215]. Moreover, Enholm also reported the sequential SmI2-promoted one- and two-electron reactions as an application of this concept (> Fig. 14, Eq. (2)) [216]. Treatment of the compounds [XVIa] and carbonyl compounds with SmI2 afforded a highly modified carbocycle. In the domino key step, SmI2 promoted a sequential one-electron (radical) cyclization of an aldehyde and an alkene, followed by a two-electron intermolecular carbonyl addition reaction to afford compounds [XVIII] (> Fig. 14).

4.2.2 Cyclization between Carbonyl Compounds and Simple Oleﬁns As the example of the type [B] reaction, the reports by Holzapfel et al. and Chiara et al. who independently reported this type of reaction are described. Initially, Holzapfel et al. reported the SmI2-mediated radical cyclization of the ring-opened hex-5-enals [XXI] derived from pyranoses [XIX] via the step of the reductive elimination of C6 iodopyranose derivatives [XX] with active Zn [217](> Fig. 15, Eq. (1)). Afterwards, they also reported the one-pot SmI2-

⊡ Figure 15 Cyclization between carbonyl compounds and simple oleﬁns (1) Carbasugars: Synthesis and Functions 9.2 1987

⊡ Figure 16 Cyclization between carbonyl compounds and simple oleﬁns (2)

mediated radical cyclization of 6-deoxy-6-iodohexopyranose derivatives [XXIV] which were derived from glycals [XXIII] [218](> Fig. 15, Eq. (2)). In each case, the cyclization itself proceeds typically with above 65% efficiency, and the stereochemistry of C1 and C5 (carbohydrate numbering) of the products shows an anti- configuration. However, as an exception, in case of the manno-type derivative of [XXI],the compound arises via initial SmI2-mediated elimination of the α-substituent (benzyloxy group) to the carbonyl (> Fig. 15, Eq. (1.3)). On the other hand, Chiara et al. also reported outcomes similar to Holzapfel’s results [219]. They attempted the one-pot, SmI2-mediated cyclization of fully functionalized 6-deoxy-6 iodohexopyranose derivatives and investigated the influence of the stereochemistry of the substrates as well as the effects of the protecting groups in this type of reaction (> Fig. 16). Basically, their results are consistent with those of Holzapfel et al., especially with respect to the stereochemical arguments given above and the chemical yield of the products. According to these studies, the TBDMS group is suitable for the reaction from the viewpoints of the ease of separation of the products and the avoidance of the β-elimination seen in the reports of Holzapfel et al. Also, they reported that 6-deoxy-hexopyranose derivatives were obtained as the by-products in the range of yields of 10–50%.

4.2.3 Cyclization between Carbonyl Compounds and Oximes Chiara et al. also reported the type [C] reaction, the cyclization between carbonyl compounds and oximes [220,221]. As illustrated in > Fig. 17, three types of substrate were synthesized from hexopyranoses and pentopyranoses, and the terminal O-benzylformaldoxime-ketones and the terminal O-benzylformaldoxime-α, β-unsaturated esters were synthesized from a series of hexopyranoses, and meanwhile the terminal O-benzylformaldoxime-aldehydes were derived from a series of pentopyranoses (> Fig. 17). The goal of this attempt is the stereochemistry of the hydroxy group and amino group generated after cyclization. In cases of cyclization of the terminal O-benzylformaldoxime-ketones and the terminal O-benzylformaldoxime-α, β-unsaturated esters, the stereochemistry is mainly the anti-conﬁguration as is also the case for the cyclization described above. However, in contrast to the latter substrates, in the case of cyclization of the terminal O-benzylformaldoxime-aldehydes, the realized stereochemistry depends on the conﬁguration or the protecting groups. 1988 9 Glycomimetics

⊡ Figure 17 Cyclization between carbonyl compounds and oximes

⊡ Figure 18 SmI2-mediated Pinacol coupling

4.3 SmI2-Mediated Pinacol Coupling

SmI2-mediated pinacol coupling is also a useful synthetic method to form carbasugars possessing a cis-diol moiety from sugars. In fact, as mentioned above, Nakata et al. utilized this method in the synthesis of cyclophelitols, and it is one of the good examples to verify the synthetic utility of SmI2-mediated pinacol coupling [20]. This coupling reaction has also been utilized in the ﬁeld of the transformation of sugars to highly functionalized 5-membered carbocycles (> Fig. 18). Initially, application of this method to the formation of 5-membered carbocycles was reported by Sinäy et al. [222]. In their case, a dialdehyde was used as the substrate, and cyclization occurred with a comparable overall yield of carbocyclic cis-diols but with much lower diastereoselectivity. Later, Iadonisi et al. reported the results of the application using keto-aldehydes as the substrates [223]. Compared to Sinay’s results, cyclization of the keto-aldehydes occurred with a relatively high stereoselectivity, and it should be suggested that, in particular, electro- static interactions play an important role in the stereocontrol of this reaction.

4.4 SmI2- or Zirconium-Mediated Ring Contraction of Hexapyranoside Derivatives to 5-Membered Carbosugar

As mentioned in the above sections on the syntheses of natural glycosidase inhibitors, the ring contraction reaction of a 6-membered ring system to a 5-membered ring system is deﬁnitely Carbasugars: Synthesis and Functions 9.2 1989

⊡ Figure 19 SmI2-mediated ring contraction of hexapyranoside derivatives to 5-membered carbasugars

a significant aspect in synthetic chemistry, and the further development of this type of method should pave the way to the syntheses of the complex natural products and artificial chemicals including clinical drugs as well as a series of glycosidase inhibitors. Here, the SmI2- or zirconium-mediated ring contraction of hexapyranoside derivatives to 5-membered ring carbosugars will be described. Sinäy et al. presented the SmI2-mediated ring contraction of methyl 6-unprotected-hexopyranoside derivatives to fully functionalized 5-membered carbosugars via the intermediate aldehyde shown in > Fig. 19 [224]. The aldehyde [XXXVII], which was derived from the methyl 6-unprotected-hexopyranoside derivative [XXXVI] via Swern oxidation, was treated with SmI2 (5 equiv.) in THF in the presence of HMPA and t-BuOH to afford the cis-cyclopentane [XXXVIII] exclusively. Interestingly, the cis-configuration between C1 and C5 (carbohydrate numbering), which is the new stereocenter generated in this reaction, does not depend on the substrates, and each compound can be obtained in moderate yield (> Fig. 19). Sinäy et al. anticipated the mechanism of the reaction illustrated in > Fig. 20.Thefirst equivalent of SmI2 reduces the aldehyde 326, which was derived from 325,tothesamari- umketyl 327. The second equivalent of SmI2 reduces the corresponding ketyl 327 to disamar- ium species 328, which then undergoes ring opening followed by methoxide elimination to give the key intermediate 330. Subsequently, this reaction uniquely generates a system which is ideally suited for a following aldol cyclization reaction involving intramolecular nucleophilic attack of the samarium enolate onto aldehyde through a 5-enol exo-exo-trig process. With respect to the stereoselectivity of this reaction, they concluded that it was expected to ensue from a samarium-linked, medium-sized chelate, from which the carbon–carbon bond formation would take place as a ring contraction (> Fig. 20). Recently, Aurrecoechea et al. reported the synthesis of 2-vinylcyclopentanols by SmI2/Pd(0)- promoted carbohydrate ring-contraction [225]. On the other hand, Ito and Taguchi et al. independently presented the Cp2Zr/BF3·Et2O-mediated carbohydrate ring-contraction [226,227]. The contrast regarding the stereochemistry of products in each methodology is interesting as illustrated in > Fig. 21. First, the SmI2/Pd(0)-promoted carbohydrate ring-contraction will be described. As illustrated in > Fig. 21, substrates [XXXIX], which were derived from methyl 6-unprotected hexapyranoside derivatives via Swern oxidation followed by Wittig olefination, were treated with SmI2 and a catalytic amount of Pd(PPh3)4 to afford the 2-vinylcyclopentanols [XXXX] possessing the trans-stereochemistry between the vinyl and the newly generated hydroxyl group. The authors speculated on the mechanism of this reaction and the cause of the predominant trans-stereoselectivity of the resulting compounds as illustrated in > Fig. 22.Thefor- 1990 9 Glycomimetics

⊡ Figure 20 Proposed mechanism of SmI2-mediated ring contraction

⊡ Figure 21 SmI2-Pd(0)- or Zr-mediated ring contraction of hexapyranoside derivatives to 5-membered carbasugars

mation of cyclopentane products can be rationalized by a Pd(0)-promoted ring-opening of [XXXIX] leading to an intermediate [XXXXII] that contains both a p-allylpalladium complex and an aldehyde moiety. Reduction of the palladium complex by SmI2 and carbonyl addition of the resulting allylsamarium species [XXXXIII] would then lead to the observed products [XXXX]. With respect to the stereochemistry of the products observed in these cyclizations, the predominant trans-stereoselectivity is surprising when compared to the relat- Carbasugars: Synthesis and Functions 9.2 1991

⊡ Figure 22 Proposed mechanism of SmI2-Pd(0)-mediated ring contraction

⊡ Figure 23 Proposed mechanism of Zr-mediated ring contraction

ed SmI2/Pd(0)-promoted intramolecular cyclization of a ketone containing a pendent viny- loxirane moiety. This latter case is also assumed to proceed through allylsamarium intermediates, but affords preferentially a cis-product, presumably through a chelated cyclic transition structure. If a similar mechanism is indeed operating, the different stereochemical outcomes described above could be due to a combination of factors derived from differences in reaction conditions and substrate structures. Interestingly, in addition to the preferred trans-relationship observed between the vinyl and hydroxyl groups, the glucose- and mannose-derived substrates also displayed preference for a trans-relationship between the hydroxyl group and the adja- cent benzyloxy group. Whereas for the more selective galacto derivatives, the opposite cis- relationship was found (> Fig. 22). On the other hand, the zirconium-mediated ring contraction of hexapyranoside derivatives, which was reported by Ito and Taguchi et al. afforded the 2-vinylcyclopentanols, and the 1992 9 Glycomimetics resulting 2-vinylcyclopentanols possess cis-stereochemistry between the vinyl group and the newly generated hydroxyl group. They explained that the derived stereochemistry would be induced through the intermediates illustrated in > Fig. 23. Treatment of [XXXIX] with Cp2Zr in THF provided intermediate [XXXXV], which was conﬁrmed by NMR analysis. The proof of [XXXXV] as an intermediate in the reaction was further conﬁrmed by the conversion of [XXXXV] to [XXXXI] upon addition of BF3·Et2O. It is probable that BF3·Et2O acceler- ates the elimination of the methoxy group through coordination to the methoxy oxygen of [XXXXV] to form the sterically favored oxocarbenium ion [XXXXVI]. Finally, this intermediate was cyclized to afford the resulting cis-2-vinylcyclopentaol [XXXXI] (> Fig. 23).

5Conclusion

As mentioned above, a variety of natural glycosidase inhibitors have been isolated, and they show interesting, strong, and enzyme-speciﬁc inhibitory activities. Recently, it was recognized that these compounds are important molecules to elaborate the pivotal roles of glycoconjugates in living systems, and have potential use as clinical drugs, and, in the ﬁeld of biochemistry, the synthesis and supply of the glycosidase inhibitors and their analogues occupies a quite significant position. In this chapter, a variety of syntheses of glycosidase inhibitors were described, and also applications and developments of the new synthetic methodologies for transformation of sugars to carbasugars, ranging from the typical Ferrier reaction to the recent SmI2/Pd(0)- mediated ring-contraction, could be introduced through the synthesis of a variety of glycosidase inhibitors. I hope that a variety of studies in glycoscience using carbasugars will pave the way to further elucidation of the functions of glycosidases and glycoconjugates in living systems.

Acknowledgements

Finally, I thank Dr. Masao Shiozaki, currently at RIKEN, for helpful discussions and sugges- tions during the preparation of this chapter.

References

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