PART 2
Synthesis of aminosugars as new Natural Killer
cell activators
1. Introduction
1.1. Natural killer cells
Natural killer (NK) cells represent a distinct subset of lymphoid cells that have innate immune functions.1 Derived from the bone marrow, NK cells circulate in the blood and become activated by cytokines or upon encountering target cells that express ligands for NK cell receptors.2 NK cell receptors are encoded in the germline and do not undergo somatic recombination like B-cell and T-cell antigen receptors; it is the balance of signals from activating and inhibitory receptors that determines the outcome of NK cell activity.3 Some inhibitory receptors recognize MHC class I, which is present on virtually all healthy cells, and prevent NK cell attack against these cells. Loss of MHC class I from cells owing to infection or transformation can lead to NK cell activation provided that an activating receptor is engaged. These activating NK receptors bind to host-derived or pathogen-encoded ligands that are up-regulated on “stressed” or infected cells (Fig. 1.1). Figure 1.1
Upon activation, NK cells directly lyse target cells by exocytosis of perforin and granzymes and secrete cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which mediate their immune response to infection. In this manner, NK cells function as important sentinels of the immune system, working as primary responders and alerting the host to the presence of infectious organisms. Through the mechanism described above NK cells play important roles in host defense against viruses (MCMV, HCMVI, Sendai virus, Influenza A virus, HIV, MHV, Ebola virus),4 parasites (P. falciparum, T. cruzi, P. berghei ANKA),5 bacteria (Shigella flexneri, M. tuberculosis)6 and tumors, and other therapeutic uses are currently under investigation.7
1.2. Natural killer cell receptors for carbohydrates
The major activating receptor at the surface of rat natural killer cells is the NKR-P1A protein which has been cloned and characterized, and belongs to a superfamily of animal C-lectins. The structural requirements to prepare optimal saccharidic NKR-P1 ligands have been determined8 and are here reported.
1.2.1. Type of sugar unit
In Figure 1.2, the affinities of carbohydrate ligands to the NK cell activation receptor are shown, NKR-P1, expressed in logarithmic scale (-logIC50). In particular the relative potencies to inhibit the bind of the receptor to its high affinity ligand GlcNAc23BSA neoglycoprotein are compared and allow to take some conclusions about the optimal structure of the sugar unit.
Figure 1.2
One important aspect is that (a) 2-acetamidosugars bind in the following order: ManNAc > GalNAc > GlcNAc >> TalNAc. ManNAc was identified as a superior monosaccharide ligand for the receptor, about an order stronger ligand than GalNAc. The inhibition activity of N-acetyltalosamine is considerably weaker (5 vs 8). This can be caused by the fact that TalNAc in aqueous solutions mainly occurs (contrary to the other common aminosugar) in its furanose form. All good saccharide ligands identified up to now are pyranoses, and it is possible to assume that furanoses are not suitable for the binding. (b) A 2-acylamido-2-deoxy group is crucial for the binding, but the length
of the acyl group is not important. The importance of the acetylation of the 2-amino group in the pyranose structure for the binding has evoked a question whether the acetylation is the most convenient acylation. Therefore, the influence of the length of acyl was investigated. All the N-acyl derivatives e.g. 2-deoxy-2-propionylamino-D-glucopyranose, 2-deoxy-2-butyrylamino-D -glucopyranose, 2-deoxy-2-isobutyrylamino-D-glucopyranose exhibited the –log IC50 value 6.4 which is a slightly worse value than that of acetyl derivative (GlcNAc = 6.7). (c) In the C-6 position, the presence of a group with hydrogen bond accepting properties is important (-OH, O-acyl, carboxyl). Its removal abolishes the binding. Position C-6 is important as it is often sulfated in natural glycostructures or converted into carboxy group and thus negatively charged. Negatively charged carbohydrate structures (chondroitin sulfates, dermatan derivatives and heparans – all being sulfates and/or carrying carboxy groups) have been shown to strongly bind NKR-P1 where they may interact with the Ca2+ ions (C-type lectin). Indeed, while 6-deoxy-D-glucose displayed no activity (elimination of H-bonding or ionic bonding) but D-glucuronic acid was a very good ligand with its IC50 being three orders of magnitude lower than that of D-glucose itself. (d) Hexopyranose structures seem to be optimal for the binding. The stereochemistry at C-2 and C-4 positions is important, however, its changes influence the affinity within one order only. Furanose structures do not seem to be favourable for the binding.
1.2.2. Type of sugar linkage
To understand the importance of the type of linkage, Kren et al.8 tested both p-nitrophenyl α- and β- 2-acetamido-2-deoxy-D-galactopyranosides and 2-acetamido-2-deoxy-D-glucopyranosides (Fig. 1.3). The –logIC50 values (1αααα=5.2; 1ββββ=8.2; 2αααα=4.0; 2ββββ=7.5) clearly show an influence of the anomeric linkage on the binding properties.
Figure 1.3
In particular, the linkage must be β and β-(14) in case of di and polisaccharides as obtained
comparing chitobiose [GlcNAc-β−(14)-GlcNAc] and its regioisomer GlcNAc-β(16)-GlcNAc.
Furthermore, the same authors studied the effect of the carbohydrate chain length on the binding properties and pointed out its crucial role; four carbohydrate units being the optimum length of the oligosaccharidic chain.
1.3. Preparation of ββββ-D-MacAc-(14)- D-Glc and ββββ-D-TalNAc-(14)- D-Glc disaccharides as agonists of NKR-P1 and CD69 receptors
On the basis of the structural requirement of optimal NK activators, recently our research group studied the synthesis of two disaccharides β-D-TalNAc-(14)-D-Glc (5) and β-D-ManNAc-(1 4)-D-Glc (10) starting from cheap commercially available D-lactose and avoiding the difficult β -glycosylation step.9 The talo derivative 4 (Scheme 1.1) was prepared through an acetonation reaction (DMP, TsOH) which allow to obtain a selectively 2’-O-deprotected compound easily oxidised with excellent yield using the TPAP-NMO system. Derivative 3 was reacted with hydroxylamino hydrochloride reactants and the oximes obtained were reduced with LiAlH4 affording to 4 with good or complete stereoselectivity.
Scheme 1.1 O O O NHAc OR O O O O (MeO)2HC O O OH HO OH OH O OHO OH OH OH Lactose O O O O O O (MeO)2HC O O O OR O OH HO NHAc OH O OHO OH OH OH 3 4 5
The final acid acetal function removal gave the target compound 5 in good yield. Derivative 6, through an innovative C-4’ epimerization strategy, was used also for the preparation of the ManAc disaccharide 10 (Scheme 1.2).10
Protective groups manipulations had easily permitted the preparation of selectively C-4’ deprotected derivative 7, that treated with NaH, Im2SO2 in DMF, gave hex-3’-enopyranoside 8 in almost quantitative yield. The complete regioselectivity in the elimination of the putative intermediate 4’-O-imidazole-1-sulfonate was ascribed to the stereoelectronic assistance of the C-2’ axial substituent favouring the anti elimination of the axial H-3’ to a greater extent than to the axial H-5’.11 The specific role of the axial orientation of the C-2’ substituent of the talo derivatives, was demonstrated by the complete lack of regioselectivity observed in the same reaction of the galacto derivatives.
Scheme 1.2
The second step of the epimerization procedure was the regio- and stereoselective hydroboration-oxidation of the enol ether 8 (BH3
.
Me2S/Et2O) which gave 9 in good yield. Final catalytic debenzylation and acid hydrolysis brought to the other target product 10. The potencies of the prepared disaccharides to inhibit the binding of two natural killer cell receptors, rat NKR-P1A and human CD69, to their high affinity ligand, GlcNAc23BSA bioglycoprotein were tested (Table 1.1).
Table 1.1. Affinity of carbohydrate ligands to rNKR-P1A and hCD69 receptors(-logIC50)
Compound NKR-P1 CD69 TalNAc-β-OMe 6.7 5.0 TalNAc-β-(14)Glc 6.7 4.5 ManNAc-β-(14)Glc 7.4 3.2 ManNAc 8.0 3.0 GlcNAc 6.7 5.0
While none of the newly prepared compounds proved to be an extremely good inhibitor for CD69, ManNAc disaccharide display quite high affinities for NKR-P1 receptor, for which, as seen before, ManNAc is the highest-affinity monosaccharide ligand.
1.4.Aim of this research
On the basis of the biological results shown in Par. 1.3, our interest was first directed to the preparation of new structures as new possible NK cells activators. In particular, we wanted to prepare a serie of iminosugars 11 (Figure 1.4), where an N-acetylhexosaminyl unit is β-(14)
linked to a D-deoxynojirimycin (DNJ). Figure 1.4
These types of derivatives present the optimal structure requirements for NK cells agonists in the sacchare unit (2’-acetamido group, β-(14) anomeric connection, C-6’ group with hydrogen bond
accepting properties, hexopyranose structure), while in the azapyranose one the replacement of the ring oxygen atom with a nitrogen one may produce new interesting characteristics and possibilities. On one hand azapyranose derivatives are good glycosidase inhibitors12 and they could stop the hydrolysis of the glycosidic bond promoted by that enzymes in vivo thus enhancing the receptor affinity response (see Par. 1.6), on the other hand the nitrogen atom may be used as a connecting point to construct glycocluster, if X in compounds 11 is an useful substituent as a functionalized spacer. About this latter point, the synthesis of GlcNAc polyamidoamine glycoclusters (GlcNAc3 -cluster, GlcNAc4-cluster, GlcNAc6-cluster, GlcNAc8-cluster) has been reported.13 It involves the reaction of glycosyl isothiocyanates with branched and hyperbranched tri-, tetra-, hexa- and octaamines to provide the corresponding multiantennary thiourea-bridged glycocluster. These compounds, and in particular GlcNAc8-cluster, are potent inhibitors14 of binding of the NKR-P1A receptor to its high affinity ligand, GlcNAc23BSA, much more efficient with respect to the monomeric unit. Despite their low molecular mass, these highly branched GlcNAc-terminated glycocluster are well comparable with traditional high molecular mass carbohydrate ligands for NKR-P1A, such as natural glycoproteins15 or neoglycoproteins.16 The strong reactivity of these compounds with NKR-P1A may relate to the degree of conformational freedom and accessibility conferred to the terminal monosaccharide residues.
1.5. Iminosugars: past and present
Iminosugars are sugars in which the endocyclic oxygen is replaced by a basic nitrogen atom. This apparently simple substitution raises many synthetic challenges and opens the way to interesting
biological properties. For these reasons iminosugars form the most attractive class of carbohydrate mimics reported so far.
The origin of their therapeutic use goes back to ancient times and traditional Chinese phytomedicines. In Occident, Haarlem oil, the first medication produced on an industrial scale in the 17th century, was recommended for the treatment of diabetes and for whitening the skin. One of the major constituents of Haarlem oil was an extract from leaves of Morus alba, the white mulberry, an extremely rich source of iminosugars.17 The scientific history of iminosugars began in the early 1960s with the almost simultaneous reports of the synthesis of sugar derivatives containing a nitrogen atom in the ring by many research groups as the Paulsen one that in 1966 published the first synthesis of 1-deoxynojirimycin or DNJ (12, Fig. 1.5),18 that were isolated from natural sources and found to act as an α-glucosidase inhibitor by Bayer chemists only in 1976. This discovery triggered an enormous amount of interest in imino analogues of carbohydrates.19
Figure 1.5
In the last decade the rate of discoveries has increased dramatically. Original structures have been designed and synthesized such as seven- or eight-membered iminoalditols, conformationally constrained analogues of iminosugars and complex glycoconjugate mimetics (Fig. 1.6).20 Innovative synthetic strategies have been developed, including combinatorial approaches to iminosugar libraries. This new concept of dynamic combinatorial chemistry has been applied to accelerate the discovery of iminoalditol-based glycosidase inhibitors.21
Starting from the discovery of DNJ (13) as a α-glucosidases inhibitor, the scope of the biological activity of iminosugars has been extended to the inhibition of a large number of enzymes of medicinal interest from glycosyltransferases22 to metalloproteinases23 (Fig. 1.7). Thanks to iminosugars, significant progress has thus been made in glycobiology in the past 10 years. The amazing diversity of enzymes inhibited by those compounds promises a new generation of medicines useful in a wide range of diseases such as diabetes, viral infections, lysosomal storage disorders or tumour metastasis (Fig. 1.7).24
Figure 1.7. A recent timeline of iminosugars
1.6. Biological activity of 1-deoxynojirimycin (DNJ) and its derivatives
1-deoxynojirimycin or DNJ (13) is one of the most investigated iminosugars for its multivalent therapeutic use in a wide range of diseases (see Fig. 1.7). Over 40 years have passed since nojirimycin (NJ, 12, Fig. 1.5) was discovered as the first natural glucose mimic and originally described as an antibiotic produced by Streptomyces species.25 However, because this iminosugar is fairly unstable, it is usually stored as a bisulfite adduct or may be reduced by catalytic hydrogenation with a platinum catalyst or by NaBH4 to 1-deoxynojirimycin (DNJ, 13, Fig. 1.5).26 DNJ was later isolated from the roots of mulberry trees and called molanoline,27 and was further found to be produced by many strains in the genera Bacillus and Streptomyces.28 Talking about the biological activity of DNJ and its derivatives, the most important is the seen inhibition of glycosidases. Glycosidases are involved in a wide range of important biological processes, such as intestinal digestion, post-translational processing of the sugar chain of glycoproteins,
quality-1970 1980 1990 2000 Enzymatic targets Therapeutic targets 1992: Glycosyltransferases Glycosidases 1993: Nucleoside-processing enzymes
1997: UDP Gal mutase
2004: Metalloproteinases 1997: Glycogen phosphorylases Diabetes Cancer Lysosomal diseases 2006: Cystic fibrosis
Viral diseases Psoriasis
control systems in the endoplasmic reticulum (ER), ER-associated degradation mechanisms and the lysosomal catabolism of glycoconjugates. Hence, inhibition of these glycosidases can have profound effects on carbohydrate catabolism in the intestine, maturation, transport and secretion of glycoproteins, and can alter cell–cell or cell–virus recognition processes.
1.6.1. αααα-glucosidases inhibition
The intestinal oligo- and disaccharidases are fixed components of the cell membrane of the brush border region of the wall of the small intestine. Glycosidases digest dietary carbohydrate to monosaccharides which are absorbed through the intestinal wall. They include sucrase, maltase, isomaltase, lactase, trehalase and hetero-α-glucosidase. In the late 1970s, it was realized that inhibition of some, or all, of these activities by inhibitors could regulate the absorption of carbohydrate and these inhibitors could be used therapeutically in the oral treatment of the non-insulin-dependent diabetes mellitus (NIDDM or type 2 diabetes).28a The strong inhibition of DNJ on digestive α-glucosidases attracted the interest of various research groups and a large number of
substituted DNJ derivatives were prepared in the hope of increasing the in vivo activity.
N-hydroxyethyl-DNJ or Miglitol (Fig. 1.8) was identified as one of the most favourable candidates showing a desired glucosidase inhibitory profile29 and is almost completely absorbed from the intestinal tract with possible systemic effects in addition to the effects in the intestinal border. In 1996, Glyset (miglitol) tablets were accepted by the US Food and Drug Administration (FDA) and introduced onto the market in 1999 as a more effective second-generation α-glucosidase inhibitor with fewer gastrointestinal side effects.
Figure 1.8
.
1.6.2. Antiviral action
The viral envelope glycoproteins are often essential for virion assembly, secretion and/or infectivity. Compounds that interfere with the glycosylation processes of viral glycoproteins can be expected to be antiviral agents. In fact, α-glucosidase inhibitors such as DNJ and N-butyl-DNJ (Zavesca, Fig.1.8), inhibit human immunodeficiency virus (HIV) replication and HIV-mediated syncytium formation in vitro.30 The in vivo data obtained do not promise practical use of processing
α-glucosidase inhibitors as anti-HIV agents. Problems exist in achieving therapeutic serum concentrations of inhibitors needed to inhibit α-glucosidase sufficiently and side effects such as diarrhoea occur. In contrast to the heavily glycosylated HIV envelope glycoproteins, the envelope glycoproteins of the hepatitis B virus (HBV) contain only two glycosylation sites.31 However, the HBV glycoproteins are sensitive to inhibitors of the glycosylation pathway. In this virus, correct glycosylation appears to be necessary for processes involved in transport of the virus out of the host cell. In vitro treatment of HBV with N-butyl-DNJ (Fig. 1.8) results in a high proportion of virus particles being retained inside the cells.32 Block et al.33 reported that N-nonyl-DNJ reduces the
viremia in chronically infected woodchucks in a dose dependent manner. N-Nonyl-DNJ is 100–200 times more potent than N-butyl-DNJ in inhibiting HBV in cell based assays. A single drug against HBV and HCV may be of great therapeutic value. However, when processing α-glucosidase inhibitors are used as antiviral agents, it remains to be determined what effects occur on host cell glycoprotein processing and/or glycoprotein transport.
1.6.3. Gaucher’s disease
Gaucher’s disease, the most common glycolipid storage disease, is a relatively rare hereditary disorder due the deficiency in a specific β-glucosidase (β-glucocerebrosidase) involved in the catabolism of glycosphingolipids in lysosomes.34 Defects in the catalytic activity of this enzyme lead to the accumulation of undegraded glucosylceramide (GlcCer) in macrophages and to severe symptoms as enlarged spleen and liver, liver malfunction, skeletal disorders, bone lesions that may be painful and severe neurologic complications. The treatment of that disease is based on the use of Zavesca (N-butyl-DNJ, Miglustat, Fig. 1.8), a DNJ derivative. The primary pharmacological activity of Zavesca is the inhibition of the enzyme glucosylceramide synthase, catalyzing the first step in the biosynthesis of glycosphingolipids (GSL), i.e., the formation of glucosylceramide (GlcCer). Reduced formation of GlcCer will lead to decreased biosynthesis of more complex GSL. This therapeutic principle, called substrate reduction therapy (SRT), may be useful in disorders of intracellular (predominantly lysosomal) accumulation of GSL either due to their deficient breakdown or intracellular transport/trafficking. Miglustat exhibits a large volume of distribution and has the capacity to access deep organs such as the brain, bone and lung.
1.7. Synthesis of ββββ-(14) azadisaccharides in literature
Three different approaches are reported in literature which could be followed to prepare target aza-disaccharide compounds 11. (i) Pseudo-aza-disaccharides could be obtained through chemical
glycosylation reactions between a glycosyl donor and an iminosugar acceptor. The problems of this strategy are connected with the hydroxy functions protection, necessary to prepare suitable donor and acceptor compounds, and to the low stereocontrol in the glycosidic bond formation. Here is reported as an example (Scheme 1.3), studied by Martin et al.,35 the synthesis of 4-O-(β-D -glucopyranosyl)-N-butyl-1,5-dideoxy-1,5-imino-D-glucitol (16) as possible inhibitor of the glucosylceramide β-glucosidase which is involved in the Gaucher’s disease. The glycosylation reaction between iminosugar 15 and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide 14 promoted by AgOTf afforded protected 16 in 34% yield. The free cellobiose mimic was obtained in 9% overall yield after protecting groups removal.
Scheme 1.3 N HO BnO OTBDMS OBn O AcO AcO OAc OAc Br O HO HO OH OH N O HO OH OH 14 15 i-iv 16
Reagents and conditions. i: AgOTf, 4 Å MS, DCM, -78°C to rt (34%); ii: TBAF, THF, 0°C to rt (70%); iii. MeONa, MeOH (86%); iv. H2, Pd-C, iPrOH-AcOH (44%).
(ii) The enzymatic glycosylation could represent a valid alternative for the synthesis of aza-disaccharides and in Scheme 1.4 is described the transgalactosylation reaction36 between lactose and 1-deoxynojirimycin 13 using a β-galactosidase from Bacillus circulans. The enzymatic glycosylations are extremely interesting because they don’t need any hydroxyl group protection. The problem of this reaction is often the low regiocontrol; in fact, the target 4-O-β-galactopyranosyl-1-deoxynojirimycin 17 (26% yield) is obtained as a mixture with the 2-, 3-, 6-regioisomers (18a, 18b and 18c), and that requires difficult purification procedures.
A third different approach (Scheme 1.5) starts with the preparation of 1,5-dicarbonyl disaccharides to be submitted to a double reductive amination with some primary amines in MeOH using NaBH3CN as reducing agent,
37
obtaining azamimic 17 avoiding the glycosylation step. Scheme 1.5
Reagents and conditions. i: Bu2SnO, toluene, reflux then NBS, CHCl3 (98%); ii: a) 90% aq TFA; b) NH4OAc, NaBH3CN, MeOH, 60°C (60% from 20); iii. H2, Pd-C, MeOH-HCl (quant.).
The starting diol 19, readily obtained from lactose through a method developed by Catelani et al.,38 was selectively C-5 oxidize to 20 through the starting formation of a O-dibutylstannylidene intermediate (Bu2SnO in refluxing toluene) followed by, after a change of the solvent from toluene to chloroform, a direct oxidation with NBS. The complete removal of the acetal groups of 20 was easily achieved with 90% aq trifluoroacetic acid, to give the 1,5-dicarbonyl disaccharide as crude material complicated by complex mixtures of tautomeric forms. The crude product was subjected to a double reductive amination under conditions analogous to those previously reported for the monosaccharide series39 giving stereoselectively the corresponding azadisaccharide that was finally deprotected to 17 in acceptable yields (60% based on the uloses 20).
On this basis, we have chosen this latter approach for the preparation of the azamimic serie 11 with its many advantages. First of all the use of the cheap and easy available lactose as starting material: lactose have a preformed β-(14) linkage and that permits to avoid any β-glycosylation
step. The aminocyclization gives stereoselectively a single product, avoiding in this way difficult purification procedures. Furthermore, using different types of amine in the aminocyclization reaction (in particular ω-functionalised amine), it allows also the synthesis of useful monomers for glycocluster construction.
2. Results and discussion
For the preparation of azadisaccharide serie 11, a common intermediate 21 (Scheme 2.1) has been used, readily obtained through double acetonation of commercial lactose.40 Selectively deprotected 21 has been subjected to C-2’ epimerization by an oxidation-stereoselective reduction sequence11b on that position, giving talo derivative 22. From this key intermediate GalNAc and GlcNAc containing azadisaccharide have been obtained.
Scheme 2.1 O O O OH OC(OMe)Me2 O O O O (MeO)2HC O Ref .40 Lactose 21 O O O OH OC(OMe)Me2 O O O O (MeO)2HC O 22 Ref . 11b O O O NHAc OBn O O O O (MeO)2HC O Ref. 9b 6 O HO HO NHAc OH N H OHO OH OH O OH HO NHAc OH N H OHO OH OH GalNAc-DNJ GlcNAc-DNJ TalNAc-DNJ ManNAc-DNJ O OH HO NHAc OH N H OHO OH OH O HO HO NHAc OH N H OHO OH OH
For the preparation of TalNAc and ManNAc containing azadisaccharide, the key intermediate has been represented by protected talosamine 6, also prepared from 21.9b In a general procedure all compounds have been elaborated for first on the non reducing end then subjected to a common procedure of protected glucopyranoside trasformation to DNJ: (a) selective deprotection of isopropylidene bridges; (b) C-5 regioselective oxidation; (c) final acetal removal; (d) stereoselective aminocyclization; (e) for GalNAc and GlcNAc containing azadisaccharides azide reduction and N-acetylation; (f) final protection removal. The obtained hydrochloride analogues of that azamimic serie have been tested as activator of NKR-P1 (rat) and CD-69 (human) receptors, comparing that results with the “oxygenated” analogues. As an interesting elaboration of this
argument, PAMAM-glycoclusters (see Section 1) loaded with different types of (potential) NK cell activators have been synthesized to evaluate possible multivalent effects on biological targets.
2.1. Preparation of GalNAc-DNJ precursor 24
Talo derivative 22, prepared from easy available lactose,11b has been transformed into imidazilate 23 as recently reported in literature (Scheme 2.2).41 The imidazyl group introduction has been made to “activate” C-2’ position in a sequence of amination with inversion. The activation of 22 has been achieved by treatment with imidazyl sulfate (Im2SO2) and NaH in DMF at -30 °C, resulting in the corresponding imidazylate 23. Reaction quencing at -40 °C has been made to avoid the 2’,3’-enolether formation (see Section 1, Par. 2.1). Compound 23 has been subjected to a starting SN2 substitution reaction, with NaN3 in DMF and, during acid work-up (5% aq HCl/DCM extraction), to 6’-O-MIP removal. The crude has been directly C-6’ protected as a benzyl ether, using the system BnBr/NaH in DMF. Finally desired azide 24 has been isolated in a good yield after 3 synthetic steps (77% from 23). This result confirms the usefulness of the imidazylate leaving group for performing efficient substitution in position 2 of a pyranoside, where other aryl and alkyl sulfonates are known to give unsatisfactory results.42 For GalNAc precursor 24 (and also for GlcNAc one) it has been decided to insert azide moiety as a stable acetamido group precursor, that has been converted to amide only during the final steps of the synthetic way. For that reason problems in reactivity and purifications steps of hexosaminyl derivatives have been avoided. Scheme 2.2
Reagents and conditions. i: a) Im2SO2, NaH, DMF, -30°C (92%); ii: a) NaN3, DMF, 100°C then 5% aq. HCl/DCM; b) BnBr, NaH, DMF (77%, from 23).
2.2. Preparation of GlcNAc-DNJ precursor 30
The transformation of talopyranoside core of 22 to gluco one has been achieved through elaboration of C-2’ and C-4’ positions. The sequence has been started (Scheme 2.3) with selective protection of C-2’ as p-methoxybenzyl ether, using standard alkylation conditions (PMBCl, NaH, DMF), followed by acid work-up (5% aq HCl/DCM extraction) and crude 6’-O benzylation as seen
for the preparation of 24. Protected derivative 25 has been isolated, after chromatographic purification, in a overall 83% yield calculated from 22 (3 reactions, 1 purification). PMB has been chosen as C-2’ protection group because it is possible to remove using reagents and conditions that do not affect the other protecting groups of 25.
Scheme 2.3
Reagents and conditions. i: a) PMBCl, NaH, DMF, then 5% aq. HCl/DCM; b) BnBr, NaH, DMF (83%, from 22); ii: a) t-BuOK, THF, reflux; b) BnBr, NaH, DMF (77% from 25); iii: BH3
. Me2S, THF then H2O2, NaOH, H2O (80%); iv: BnBr, NaH, DMF (93%); v: DDQ, DCM/H2O (76%); vi: Im2SO2, NaH, DMF, -30 °C (83%); vii: NaN3, DMF, 100 °C (92%);
Protected 25 has been subjected to C-4’ epimerization, using the method applied in Section 1 Par. 2.1 for the preparation of ManNAc unit containing glycosil donors. After base catalyzed (t-BuOH, refluxing THF) elimination of acetone and C-3’ protection of the crude (BnBr, NaH, DMF), 4,5-enolether 26 has been selectively transformed into mannopyranoside 27 through a hydroboration-oxidation sequence. Compound 27 has been protected on C-4’ as benzyl ether and, after selective PMB group removal, subjected to an amination with inversion sequence as seen for the preparation of GalNAc analogue 24. This selective cleavage of C-2’ protection has been made following a method developed by Horita et al43 based on the use of DDQ (2,3-dichloro-5,6-dicyanobenzoquinone, Scheme 2.4), an oxidizing agent. The reaction proceeds via initial formation of a charge-transfer (CT) complex between an electrondonating aromatic ring and electron-accepting DDQ followed by benzylic dehydrogenation.
Scheme 2.4 OMe RO O O Cl Cl CN CN CT-complex OMe RO OMe RO HO DDQ OH OH Cl Cl CN CN DDQH OMe HO ROH H2O
Therefore, when an PMB ether is treated with DDQ in the presence of water, the oxidation is expected to take place affording an alcohol and anisaldehyde as shown in Scheme 2.4. This implies that an PMB protection is removed under mild DDQ oxidation conditions. Intermediate 28 has been threated with 1.1 eq of DDQ in DCM-H2O mixture (18:1) and C-2’ deprotected mannopyranoside has been isolated after 1h in good yield (76%). This particular mixture of solvents has had two merits: (a) the deep green color of the CT complex has been faded quite rapidly to colourless and reaction has been completed within 40’-1h; (b) weakly acidic DDQH (2,3-dichloro-5,6-dicyanohydroquinone) precipitated from the solution as the reaction proceeded because DDQH is almost insoluble in both dichloromethane and water, and the reaction medium was consequently kept almost neutral all through the reaction. This is sometimes very important in the case of substrates bearing acid-sensitive protecting groups as isopropylidene bridges on primary position. Obtained C-2’ alcohol has been finally subjected to the amination with inversion sequence described for GalNAc analogue 24: synthesis of imidazylate 29 in standard condition and efficient SN2 reaction performed with NaN3 in DMF (76% over 2 steps). The C-2’ inversion has been confirmed by 1H-NMR, analyzing coupling constant values for H-1’, H-2’ e H-3’ of manno 28 and
gluco 30 (Table 2.1).
Table 2.1
Compound J1΄,2΄ (Hz) J2΄,3΄ (Hz) J3΄.4΄ (Hz)
28 0.6 2.3 10.5
2.3. Preparation of ManNAc-DNJ precursor 9
The TalNAc- and ManNAc-DNJ have been also prepared, as shown in Par. 2.1, from lactose through a key intermediate 6 (Scheme 2.1). This compound has been the direct precursor of talosamine containing azadisaccharide which has been subjected to elaboration of protected gluco portion. For the synthesis of ManNAc-DNJ precursor, C-4’ epimerization has been necessary for first (Scheme 2.5) in the conditions seen for the preparation of 30. The mechanism of this epimerization method has been extensively discussed in Section 1, Par. 2.1.
Scheme 2.5 O O O NHAc OBn O O O O (MeO)2HC O O NHAc OBn BnO O O O O (MeO)2HC O O HO BnO NHAc OBn O O O O (MeO)2HC O i ii 6 31 9
Reagents and conditions. i: a) t-BuOK, THF, reflux; b) BnBr, KOH, 18-crown-6, THF, 0 °C (91% from 6); ii: BH3
.
Me2S, THF then H2O2, NaOH, H2O (80%).
2.4. Regioselective acid hydrolysis of N-acetylhexosaminyl-ββββ-(14)-DNJ precursors With all precursors (6, 9, 24 and 30) of desired azadisaccharide serie in hand, the first step of protected glucose elaboration has been approached: the selective removal of 5,6-O-isopropylidene. The isopropylidene group removal could be achieved through acid hydrolysis employing a lot of either protic or Lewis acids.44 One of the most used system is the treatment with acetic acid aqueous solutions at different concentrations and temperatures. Good results in terms both of yields and selectivities have been obtained for polyacetonide disaccharide compounds such as 6, 19 and 32 (Scheme 2.6) using 80% aq AcOH solution and heating to 40°C.
Scheme 2.6 O O O X OBn O O O O (MeO)2HC O Y O OH HO X OBn O OH O OH (MeO)2HC O Y 80% aq AcOH, 40 °C 19: X=H, Y=OBn 32: X=OBn, Y=H 6: X=NHAc, Y=H 33: X=H, Y=OBn 34: X=Bn, Y=H 35: X=NHAc, Y=H
In all cases the corresponding tetraols 33, 34 and 35 were isolated in yield up to 70% in good agreement with the supposed isopropylidene groups reactivities.38 In fact, the most reactive one is that employing a primary hydroxy function (5, 6 positions), followed by the tense one fused with
the pyranose ring (3’, 4’ position) and, finally, by the other isopropylidene group on the acyclic glucose unit (position 2, 3). This optimal condition of acid hydrolysis (80% aq AcOH, 40 °C) has been applied to all precursors and the results are summarized in Table 2.2. As shown in this Table, tetraols 35 and 37 and diols 36 and 38 have been isolated in a good yield (up to 65%) from their protected precursors. Main subproducts of hydrolysis reaction, that have been also isolated and analyzed by NMR, have been constituted by mixtures of 5,6- and 3’,4’-diols as a confirmation of the different reactivity of isopropylidene bridges of these substrates.
Table 2.2
Reagent Product Time Yield
1h 45’9a 79%9a 4h 80% 3h 30’ 68% O BnO BnO N3 OBn O OH O OH (MeO)2HC O 38 3h 30’ 74%
As a further study on different reactivity of isopropylidene protections, selective removal of 5,6-O protection has been attempted using the “sacrificial glycol approach”. As shown in Fig. 2.1, this method concerns selective isopropylidene removal through an acid catalyzed transacetalization between a protected sugar and a 1,2-diol as propylene glycol.38
Figure 2.1 OH OH O O O O OH OH
Using a low polar solvent as dichloromethane, it is possibile to precipitate polar diol in the reaction ambient and therefore to move equilibrium towards deprotection. Also an excess of glycol might work in that direction. On this basis, diol 39a has been attempted to obtain from 24, using a large excess (7 eq) of propylene glycol in presence of catalytic TsOH in CH2Cl2 Scheme 2.7). After 2 h, 3 main products have been detected by TLC analysis, that have been analyzed by NMR after chromatographic purification.
Scheme 2.7
Desired diol 39a has been isolated in mixture with 3’, 4’ diol 39b (20% yield), that has been also obtained pure (13%). Starting material has been isolated as the main reaction product (26%) and tetraol 37 has been obtained in traces (less 10%). These results pointed out that this approach can’t be applied to our substrates.
2.5. Regioselective C-5 oxidation reaction
Two different couples of diols, as the one which is present in our structures, could be selectively oxidised using the stannyliden acetal method.45 The reaction starts with the formation of intermediate 40 which is then open by the oxidising agent that extracts an hydride from the more hindered position through a cyclic mechanism. (Fig. 2.2).
Figure 2.2 O R O Sn Bu Bu H Br Br O R O Sn Bu Bu Br 40 41 5 6 5 6
In particular in the tetraols 35 and 37, using a stochiometric amount of Bu2SnO, the more reactive primary hydroxy function might react for first and is thus possible to obtain the intermediate 40 on C-5 and C-6 with high regioselectivity. All the obtained alcohols solved in toluene have been refluxed overnight under azeotropical water removal conditions, favouring stannyliden acetal formation. The solvent has been removed and the crude added with CHCl3 and the oxidizing agent,
N-bromosuccinimide (NBS). As reported in the Table 2.3, the 5-ulosyl derivatives 43, 44 and 45
have been obtained in good yields (up to 65%), after chromatographic purification. Scheme 2.8
Table 2.3
Alcohol X Y R1 R2 R3 Yield
Talo 35 NHAc H OH H OH 67% (42)
Manno 36 NHAc H OBn OH H 94% (43)
Galacto 37 H N3 OH H OH 28% (44)
Gluco 38 H N3 OBn OBn H 77% (45)
Problems have been occurred in the preparation of 44 from tetraol 37: TLC analysis of oxidation crude shown two main spots at Rf 0.44 (C-5 ulosyde 44) and Rf 0.50. The main product (Rf 0.50) has been constituted by a complex mixture of ulosydes, not separable on silica gel, probably the mono 4’-keto compound and the 5,4’-diketo derivative. This problem is probably caused by equatorial C-2’ azido moiety that might interfere with the selective stannyliden formation on C-5 and C-6. A different synthetic strategy has been approached to overcome this problem: the synthesis of C-5, C-6 deprotected disaccharides using different approches. First attempt to selective protection at C-6 (primary alcohol) has been made for the preparation of 46 and 47 that afterwards can be protected on C-3’ and C-4’ (Scheme 2.9). Unfortunately selectively C-6 etherification with trityl chloride in pyridine or C-6 pivaloylation with pivaloyl chloride in piridina-dichloromethane
have been not exhaustive, with the isolation of 46 and 47 in poor yield (19% and 25% respectively).
Scheme 2.9
Reagents and conditions. i: for 46: TrCl, Py (19%); for 47: PivCl, Py-DCM, -30°C to rt (25%). Another approach to complete protection of galacto portion of 37 has been planned to take advantage from higher reactivity of C-5 and C-6 diol compared to C-3’ and C-4’ one. As shown in Scheme 2.10, tetraol 37 was converted to C-3’, C-4’diol using 2-methoxypropene (2-MP) as acetonizing agent and pyridinyl tosylate (PyHOTs) as acid catalyst,46 a well-known method for selective protection of vicinal diols.
Scheme 2.10
Reagents and conditions. i: a) 2-MP, PyOHTs, DCM; b) BnBr, NaH, DMF (73% from 37) ; ii: 80% aq AcOH, 40 °C (73%); iii: Bu2SnO, toluene, reflux then NBS, CHCl3 (89%).
The reaction mechanism starts with vinyl ether attacked by diol 53 primary alcoholic function, achieving the acyclic acetal 54. This intermediate is subjected to intramolecular nucleophilic attack from the nearest OH function giving final cyclic acetal 55 (Scheme 2.11).
Scheme 2.11
Desired 3’,4’-diol has been obtained in quite good yield (66%) in presence of 27% of protected starting product and subjected to complete protection of C-3’ and C-4’ as benzyl ethers (BnBr, NaH, DMF, 98%). Protected 50 has been also prepared without diol purification in a 73% overall yield from 37. After selective removal of 5,6-O-acetal protection (80% aq AcOH, 40 °C), C-5 oxidation conditions have been applied to diol 51, leading to 5-keto derivative 52 in 89% yield.
2.6. Stereoselective double reductive amination
To perform the aminocyclization step the aldehydo function must be deprotected: all 5-keto derivatives have been treated with 90% aq CF3COOH at room temperature (Scheme 2.12). All deprotected compounds have been obtained as complex mixtures of tautomeric forms and directly used in the next reactions without further purifications
Following the synthetic approach described in Par. 1.7, all compounds have been subjected to double reductive amination with some primary amines in MeOH using NaBH3CN as reducing agent. The reaction starts (Fig. 2.3) with the first amine attack on the more reactive aldehydo group with the formation of the imine 56b which is in situ reduced to 56c.
The secondary amine now attacks the keto function with the formation of the cyclic iminium ion intermediate 56f after water elimination. The last reduction brings to the azapyranose derivatives 57a and 57b. As primary amines in the double reductive amination of those deprotected compounds, benzylamine hydrochloride has been chosen for the preparation of the azadisaccharide serie and N-Boc ethylenediamine for the synthesis of azamimics to be loaded on PAMAM dendrons. The reaction has been performed in anhydrous MeOH, under argon, heating to 60°C and adding NaBH3CN, a reducing agent able to reduce imine but not carbonyl functions. Azadisaccharides prepared from 42 and 43 have been finally submitted to an acetylation reaction to simplify the purification procedures (Scheme 2.12).
Scheme 2.12
Reagents and conditions. i: 90% aq TFA; ii: a) BnNH3+Cl- or N-Boc(CH2)2NH2, NaBH3CN, MeOH, 60°C (see table below); b) Ac2O, Py (see table below).
Table 2.4. Double reductive amination results.
C-5 keto X Y W R R1 R2 R3 Yield
Tal 42 NHAc H Bn Ac OAc H OAc 42% (58)
Man 43 NHAc H Bn Ac OBn OAc H 44% (59)
Man 43 NHAc H N-Boc-(CH2)2 Ac OBn OAc H 70% (60)
Gal 52 H N3 Bn H OBn H OBn 58% (61)
During this study talosamine containing azadisaccharide 66 has been also prepared from key intermediate 6. In the synthetic sequence (Scheme 2.13) 6’-O-Bn protection has been removed for first through hydrogenolysis (95% yield) and substituted with an acetyl ester (2:1 py/Ac2O, 96% yield). Derivative 63 has been subjected to the sequence of protected gluco portion transformation into N-functionalized DNJ: selective acid hydrolysis to tetraol 64, regioselective C-5 oxidation and final acetal removal with 90% aq TFA. Aminocyclization step has been performed using N-Boc ethylenediamine as amine: the obtained iminosugar has been finally peracetilated (67% yield from 65) and loaded on PAMAM dendron (see next Par.). This intermediate has been prepared in a different manner to have same protections on each OH group of the iminosugar (one reaction for final deprotection) and to avoid hydrogenolysis step on final glycodendrimer (catalyst poisoning problems).
Scheme 2.13
Reagents and conditions. i: H2, 10% Pd-C, MeOH (95%); ii: Ac2O, Py (96%); iii: 80% aq AcOH, 40 °C (75%); iv: Bu2SnO, toluene, reflux then NBS, CHCl3 (40%); v: a) 90% aq TFA; b) N-Boc(CH2)2NH2, NaBH3CN, MeOH, 60°C; c) Ac2O, Py (67% from 65).
As a further study on aminocyclization reaction, DNJ derivative 69 (Scheme 2.14) has been prepared from C-5 keto-D-glucose 6747 that has been subjected to acid hydrolysis and the resulting 1,5- dicarbonilic compound 68 has been transformed using mono benzyloxycarbamate of ethylenediamine hydrochloride in the aminocyclization. Functionalized DNJ 69 has been prepared in 27% yield from 67, after complete protection of hydroxy functions as acetyl esters. Problems in iminosugar TLC detection and isolation have been occured using benzylamine or N-Boc mono protected ethylenediamine as amines in aminocyclization step.
Scheme 2.14
Reagents and conditions. i: 90% aq TFA; ii: a) Z-NH(CH2)2BnNH3+Cl-, NaBH3CN, MeOH, 60°C; b) Ac2O, Py (27% from 67).
As reported in the Table 2.4, a single product has been stereoselectively obtained in each aminocyclization reaction and every final structure has been confirmed and assigned on the basis of NMR spectra (Table 2.5). Figure 2.4 H5 N RO RO OR OR X H1ax H1eq H2 H3 H4 N H1eq H2 H3 X H5 OR H4 OR OR H1ax RO 4C 1 D-gluco 1C4 N RO RO OR H5 X H1ax H1eq H2 H3 H4 N H1eq H2 H3 X OR H4 OR OR H1ax H5 4C 1 L-ido 1C4 OR RO
Table 2.5. Coupling constants (Hz) of the azapyranose unit.
Compound
J
1ax,1eqJ
1ax,2J
1eq,2J
2,3J
3,4J
4,5J
5,6aJ
5,6bJ
6a,6b69 (Mono)
11.4
10.1
4.9
9.7
-
-
-
-
-
61 (Gal)
11.2
10.1
4.5
8.8 8.8 9.7
2.1
2.1
12.3
62 (Glc)
11.2
10.2
4.7
8.9 8.9 9.2
1.8
2.2
12.4
58 (TalB)
11.8
10.5
5.2
9.4 9.4 9.6
3.1
2.3
12.7
66 (TalBoc)
11.7
10.3
4.9
9.4 9.1 9.6
3.4
2.5
12.7
59 (ManB)
11.3
10.7
5.1
9.4 9.4 9.4
2.7
2.7
12.8
60 (ManBoc)
11.7
11.0
5.1
9.6 9.3 9.5
3.1
2.8
12.8
The aminocyclization step that gives the complete stereoselectivity to this reaction is the hydride attack on the cyclic iminium ion intermediate (Fig. 2.5). This charged intermediate has two conformers 3H2 and
2
H3 in a twisted semi-chair structure: the conformer 2
H3 is more stable then 3
H2 because of its equatorial substituents. The “pseudo-axial” hydride attack on 2H
3 iminium ion is on
α-side for stereoelettronic interactions. In this case there is the formation of a “chair-like”
transition state (low energy) where partial formed σ-bond between hydride and C-5 is antiperiplanar to N lone pair.
The result is the formation of a pseudo D-gluco iminosugar (N-substitute DNJ). The “pseudo-axial” hydride attack on β-side should be on the less stable conformer 3H2, with the formation of a “chair-like” transition state containing three axial substituents (high energy) and therefore unfavourable 1,3-syn diaxial interactions with incoming hydride: the formation of pseudo L-ido iminosugar was not detected.
Figure 2.5
2.7. Conversion of azido iminosugars 61 and 62 to acetamido ones and final deprotections
At this point selective transformation of 2’-azido moiety of 61 and 62 to acetamido one has been attempted. Reduction of azide group to amine has been the first step for the introduction of an acetamido moiety on C-2’ (Scheme 2.8). Different azide reduction methods were reported but not all are good for those compounds. For example hydrogenolysis with base poisoned 10% Pd-C isn’t usable in this substrate because N-Bn on DNJ portion will be also removed, with selectivity problems in N-acetylation step. Problems occured using PPh3 supported on resin in aq THF,48 an optimal method applied on SP19F repeating unit (see Part 1, Section 1, Par. 2.2). Finally ammides
70 and 71 have been prepared using the well-known NiCl2·6H2O/NaBH4 method49 for the azide reduction, followed by selective N-acetylation (Ac2O-MeOH, Scheme 2.15).
Scheme 2.15
Reagents and conditions. i: a) NiCl2.6H2O, NaBH4, MeOH; b) Ac2O, MeOH.
In this “simple” reaction, a mixture of desired iminosugar and 6-O acetyl analogues as 72 and 73 have been always obtained, also with a low temperature stirring. The only found solution has been the selective 6-O deprotection applying standard Zémplen condition at 0°C (NaOMe in MeOH) to the mixture: desired 70 and 71 has been finally prepared in an overall good yield (up to 80%) from azide 61 and 62. Probably the formation of 72 and 73 has been caused by the cationic specie 74 that
in situ permits the N→O acyl shift through a stable cyclic intermediate 75 (Scheme 2.16).
Scheme 2.16
With the four representatives of azadisaccharide serie in hand, complete removal of protections (acetyl esters and benzyl ethers) has been performed to prepare deprotected hydrochlorides to be tested as NKR-P1 (rat) and CD-69 (human) receptors of Natural Killer cells. For first compounds 58 and 59 have been deacetylated using standard Zémplen conditions (NaOMe in MeOH) and, after chromatographic purification, isolated in good yield (up to 80%, Scheme 2.17). Final deprotected hydrochlorides have been obtained in nearly quantitative yield applying hydrogenolysis (H2, 10% Pd-C) in presence of 1% methanolic HCl to benzyl protected compounds.
Scheme 2.17
Reagents and conditions. i: NaOMe, MeOH, 0°C; ii: H2, 10% Pd-C, 1% HCl-MeOH, MeOH. All final compounds have been characterized with 600 MHz NMR monodimensional and bidimensional experiments (see Fig. 2.6-2.9). Hydrochlorides 79 and 80 have been purified through recrystallization from MeOH-iPrOH mixture. Attempting of crystallization on 81 and 82 have been failed because of their hygroscopicity.
2.8. Preparation of dendrimers loaded with potential NK activators
In this study on NK cell activators, it has been decided to include the preparation of glycodendrimers that have on their surface sugars or iminosugars, to study a possible multivalence effect on NK receptors (see introduction of Section 1). PAMAM dendrons (see Section 1, Par. 2.3) have been used as dendritic matrix following the thioureidic bridge approach as loading method of saccharides.
The sugars, thaT have been chosen to be elaborated and loaded on PAMAM are (Fig. 2.10): a) the functionalized DNJ 69, with a N-protected spacer,
b) two different azadisaccharides, 66 and 83, with the same protected spacer;
Figure 2.6a. 1H-NMR of 79 (CD3OD-D2O, 600 MHz). O OH HO NHAc OH N O HO OH OH H .HCl
Figure 2.6b. 13C-NMR of 79 (CD3OD-D2O, 600 MHz). O OH HO NHAc OH N O HO OH OH H .HCl
Figure 2.7a. 1H-NMR of 80(CD3OD-D2O, 600 MHz). O HO HO NHAc OH N O HO OH OH H .HCl
Figure 2.7b. 13C-NMR of 80 (CD3OD-D2O, 600 MHz). O HO HO NHAc OH N O HO OH OH H .HCl
Figure 2.8a. 1H-NMR of 81 (CD3OD, 600 MHz). O OH HO NHAc OH N OHO OH OH H .HCl
Figure 2.8b. 13C-NMR of 81 (CD3OD, 600 MHz). O OH HO NHAc OH N OHO OH OH H .HCl
Figure 2.9a. 1H-NMR of 82(CD3OD, 600 MHz). O HO HO NHA c OH N O HO OH OH H .HCl
Figure 2.9b. 13C-NMR of 82 (CD3OD, 63 MHz). O HO HO NHA c OH N O HO OH OH H .HCl
Figure 2.10
The first step to coupling reaction has been the introduction of isothiocyanate moiety on the spacer of sugars and iminosugars, that permits the formation of the thioureidic bridge with the amino groups on the dendritic surface.
Scheme 2.18
For that reason, amino functionality of 69 has been deprotected (H2, 10% Pd-C, acid MeOH) and finally transformed into isothiocyanate one using dipyridil-thionocarbonate (DPT, Scheme 2.19),50 a “thiocarbonyl transfer” reagent, good alternative to thiophosgene (27% yield from 69). Azamimic 83, containing a ManNAc unit, has been simply obtained from the product of aminocyclization 60 through an exchange from benzyl protections to acetyl ones: compound 60 has been subjected to hydrogenolysis (10% Pd-C in MeOH) followed by standard acetylation (py/Ac2O), giving 83 in 89% yield from 60. Completely acetylated 66 and 83 have been converted into isothiocyanate analogues after standard Boc removal (TFA, DCM) and reaction using DPT in DCM (52% and 75% respectively).
The two disaccharides 84 and 85 have been both synthesized from glycosyl donor 86, prepared from commercial lactose as shown in Section 1 Par. 2.1. Donor 86 has been glycosylated with 3-O-tosylpropandiol (easily synthetized from 1,3-propandiol and tosyl chloride) as acceptor and NIS/AgOTf as activating system (Scheme 2.19). α-glycoside 87αααα has been isolated in 44% yield
and β-saccharide in 38% yield (α/β≈1:1). Applying SN2 reaction condition to this derivative (NaN3, TBAI, DMF) azide 85αααα and 85ββββ have been prepared (up to 90% yield) and trasformed into isothiocyanate analogues following the seen sequence azide reduction-DPT reaction (Scheme 2.19). Isothiocyanates 88ββββ has been obtained from 85ββββ in 56% yield.
Scheme 2.19
Reagents and conditions. i: 3-tosylpropandiol, NIS, AgOTf , DCM, -30°C to rt (38% for 87αααα, 44% for 87ββββ); ii: NaN3, TBAI, DMF, reflux (96% for 85αααα, 91% for 85ββββ); iii: a) H2, 10% Pd-C, TEA, MeOH; b) DPT, DCM (56% for 88ββββ); iv: H2, 10% Pd-C, (Boc)2O, EtOAc-EtOH then Ac2O, Py (65% for 84αααα, 84% for 84ββββ); v: DCM-TFA-H2O then DPT, DCM (78% for 89αααα, 71% for 89ββββ).
The preparation of peracetylated analogues 84αααα and 84ββββ has been performed also from azide 85α,βα,βα,βα,β through a reaction sequence that permitted azide reduction to protected amine and exchange from benzyl ether protections to acetyl ones.51 After a starting catalytic hydrogenation (H2, Pd-C in presence of Boc2O) that at the same time afforded to debenzylation, azide reduction and N-Boc protection, the final complete acetylation gave 84αααα and 84ββββ. Those derivatives have been transformed to isothiocyanate analogues through standard Boc removal (TFA, DCM) and reaction with DPT in DCM. All the prepared isothiocyanates have been coupled with the smallest PAMAM-(NH2)2 (Fig. 2.11), performing the reaction at 40°C in a optimized solvent mixture (DCM-DMF) that can solv both reagents. In the Table 2.6 are reported the loading results.
In the Table 2.6 the loading results are reported. Figure 2.11
Table 2.6
Compound Coupling Time Coupling Yield
88 96 h 32%
89 72 h 65%
90 72 h 61%
91 30 h 63%
92 24 h 54%
As reported in the Table 2.6, coupling reactions with PAMAM dendron have been achieved to desired glycodendrimers in quite good yield after chromatographic purifications: as observed for the failed loading of SP19F trisaccharide on the same dendritic substrate (Section 1 Par 2.2) with no perbenzylated loading products, the use of benzyl protected saccharides have been resulted in a drop of yield (from 50% to 30%) faced to peracetylated analogues. Comparison of signal intensity (1H-NMR) of the sugar moiety and of the terminal t-butoxy group allowed to confirm the reaction of both amino groups. The next step that will be faced is the deprotection of saccharide moieties and the biological evaluation of a multivalent effect of prepared glycodendrimers on NK cell receptors .
2.9. Evaluation of compounds 81 and 82 as activators of NKR-P1 and CD-69 receptors52
New compounds 81 and 82 have been tested for binding affinity towards the activation receptors of NK cells, rat NKR-P1A and human CD69. As controls for assessing the structure–activity relationship, the respective building units have been also included in the plate inhibition assays as well as the standard positive control GlcNAc (Figure 2.12).Surprisingly, DNJ gave comparable results to GlcNAc, used in these tests as a benchmark (Table 2.7). Coupling the two units increases the binding, but with NKR-P1A it only has an additive and not multiplicative effect. A different situation, however, has been found with CD69 in compound 81, where the attachment of ManNAc (that is per se inactive), to DNJ increases the binding by more than one order of magnitude. Nevertheless, the most interesting result from a practical point of view seems to be the binding of DNJ to the activation receptors of NK cells. As described in the introductive section, it was clearly demonstrated that NKR-P1 receptor interacts strongly with N-acetylhexosamines. The binding groove optimally binds linear oligosaccharides (typically chitoteraose); GalNAc and ManNAc being stronger ligands than GlcNAc. In contrast, CD69 only binds well to GlcNAc, but linked to the branched, multiantennary structures, where multivalency plays a crucial role.53This is also the
reason why the binding affinities with simple oligosaccharidic ligands (see Table 2.7) are much weaker in CD69, where the presence of multivalent ligands improves the binding by several orders of magnitude. Typically, binding affinity correlates well with the killing activity of the respective NK cells towards tumor or virally infected somatic cells. In summary, probably the most interesting result is the binding of DNJ itself to NK cell activation receptors.
Figure 2.12 Table 2.7 Compound NKR-P1A CD69 TalNAc-DNJ·HCl (81) 5.5 3.3 ManNAc-DNJ·HCl (82) 6.5 4.5 DNJ·HCl (13) 5.0 3.2 TalNAc (94) 4.6 0 ManNAc (95) 6.7 0 GlcNAc (93) 5.6 3.5
The immunomodulatory activity of these iminosugars has rarely been studied and only a few reports can be found in the literature, mostly describing the immunosuppressive activity of such compounds.Van den Broeck54tested a series of DNJ-N-alkyl derivatives and found that an N-7-octadecyl derivative inhibited PBMC (lymphocyte)-induced proliferation. Unfortunately DNJ was not tested. Ye et al.55atested a series of newly prepared iminosugar derivatives for their effects on the secretion of IL-4 and IFN-c from mouse splenocytes and found that some compounds are strong immunosuppressants with potential for use in the treatment of autoimmune diseases. Recently, Zhou et al.55bprepared a series of 1,6-dideoxy-N-alkyl iminosugars and demonstrated IFN-c and IL-4 inhibition activity in some of them. None of the above studies tested non-derivatized DNJ as a control (benchmark) (13), which we consider a drawback. These new results on the potential immunoactivation effect of DNJ (13) which could be of significant importance for the treatment of viral infections, during which the activation of NK cells represents one of the critical elements of the immune response.56This pilot study indicates that DNJ and mainly its derivatives currently used in therapy should be further tested for their immunostimulating activities in more complex cellular systems, and in vivo.
3. Experimental.
General methods
NMR spectra were recorded with a Bruker AC 200 instrument operating at 200.13 MHz (1H) and 50.33 MHz (13C) and with a Varian INOVA600 spectrometer operating at 600 and 150 MHz for 1H and 13C respectively using Me4Si as internal reference. Assignments were made, when possible, with the aid of DEPT, HETCOR, HSQC, by comparison of values for known compounds and applying the additivity rules. In the case of mixtures, assignments were made by referring to the differences in the peak intensities.All reactions were followed by TLC on Kieselgel 60 F254 with detection by UV light and/or with ethanolic 10% phosphomolybdic or sulphuric acid, and heating. Kieselgel 60 (E. Merck, 70-230 and 230-400 mesh, respectively) was used for column and flash chromatography. Solvents were dried by distillation according to standard procedure, and storage over 4Å molecular sieves activated at least 24 h at 400 °C. MgSO4 was used as the drying agent for solns.
3.1. Elaboration of disaccharide non-reducing end
2-azido-6-O-benzyl-2-deoxy-3,4-O-isopropilidene-ββββ-D-galactopyranosyl-(1
4)-2,3:5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (24)
A soln of 2341 (3.5 g, 4.92 mmol) and NaN3 (641 mg, 9.85 mmol) in dry DMF (32 mL) was stirred under argon atmosphere at 100 °C. After 1 h and 15’ TLC analysis (EtOAc) revealed the complete disappearance of the starting material, mixture was cooled to rt and partitioned between CH2Cl2 (50 mL) and 5% aq HCl (v/v, 50 mL), the organic phase separated when TLC analysis showed the complete 6’-O-MIP group removal (EtOAc) and neutralized with satd aq NaHCO3 (30mL). The collected organic phases were dried (MgSO4
.
H2O), filtered and concentrated under dimished pressure. The crude residue was used in the next reaction without further purification procedures. To a suspension of NaH in mineral oil (60%, 394 mg, 9.84 mmol) pre-washed with hexane under argon atmosphere and cooled at 0°C, a soln of the crude in dry DMF (35 mL) was slowly added. The mixture was stirred at 0°C for 30 min and treated with BnBr (0.73 mL, 5.90 mmol). After 15 h under room temperature stirring, starting compound was completely reacted (2:8 hexane-EtOAc) and the reaction mixture was then cooled to 0°C, excess of NaH was destroyed by addition of MeOH (3 mL) followed by 10 min stirring and MeOH removal under dimished pressure. The solution was partitioned between Et2O (50 mL) and H2O (50 mL), the organic phase separated and
(MgSO4.H2O), filtered and concentrated under dimished pressure. The flash chromatographic purification over silica gel of the reaction product (2:8 hexane-EtOAc) gave pure 24 (2.36 g, 77% yield from 23) as a colourless syrup, Rf 0.74 (2:8 hexane-EtOAc), [α]D –26.7 (c 1.05; CHCl3); 1 H-NMR (250 MHz, CD3CN): δ .38-7.27 (m, 5H, Ar-H), 4.66 (d, 1H, J1’,2’ 8.5 Hz, H-1’), 4.57, 4.51 (AB system, 2H, JA,B 12.1 Hz, CH2Ph), 4.33 (m, 2H, H-1, H-2), 4.25 (dt, 1H, J4,5 4.6 Hz, J5,6a=J5,6b 6.5 Hz, H-5), 4.13 (dd, 1H, J3’,4’ 5.3 Hz, J4’,5’ 2.0 Hz, H-4’), 4.09 (dd, 1H, J6a,6b 8.5 Hz, H-6b), 4.03 (m, 1H, H-3), 4.00 (dd, 1H, H-6a), 3.97 (m, 1H, H-4), 3.93 (m, 1H, H-5’), 3.90 (dd, 1H, J2’,3’ 8.2 Hz, H-3’), 3.67 (dd, 1H, J6’a,6’b 10.0 Hz,
J5’,6’b 5.8 Hz, H-6’b), 3.62 (dd, 1H, J5’,6’a 6.6 Hz, H-6’a), 3.32, 3.30 (2s, each 3H, 2 × OMe-1), 3.31 (dd, 1H, H-2’), 1.48, 1.40, 1.33, 1.32, 1.30, 1.29 (6s, each 3H, 3 × CMe2);
13
C-NMR (63 MHz, CD3CN): δ 139.6 (Ar-C), 129.4-128.7 (Ar-CH), 111.1, 110.9, 109.1 (3 × CMe2), 106.3 (C-1), 102.1 (C-1’), 78.2 (C-3, C-5), 78.0 (C-3’), 76.7 (C-2), 76.1 (C-4), 74.2 (C-4’), 74.0 (CH2Ph), 72.9 (C-5’), 69.8 (C-6’), 67.5 (C-2’), 66.2 (C-6), 56.3, 54.4 (2 × OMe), 28.6, 27.7, 27.3, 26.9, 26.6, 25.3 (3 × CMe2).
Anal. Calcd for C42H54O12 (623.69): C, 57.77; H, 7.27; N, 6.74. Found C, 57.70; H, 6.68; N, 6.69.
2-azido-6-O-benzyl-2-deoxy-3,4-O-isopropilidene-ββββ-D-galactopyranosyl-(1
4)-2,3-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (37)
A soln of 24 (2.4 g, 3.79 mmol) in 80% aq. AcOH (78 mL) was stirred at 40°C until the starting compound was completely reacted (3.5 h, TLC, EtOAc) with formation of slower moving products. The reaction mixture was concentrated at reduced pressure and repeatedly co-evaporated with toluene (5 × 30 mL). The crude residue was directly applied to a flash chromatography column (93:7 CHCl3-MeOH) to give pure tetraol 37 (1.4 g, 68% yield) and mixture of partially deprotected diols 38 and 39 (524 mg, 23% yield, 38:39≈4:6).
Tetraol 37 was collected as a white solid, Rf 0.38 (EtOAc), m.p. 130-133°C (crom.), [α]D -1.57 (c 1.03, MeOH); 1H-NMR (250 MHz, CD3CN-D2O): δ 7.36-7.27 (m, 5H, Ar-H), 4.51, 4.45 (AB system, 2H, JA,B 11.9 Hz, CH2Ph), 4.43 (d, 1H, J1’,2’ 7.9 Hz, H-1’), 4.41 (t, 1H, J1,2 6.5 Hz, J2,3 6.2 Hz, H-2), 4.33 (d, 1H, H-1), 4.26 (dd, 1H, J3,4 0.9 Hz, H-3), 3.78-3.60 (m, 5H, H-3’, H-4’, H-5’, H-6a, H-6b), 3.58 (m, 3H, H-6’a, H-6’b, H-4), 3.42 (m, 2H, H-2’, H-5), 3.28, 3.27 (2s, each 3H, 2 × OMe), 1.35, 1.33 (2s, each 3H, CMe2);
13 C-NMR (63 MHz, CD3CN-D2O): δ 139.0 (Ar-C), 129.3-128.7 (Ar-CH), 111.3 (CMe2), 105.8 (C-1),
O O O N3 OBn O O O O (M eO)2HC O O OH HO N3 OBn O OH O OH (M eO)2HC O
