RESULTS AND DISCUSSION

RESULTS AND DISCUSSION

1.

State of Art

Carbohydrate multivalent compounds should be an adequate strategy to interact with human lectins, for inhibition of interactions with viruses or bacteria. The discovery of the binding events between virus and host, which concerns utilization of only a fraction of the Man9(GlcNAc)2 epitope, has interested many scientists. Their object is to develop a glycan mimetic of Man9(GlcNAc)2 that potentially binds to carbohydrate-specific receptor, as DC-SIGN.34 The design of DC-SIGN high affinity ligands which mimics the terminal two or three mannose residues is one of main topic studied by Professor Anna Bernardi.35

Figure 1.1 Structure of pseudo-1,2-mannobioside with ester functionality.

In 2007, Professor Bernardi reported the synthesis36 of the 1,2-mannobioside mimic reported in Figure 1.1 as very interesting DC-SIGN ligand. This mannobioside mimic presents an enzymatic stability higher than the corresponding natural disaccharide and represents an important class of glycomimetics with a “carbasugars” portion, analogue of a true sugar in which the ring oxygen has been replaced by a methylene group (-CH2-). The structural substitution of the endocyclic oxygen atom with a methylene group makes the compounds more stable toward endogenous degradative

enzyme. Indeed, sugar mimics are generally more soluble and membrane penetrant, less hydrophilic and more metabolically stable than the sugar themselves. For these reason, the synthesis of mimics of nonreducing unit Manα1-2Man is considered as desirable design of anti-infective agents.36

Starting from these pseudo-1,2-mannobiosides bearing an ethoxy amino or azido chain on pseudo anomeric position, Bernardi’s group (Obermajer et al.)37

continued to pursue the idea of increasing the binding affinity by identifying two binding areas around Phe313 in the DC-SIGN binding site that were only partially occupied by cocrystallized tetramannoside Man4. These hydrophobic areas were targeted by attaching different hydrophobic moieties. A number of mannose-based DC-SIGN antagonists were synthesized (an illustrative example is the bis-benzylamide in Figure 1.2), and the majority of them inhibited DC adhesion at low micromolar concentrations.

Figure 1.2 Increasing potency of DC-SIGN antagonists by attaching hydrophobic moieties to deprotected carboxylates and its derivative.37

Recently, in laboratory where I carried out my thesis, in collaboration with Professor Bernardi, two new pseudo-disaccharide, real mimics of the natural Man1-2Man, were synthetized. The 1,2-mannobioside 1.01a and 1.01b are conceptually analogues to the previously synthetized compound 14 (Figure 1.1). Hydrophobic functionality R=-NO2; =-OMe; =-Me; =-H; Attachement of lipophilic fragments R=CH2CH2NH2 =CH2CH2N3

However, instead of a more simple mimic structure, they present a real D-carbamannose unit as mimic of the reducing mannose unit as represented in Figure 1.3.

Figure 1.3 Structure of real carba mimetics of Manα1,2-Man, main component of Man9.

The synthesis of compounds 1.01a and 1.01b proceeds through the construction of the disaccharide skeleton by glycosylation of azido derivatives 1.02, as the glycosyl acceptor, by the classic trichloroacetimidate (TCA) (-)-1.00 (Scheme 1.1), as the glycosyl donor.

Scheme 1.1 Synthesis of real carba mimetics 1.01 of Manα1,2-Man.

The amino 1.01a and azido 1.01b derivatives originate from the same key intermediate, the 6-O-benzyl-1,2-anhydro-5a-carba-β-D-mannopyranose (+)-1.03, which was subjected to two different types of elaboration to afford the tri-O-benzyl-β-epoxide (+)-1.04a and the tri-O-acetyl-β-epoxide

(+)-1.05b. Subsequent elaboration allowed to obtain pseudomannobiosides (+)-1.01a and (-)-1.01b

Scheme 1.2 Key intermediate (+)-1.03 for the preparation of pseudomannobiosides (+)-1.01a and (-)-1.01b.

The crucial steps in the pathway towards the synthesis of (+)-1.01a and (-)-1.01b are: the transformation of the commercially available Tri-O-Acetyl-D-glucal (+)-2.12 into the primary alcohol (-)-2.16 and the switch of them into the corresponding carba analogue (-)-2.20 (Scheme 1.3), in which a methylene group replaces the endocyclic oxygen. Then, after appropriate elaborations, the residual C(1)-C(2) double bond presents in (-)-2.20, was subjected to a stereoselective epoxidation to afford the pivotal epoxy diol (+)-1.03.

Scheme 1.3 Precursors of the synthesis of key intermediate, 6-O-benzyl-1,2-anhydro-5a-carba-β-D-mannopyranose (+)-1.03.

At this point, to obtain selectively the 2-aminoethyl-pseudomannobioside, the key intermediate trans-diol oxirane (+)-1.03 was treated with NaH/BnBr in DMF for the formation of the tri-O-benzyl epoxide (+)-1.04a. Subsequent, ring-opening reaction of (+)-1.04a by the appropriate nucleophilic agent, the azido-ethanol in presence of catalytic amount of Cu(OTf)2 as necessary Lewis acid catalyst38afforded the desired glycosyl acceptor, the 2-azido-1-ethoxy-cyclohexanol derivative

(+)-1.02a (Scheme 1.4).

Scheme 1.4 Synthesis of azido-ethoxy-glicosyl acceptor (+)-1.02a.

In this way, the glycosyl acceptor (+)-1.02a was ready for the next step of the glycosylation with the appropriate mannosyl donor, the trichloroacetimidate (TCA) (-)-1.00 protected as benzoyl derivative. The reaction was carried out in dichloromethane at

-20°C for 20 minutes, in the presence of molecular sieves AWMS to provide anhydrous conditions and TMSOTf as the catalyst.

The glycoconjugate (-)-1.05a formed in a completely α-stereoselective fashion and with a yield of 85% (Scheme 1.5) was debenzylated under reducing conditions (catalytic hydrogenation H2,Pd/C 10%), to afford (-)-1.06a and in a second step, the benzoyl protective groups on mannosyl portion were removed by saponification (1.0 M solution of MeONa/MeOH), to afford pseudomannobioside

Scheme 1.5 Synthesis of 2-aminoethyl-pseudo-1,2-mannobioside (+)-1.01a.

To maintain the azido group on the chain on pseudoanomeric position, the pivotal epoxide (+)-1.03 was deprotected on benzyl functionality at C(6) position (Scheme 1.6). The reduction with H2, Pd-C 10% in EtOH allowed the formation of β-epoxy triol 1.04b, the 1,2-anhydro-5a-carba-β-D -mannopyranose,39 which was immediately submitted to acetylation in presence of acetic anhydride and pyridine to obtained the tri-O-acetyl-β-oxirane 1.05b in 40% yield. In the same condition seen before, it was treated with freshly prepared 1.3 M 2-azido-ethanol in CH2Cl2 solution, in presence of a catalytic amount of Cu(OTf)238 to obtained (2-azidoethyl)-3,4,6-tri-O-acetyl cyclohexanol derivative (+)-1.02b, which represents the new glycosyl acceptor.

Scheme 1.6 Synthesis of azido-ethoxy-glycosyl acceptor (+)-1.02b.

Glycosyl acceptor (+)-1.02b was subjected to glycosylation with the TCA (-)-1.00 as glycosyl donor to afford the fully-O-protected pseudomannobioside (-)-1.06b.

At this point, pseudodisaccharide (-)-1.06b was treated with a solution 1M of MeONa in MeOH in order to remove all the ester functionalities to afford the fully-O-deprotected product (-)-1.01b in good yield (88%).

DC-SIGN affinities and antiviral activity of these new compounds were examined by Professor Frank Fieschi at Institut de Biologie Structurale-Jean-Pierre Ebel, in Grenoble (France). The analysis was carried out on infection model based on Ebola envelope-pseudotyped viruses and Jurkat cells expressing DC-SIGN.39

The results demonstrated an inhibitor activity very similar than the activity estimated for the corresponding natural disaccharide Manα1-2Man.

Chart 1 IC50 average for compounds (+)-1.01a, (-)-1.01b, ester analogues (psdi) and

2. Purposes of the thesis

On the basis of literature data and of the previous results related to real D,D-pseudo1,2-disaccharide

1.01, which highlighted lower activity due to higher hydrophilic properties of the mimic portion, it is

clear that hydrophobic groups increase the binding affinity between pseudodisaccharide and DC-SIGN amino acids through secondary interactions. In consideration of this, we decided, thanks to the experiences acquired in our laboratory on the carba sugar synthesis of this system, to introduce an amino substituent group on C(4) position of carbamannose unit.

In a fist approach we began with the synthesis of pseudo-disaccharide with a N substitution in C(4) position, starting from the vinyl aziridine(±)-2.01, available in our laboratory when I start my thesis, as racemic building block, as shown in Scheme 2.1. Maybe the relevant D,D -pseudo-1,2-mannobioside 2.08 could interact with DC-SIGN more avidly than his analogous compound 1.01

(Figure 2.1).

The synthetic sequence to racemic glycosyl acceptor ()-2.06 (Scheme 2.1) starts from racemic aziridine ()-2.01α-Ns.

Scheme 2.1 Synthesis of glycosyl acceptor (±)-2.06 from aziridine (±)-2.01.

as the only reaction product. If the acetate ()-2.02 was immediately oxidated by MCPBA, the reaction would be a 72:28 mixture of diastereoisomeric epoxides ()-2.05β and ()-2.05α. As for the prosecution of our synthetic protocol, only β-epoxide ()-2.05β was necessary, we had to find an alternative, more advantageous protocol which would lead to the desired β-epoxide ()-2.05β, even if still in a mixture with the diastereoisomeric α epoxide ()-2.05α, in an amount higher than the unsatisfactory 28%. Based on the experience previously acquired in the functionalization of the double bond in unsaturated systems, we thought that alcohol ()-2.03, the hydrolysis product of acetate ()-2.02, may efficiently direct the epoxidation. Actually, the application of MCPBA oxidation protocol to allyl alcohol ()-2.03, obtained by saponification (MeONa/MeOH) of acetate ()-2.02, afforded the desired epoxide ()-2.04β, as the only reaction product. Subsequent acetylation (Ac2O/Py) of ()-2.04β yielded the C(3)-O-acetyl β epoxide ()-2.05β. It represents all the substituents, including a N-nosylamino group at C(4), in the required relative configuration, ready for the nucleophilic ring opening reaction (Scheme 2.1) with 2-azido-1-ethanol soolution in presence of Cu(OTf)2.

This last reaction afforded C(1)-azido-ethoxy derivative of 4-(N-nosylamino)-D,L-carbamannose (

)-2.06, as desired, through a completely regio- and anti-stereoselective trans-diaxial opening process.

Following the usual protocol, the reaction of 4-(N-nosylamino)-D,L-carbamannose derivative ()-2.06 (the glycosyl acceptor) with the mannosyl donor TCA (-)-1.00, afforded, in a absolutely clean reaction, a 1:1 mixture of diastereoisomeric all-O-protected-4-(N-nosylamino)-pseudomannobiosides (+)-2.07a and (+)-2.07b in good yield (>99%). Preparative TLC separation of this mixture afforded pure

Scheme 2.2 Synthesis of all-O-protected-4-(N-nosylamino)-pseudomannobioside(+)-2.07a/b.

At the moment, the assignment of the exact relative configuration to (+)-2.07a and (+)-2.07b and their deprotection were not possible due to the small amount of the pseudomannobiosides availble.

After studying at the outset the synthesis of the desired system in the racemic form, we decided to perform the synthesis of an analogous pseudodisaccharide 2.09 in a stereoselective manner from a starter chiral, represented by Tri-O-acetyl-carba-D,L-glucal (+)-2.11.

Figure 2.1 Structure of pseudo-1,2-Mannobioside 1.01, N-nosylamino derivative 2.08 and N-tosylamino analogue 2.09.

The synthesis of the pseudo-disaccharide 2.09 requires an appropriately protected mannose portion, like glycosyl donor, and a carbamannose unit 2.10, mimic of a reducing end mannose residue (Scheme 2.3). The latter portion has detailed structural features, essential for the purposes of the final product activity, as:

a) the “anomeric carbon”, C(1), that improved stability towards the activity of mannosidase, b) a free hydroxy functionality at C(2) for the anchorage to the glycosyl donor,

c) hydroxy functionalities at C(3) and C(6), which could allow a growing water solubility if deprotected,

d) an aromatic ring (tosyl-group) on the amino functionality in C(4) position, useful to form possible hydrophobic interactions with the binding site, and

e) a spacer-arm terminated with amino functionality (-R), suitable to generate multivalent DC-SIGN ligands.

Scheme 2.3 Synthesis of carba mimetics 2.09 of Manα1,2-Man with hydrophobic portion.

On the basis of the acquired experience by our group in the field of stereoselective synthesis of enantiomerically pure carba analogues of glycal, we had settled on a similar strategy used yet for the pseudomannobiosides (+)-1.01a and (-)-1.01b. We used a chiral starting material, the Tri-O-acetyl-carba-D,L-glucal (+)-2.11, in order to obtain only an enantiomerically pseudodissacharide. Also in this framework, the use of carbohydrate provides, indeed, important advantages to the preparation of their carbacyclic analogues, because the enantiomeric purity of the target carbasugars is guaranteed by enantiomeric purity of the starting materials.

The new project moves towards the synthesis of 6-O-benzyl-4-deoxy-4-N-tosylamino-1,2-anhydro-5a-carba-β-D-mannopyranose 2.34β to obtain the new glycosyl acceptor 2.10, instead of the carbaglycosylating agent (±)-2.04β, the pivotal compound to obtain the N-nosyl glycosyl acceptor

(±)-2.06.

Figure 2.2 Pivotal compound for the respectively synthesis of compound 1.01, 2.08 and 2.09.

The N-tosylamino carbaglycosylatin agent is structurally similar to the key intermediate (+)-1.03, which lacks a tosyl-amino substituent in C(4) position, as shown in Figure 2.2. The selective choice of the Tosyl, instead of a Nosyl group, previously used in the racemic approach as protective functionality on the nitrogen, aims to preparation of a pseudo-disaccharide stable under biological assay conditions (pH 8), due to the minor acidity of the N-tosyl group related with the tosyl group.

2.1 Synthesis of 4’-N-tosyl glycosyl acceptor

The first part of my synthetic project concerns the preparation of 6-O-benzyl-4-deoxy-4-N-tosyl-1,2-anhydro-5a-carba-β-D-mannopyranose 2.34β, pivotal precursor of our glycosyl acceptor 2.10. Starting from the carba alcohol (-)-2.20 , the synthesis of a carba aziridine (+)-2.31α represents the crucial intermediate to allow a suitable transfer of an amino substituted functionality to C(4) position of a carbapyranose system (Scheme 2.4).

Scheme 2.4 Precursors of glycosyl acceptor 2.10.

The synthetic strategies adopted to obtain carbapyranoses, and in this case modified carbapyranoses, can be broadly classified as:

a)synthetic methods in which are employed non-carbohydrates as starting material40, as previously seen for the synthesis of the pseudodisaccharide (+)-1.01a and (-)-1.01b;

b) protocols which utilize carbohydrate as the precursor.41

We decide to make use of this second strategy for our purpose. In this way, the first goal of the synthetic approach is the synthesis of the primary alcohol (-)-2.16. We started with the commercially available tri-O-acetyl-D-glucal (+)-2.11, which was subjected to a regioselective deprotection of the primary hydroxyl functionality by treatment with Lipase from Candida Rugosa (CRL, 700.0 unit/mg activity) in phosphate buffer at pH 7.0, diisopropyl ether and acetone, affording the primary alcohol

(+)-2.12 (yield of 55 %). This original procedure has been setup in our laboratory using lipase from

Candida Cilindracea.42-43 Recently Sigma-Aldrich replaced this lipase with the more active CRL, making possible the use of less amount of phosphate buffer with beneficial effects on the final work-up.

Primary alcohol (+)-2.12 was selectively protected with 3,4-dihydro-2H-pyrane (DHP) in presence of pyridinium p-toluen sulfonate (PPTS) to give the 6-O-THP-derivative 2.13. Subsequent saponification with MeONa/MeOH gave the trans diol 2.14, which was protected as p-methoxy-benzyl ethers, using PMB-Cl and NaH in DMF. The trans diol represents the first key intermediate of our synthetic process and the choice of protective groups on C(3) and C(4) was crucial for the subsequent steps of the synthesis.

Scheme 2.5 Synthesis of the primary alcohol (-)-2.16.

The protection reaction consists in the deprotonation of the diol with NaH in DMF at 0°C; then, the obtained alcoholate was treated with p-methoxybenzyl chloride and the reaction mixture was stirred at room temperature for 12h to afford the 3,4-di-O-PMB-6-O-THP-D-glucal 2.15 (89 %). The PMB-derivative 2.15 was submitted to deprotection of THP-group by treatment with AcOH/THF/H2O 1.5:2:1 mixture for 12h at 45°C to afford the primary alcohol (-)-2.16,44 in good yield (61%) after flash chromatography. The deprotection turned out to be particularly sensible to the temperature which cannot exceed 50°C and to the ratio of the mixture AcOH/THF/H2O: if the conditions don’t

correspond strictly to those described above, complex reaction mixtures are obtained due to degradation of the glycal-derived alcohol (-)-2.16.45

This point, the project proceeds with the stereoselective synthesis of carba system, the carbacyclic alcohol (-)-2.20 from the primary alcohol (-)-2.16. We decided to use a new application of the Claisen thermal rearrangement, described by Nagarajan and Sudha46 in 1998 and already used in our laboratory in the past few years.45 The original procedure described by Nagarajan and Sudan implied the use of 3,4-di-O-benzyl protection, which was removed under reducing conditions (H2/Pd-C). However, debenzylation of the protective groups under typical conditions is incompatible with the maintenance of the C(1)-C(2) double bond, necessary in our case, for the further functionalization. Thus, we decided to use the p-methoxybenzyl as protective functionality on hydroxyl groups on C(3) and C(4), because, differently from benzyl groups, it can be removed under oxidative conditions (DDQ), without reduce the olefinic carbon-carbon double bond.

Scheme 2.6 Synthesis of carba alcohol (-)-2.20.

This way alcohol (-)-2.16 was oxidized to aldehyde 2.17 through a very clean reaction by using the freshly prepared 2-iodoxy benzoic acid (IBX) as the oxidant. IBX is a mild and versatile oxidant, which is active against alcohols and vicinal diols, but maintain unchanged the double bond.

Scheme 2.7 Synthesis of IBX, precursor of the known DMP.

IBX is well known as oxidant agent also because is the precursor for the synthesis of Dess-Martin reagent (DMP), as shown in Scheme 2.7. Preparation of IBX is not simple, but recently, Frigerio47 et al. have reported an extremely effective procedure which provides oxidation of 2-iodobenzoic acid with Oxone® (KHSO5-KHSO4-K2SO4) for 3h at 70°C. This way, IBX is obtained in good yield (80%) and with high purity degree (>95%). The treatment of alcohol (-)-2.16 with dry IBX in anhydrous CH3CN at 45°C gave the desired aldehyde 2.17, pure as liquid and directly used in the next step without any purification (Scheme 2.6). In this transformation, particular attention had to be given to the reaction temperature. Actually, reaction temperatures above the indicated 45°C determined the formation of the desired aldehyde 2.17, but unfortunately accompanied by different amounts (until 50%, depending on the temperature) of p-methoxy benzaldehyde (2.17b) with a drastic reduction of the yield. The formation of p-methoxy benzaldehyde under these conditions derives from an oxidation of the benzilic carbon of the protective groups, which is particularly sensitive to attack from oxidant agent by the conjugative effect of p-methoxy group (Scheme 2.8).

Scheme 2.8: Oxidation obtained when temperature exceeded 40°C.

Aldheide 2.17 is quite instable (it could be conserved for 36 h at -78°C) and it was immediately subjected to Wittig C-1 extension (Scheme 2.6). In this case, the aldheide 2.17 was added to the phosphonium ylide obtained by deprotonation of Ph3PCH3I with KHMDS in THF, affording the glycal-derived 6-exocyclic olefin (+)-2.18 in a mixture with triphenilphosphine oxide (Ph3PO). Removal of this by product was efficiently carried out by a multi-step treatment, which included a first filtration on Celite® pad, washing the organic phase with aqueous solutions and a second filtration of the washed solution on silica gel-Fluorosil® pad with a 7:3 hexane:AcOEt mixture. Evaporation of the organic solution afforded olefin (+)-2.18 in good yield (76 %) without any residual of Ph3PO. Olefin (+)-2.18 was subjected to rearrangement to give the carbacyclic compound (-)-2.20. Thermal Claisen rearrangement is a [3,3] sigmatropic rearrangement in which a vinyl allyl ether, or a related structure, is transformed into a γ,δ-unsaturated carbonyl compound. (Figure 2.3).

Figure 2.3 [3,3] sigmatropic rearrangement.

In the present case, glycal-derived terminal olefin (+)-2.18 with an endocyclic and an exocyclic double bonds represents a peculiar vinyl allyl ether. Glycal vinyl allyl ether (+)-2.18, once subjected to the thermal rearrangement, leads to the formation of carba aldehyde 2.19 by the mechanism depicted in Scheme 2.9. In this process, the formyl carbon C(1) of aldehyde 2.19 derives from C(1) of

the endocyclic double bond of the glycal-derived olefin (+)-2.18. Since aldehyde 2.19 is instable, it is immediately reduced (with NaBH4) into the stable primary alcohol (-)-2.20, which can be purified by flash chromatography.

The rearrangement (Scheme 2.9) was carried out by warming a solution of olefin (+)-2.18 in 1,3-dichlorobenzene at 240°C in sealed vial for almost 1 h. In order to obtain the required high temperature and to keep it stable for all the time necessary for the transformation be completed, we made use of a silicone oil (AP 100, from Aldrich) perfectly suitable for the purpose, because stable and not flammable at the required temperature.

Scheme 2.9 Claisen thermal rearrangement of glycal 2.19.

This point, we had to choose an adequate protective group for the 6-hydroxyl functionality of (-)-2.20. This step turned out to be crucial, because the protective groups would be maintained during the epoxidation step, and had to be stable under deprotection conditions of 3,4-O-p-methoxybenzyl groups on C(3) and C(4). We decided to use benzyl ether which can be removed under reducing conditions.

Thus, the primary carbacyclic alcohol (-)-2.20 was orthogonally protected as a benzyl ether using a typical procedure which provides the treatment of the alcohol with NaH/BnBr in DMF, to afford fully protected unsaturated carbacycle (+)-2.21 (>99%), pure enough to be used in the next step without any purification (Scheme 2.10).

Scheme 2.10 Elaboration of carbacyclic alcohol (-)-2.20 into trans diol (+)-2.22.

The next step involved in the synthesis of key intermediate (+)-2.22 is the deprotection of p-methoxybenzyl ethers on C(3)-C(4) position,which must be carried out without affecting the O-benzyl protected primary alcoholic group on C(6) and the C(1)-C(2) double bond. Deprotection was carried out by using an electron transfer system (SET) with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) as the oxidant in a 18:1 CH2Cl2/H2O mixture, as shown in Scheme 2.11.

Scheme 2.11: DDQ-promoted deprotection of O-PMB ether functionality of compound (+)-2.22.

The deprotection mechanism involves the formation of an oxonium ion that is captured by water. Subsequent formation of a hemiacetal leads to the free alcohol ROH, in this case the trans diol

The trans carba diol (+)-2.22 had to be converted in the adequately protected trans amino-alcohol, which represents the precursor of the 3-N-Tosyl-α-aziridine (+)-2.31α (Scheme 2.12).

Scheme 2.12 Key intermediate for the synthesis of N-tosyl-α-aziridine derivative (+)-2.31α.

To obtain vinyl-β-epoxide (-)-2.25β, trans diol (+)-2.22 was treated, by following Kishi procedure,49 with the bulky PivCl which selectively protects the allyl secondary hydroxy group, affording pivaloyl derivative (-)-2.23. Pivaloate (-)-2.23 was mesylated to the fully protected 3-O-pivaloyl-4-O-mesyl derivative (-)-2.24. The subsequent saponification/cyclization of (-)-2.24 under basic conditions (MeOH/CH3CN), gives vinyl epoxide (-)-2.25β with 85% overall yield (3 steps starting from trans diol, Scheme 2.13).

Scheme 2.13 Synthesis of vinyl epoxide (-)-2.25.

Vinyl β-epoxide (-)-2.25β allows to introduce an amino group at C(3) position in a completely regio- and anti-stereoselective fashion (Scheme 2.14). Indeed, the ring-opening reaction of vinyl epoxide

(-)-2.25β carried out with NaN3 under neutral conditions in a 1:1 THF/H2O mixture, gives trans azido alcohol (+)-2.26 as the only reaction product. The azido alcohol (+)-2.26 was reduced with polimer-supported PPh3 (Aldrich)50 in a heterogeneous phase (20:1 THF/H2O) to the corresponding trans amino alcohol (+)-2.27, which was obtained in good yield (96%) and sufficiently pure to be directly used in the next step, without any further purification.

Scheme 2.14 Synthesis of trans amino-alcohol (+)-2.27.

In a first approach, as previously described for the synthesis of the racemic N-nosyl aziridine (±)-2.01, regioselective tosylation of the amino group by TsCl/Py protocol afforded N-tosyl derivative (+)-2.28. Subsequent mesylation with MsCl of the residual free hydroxyl functionality on C(4) position gave N-tosyl-O-mesylate 2.29, the ultimate precursor of aziridine (+)-2.31-Ts. Actually, the treatment of 2.29 with t-BuOK in a non-nucleophilic solvent (CH3CN) under basic conditions determined the deprotonation of the N-H bond of the N-tosyl group with subsequent intramolecular SN2-type displacement of the vicinal, appropriately anti-disposed, O-mesyl group with contemporary cyclization to the desired aziridine (+)-2.31-Ts (Scheme 2.15). This way, aziridine (+)-2.31-Ts

was obtained in 40% overall yield, through a 3 steps sequence, starting from amino alcohol (+)-2.27.

In a second moment, in view to realize a shorter synthetic sequence to obtain aziridine (+)-2.31-Ts,

amino alcohol (+)-2.27 was directly protected with TsCl (2.5 equiv) in pyridine to give trans-N,O-ditosylate (-)-2.30, which was efficiently cyclizated with t-BuOK in CH3CN to afford aziridine

Scheme 2.15 Two approach for the synthesis of N-Tosylaziridine (+)-2.31α.

This point in order to prepare the new carba glycosyl acceptor 2.10, bearing a N-tosyl substituted amino functionality introduced at C(4) in a completely region- and stereoselective fashion, we performed the acetolysis of aziridine (+)-2.31-Ts with AcONa in aqueous DMF (80%) to obtain

acetate 2.32 together with allylic alcohol 2.33. The crude of acetolysis was directly hydrolyzed (MeONa/MeOH) to afford alcohol 2.33 as the only reaction product (Scheme 2.16).

Scheme 2.16 Synthesis of 3-O-hydroxyl-4-N-tosylamino derivative 2.33.

Later on the basis of the results51previously obtained in our research group on the analogous N-nosyl compound (±)-2.03 (Scheme 2.1),we performed the epoxidation process by means of MCPBA/CH2Cl2

protocol with the idea to obtain a β-stereoselective procedure. However, much to our surprise, the

reaction of hydroxy-tosylate 2.33 was not stereoselective, affording a 40:60 mixture of the two diasteroisomeric epoxides, 2.34and 2.34respectively (Scheme 2.17).

Scheme 2.17 Butterfly mechanism of epoxidation for preparation of desired epoxide 2.34and the undesired 2.34.

We rationalize that in this case, two competitive and precedent unreported coordination processes are possible.

Indeed, typical coordination processes in form of hydrogen bind between the oriented allyl hydroxyl functionality on C(3) and the approaching peroxyacid is responsible of the formation of the epoxide 2.34pathway A).

On the other hand, in the presence of p-toluensulfonyl group on C(4) position, coordination between the sulfonyl oxygen atoms and the oxidizing agent takes place and allows the formation of epoxide

2.34pathway B).

This non stereoselective process was unexpected, especially if compared with the stereoselective results previously obtained in the epoxidation of the olephin (±)-2.03 with a N-nosyl group in C(4) position. It is clear that the electronic nature of the sulfonyl group has an effect on the stereoselectivity, with the electron donating methyl group present on Ts-group displaying higher possibility of coordination when compared to the electron withdrawing NO2 in the nosyl group. This

fact suggests that the reaction may proceed through a sulfonyl-oxygen directed pathway and the sulfonyl oxygen, instead of the amide nitrogen, plays a major role as the directing group interacting with the approaching peracid.

As reported in the literature,52 it is correct that the oxidizing agent is the protonated species of the peracid (Figure 2.4, structure B).

Figure 2.4 The ionization and protonation balance of peracid generated in reaction solution.

Figure 2.5 Transition structures involving coordination between allylic hydroxyl on C(3) and the protonated peracid a); Transition structure involving coordination between oxygen atoms of sulfonamide in C(4) and the

protonated peracid b).

Theoretical calculations carried out by Dr. Lucilla Favero from our laboratory (B3LYP/6-31+G(d)) considered this protonated form of the peracid (to simplify calculations, formic acid was used as a

model), and showed that the relative energies of the transition states involving coordination between allylic hydroxyl on C(3) and the protonated peracid (Figure 2.5, a)) and coordination between oxygen atoms of sulfonamide in C(4) and the protonated peracid (Figure 2.5, b)) are strictly related to the substituent on the aromatic ring of the sulfonamide (Table 3).

Group (σ) U‡ (kJ/mole) H‡ (0 K) (kJ/mole) G‡ (298) (kJ/mole) p-OH (-0.38) -8.74 -7.93 -1.56 p-Me (-0.17) +1.63 +0.68 +3.44 H (0) +4.19 +3.31 +7.05 p-NO2 (0.78) +13.39 +12.87 +18.95

Table 3 U: Potential Energy; H: Enthalpy; G: Free Energy, σ:Hammett constant.

Indeed, when on the aromatic ring it is present a strong electron withdrawing group, such as the nitro group in orto or para position, the coordination with the allylic hydroxyl prevails and affords preferentially the epoxide as experimentally observed.

On the other hand, when on the aromatic ring it is present a strong electrondonating group such as a hydroxyl group, the coordination with the homoallylic sulfonamide is preferred and affords preferentially the epoxide. Finally, when on the aromatic ring it is present a weak electron donating group such a methyl present in the tosyl group, the two transition states have similar energies and, in

R² = 0,9891 -5,0 0,0 5,0 10,0 15,0 20,0 25,0 -0,6 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0



G*

s

fact, epoxide andare obtained in similar amounts (40%  and 60% ), as experimentally observed.

Due to this lack of selectivity, we needed to proceed with the separation of epoxides 2.34and

2.34in order to synthesize pseudo glycosyl acceptor 2.10 (Scheme 2.19).

Subsequent acetylation (Ac2O/Py) of 2.34 yielded 3-O-acetyl -epoxide 2.35Scheme 2.19), with all the substituents, including a N-tosylamino group at C(4) position, in the required relative configuration, ready for the nucleophilic ring opening reaction (Scheme 2.18).

This point, epoxide 2.35β could be subjected to ring-opening reaction by the appropriate nucleophilic agent, to afford the desired glycosyl acceptor 2.10. As extensively reported by Ogawa,53 1,2-epoxides with β-manno configuration, such as 2.35β, effectively reacts with O- and N-nucleophiles by exclusive nucleophilic attack at the sterically and electronically favoured C(1) oxiran carbon. Indeed, C(1) is flanked by an electron-rich methylene group, whereas C(2) is flanked by a carbon bearing an electron-withdrawing group, so that, from an electronic point of view, SN2 reaction should be more favourable at C(1). Besides, the group flanking C(1) is smaller than those flanking C(2), so attack at C(1) may be favoured also from a steric point of view.54 Corresponding α-O- and α-N-carbamannopyranosides are in this way obtained through a trans-diaxial ring opening process (Scheme 2.18).

Scheme 2.18 Sterically and electronically preferred nucleophilic attack to epoxide with –manno configuration.

In this case, the nucleophilic agent required was 2-azido-1-ethanol, which could lead to the desired derivative 2.10 (Scheme 2.19). Anyway, in a first time we were acquainted with hazard associated with the preparation of low molecular weight azido compounds, because such type of compound are potentially explosive.

Scheme 2.19 Mechanism of the ring-opening reaction under Lewis acid catalytic conditions.

Hereinafter, we decided to use a particular protocol which had been suggested by Bernardi’s group. The method, safe and not hazardous, provided the preparation of a 1.3 M 2-azido-1-ethanol solution in CH2Cl2 simply based on the azidolysis of 2-chloro-1-ethanol by NaN3 in H2O at room temperature. In this way, leading the reaction in an inorganic solvent, we had avoided the use of dichloromethane as reaction solvent. Indeed, use of chlorinated solvents could results in the formation of explosively-unstable di- and tri- azidomethane. In this procedure, the reaction mixture was stirred at 70°C in H2O for three days and then eluted with dichloromethane, which extracts the desired compound from the inorganic phase; the obtained solution was conserved at 4°C under molecular sieves and without any distillation step.

The epoxide 2.35was, so, treated with the freshly prepared 2-azido-1-ethanol 1.3 M in CH2Cl2 in presence of a catalytic amount of Cu(OTf)2 as necessary Lewis acid catalyst.38 Thus, the desired C(1)-azido-ethoxy derivative of 4-(N-tosylamino)-D,L-carbamannose 2.10 is obtained through a completely regio- and anti-stereoselective trans-diaxial opening process (yield of 46%).

The crude product of the ring-opening reaction was purified by TLC preparative to give the azido derivative 2.10, pure as a white solid. In this way, the glycosyl acceptor 2.10 was ready for the next step of the protocol: the glycosylation with an appropriate mannosyl donor, the trichloroacetimidate (TCA) (-)-1.00 (Scheme 2.20).

2.2 Synthesis of the glycosyl donor

The trichloroacetimidate has been prepared in our laboratory according to the protocol largely used by Professor Bernardi’s group. The TCA derivative (-)-1.00 I used for my synthesis is a fully-O-benzoyl derivative, instead of the tetra-O-acetyl derivative used by Professor Bernardi.39 The reason is that the benzoyl protective groups allow the obtainment of a higher molecular weight product, stable and storable, in which is very easy to remove, under basic conditions, the protecting group. The derivative (-)-1.00 provide a completely α-stereoselective glycosylation, which is one of the most interesting aspect of this protocol.

The TCA derivative (-)-1.00was prepared in our laboratory starting from the commercially available (+)-D-mannose, which was treated with BzCl in pyridine, to yield the fully-O-benzoyl-mannopyranose. The penta-O-benzoyl derivative was subjected to monodeprotection using a solution of MeNH2/EtOH in THF, then treated with CCl3CN in CH2Cl2 to afford TCA (-)-1.00, pure as the only α-anomer (Scheme 2.20)55

, in which the anomeric center is active through glycosylation.

Scheme 2.20 Synthesis of glycosyl donor (-)-1.00.

The activated species (-)-1.00 are then used to release the glycosyl donor, with the help of a catalyst, to generate the glycosydic bond with the glycosyl acceptor, the azido derivative 2.10.

Glycosylation reaction of the glycosyl acceptor 2.10 by TCA (-)-1.00 was successfully carried out in dichloromethane at -20°C for 20 minutes, using molecular sieves AWMS to provide anhydrous conditions. In this settings the glycosyl acceptor, the azido derivative 2.10, reacted with 1.3 equivalents of glycosyl donor (-)-1.00, in presence of 0.3 equivalents of TMSOTf in anhydrous CH2Cl2 as the catalyst. This way the glycoconjugate 2.36 was formed in a completely α-stereoselective fashion and in 85% yield after preparative TLC (Scheme 2.21).

Scheme 2.21 Mannosylation of glycosyl acceptor 2.10 by TCA (-)-1.00.

This reaction provides necessarily for anhydrous conditions: we performed the reaction in flame-dried modified Schlenk, under a positive pressure of Argon, adding molecular sieves to avoid the presence of residual water which could interfere with the reaction, hydrolysing the α-trichloroacetimidate. The driving force of the process is the release of trichloroacetamide (CCl3CONH2) as leaving group. Due to the low basicity of this leaving group, the Lewis acid TMSOTf, essential for the activation of TCA

(-)-1.00, can be added in catalytic amount. Indeed, the catalyst is immediately released after the

reaction and is available for the activation of other molecules of glycosyl donor. The possibility to use a quite mild acid catalysis represents another significant character of this protocol.

As above mentioned, the key aspect of this glycosylation process is the possibility to exercise a stereochemical control on the anomeric center in the glycosylation step, in order to obtain the α-D -mannopyranoside as the only reaction product and in good yield. In this way, the stereochemistry of the reaction is influenced by two factors: the Anomeric effect and the Anchimeric assistance (or Neighbouring group participation). These factors are closely related to the structure of the glycosyl

donor: in particular the anomeric effect is due to the presence of an heteroatom (bearing two lone pairs) in α to the C(1), which orients its electronegative group in axial position. Indeed, the axial configuration is characterized by a partial alignment of dipoles involving heteroatoms, which repel each other. Moreover, there is a hyperconjugation effect between the orbital containing unshared electron pair on the endocyclic heteroatom and the σ*

orbital of the axial C-X bond. This causes the molecule aligns the donating lone pair of electrons antiperiplanar to the σ* orbital lowering the overall energy of the systemand causing more stability (Figure 2.6).

Figure 2.6 Anomeric effect: the conformer in which the substituent in C(1) is axial is favourite because of the opposite orientation of the two dipols and the hyperconjugation effect.

The anchimeric assistance is closely related to the presence of a substituent in C(2). The α-TCA

(-)-1.00 presents a benzoyl group in C(2) position, which is a powerful neighbouring group and is

directly involved in the glycosylation acting anchimeric assistance on the C(1) (Scheme 2.22).

Then, the axial orientation of the benzoyl group has crucial importance in leading the reaction through the obtainment of the α-anomer as the only reaction product, inducing the formation of the intermediate (-)-1.00b, which can be attacked only from the opposite face by way of a trans diaxial opening.

Scheme 2.22 Effect of anchimeric assistence on stereoselectivity of glycosylation step.

Moreover, the formation of a glycosydic bond in axial position, through an SN1 type mechanism is promoted by the presence of a quite polar CH2Cl2 and the quite strong Lewis acid TMSOTf. In this conditions, after the release of trichloroacetamide, the α-trichloroacetimidate (-)-1.00 is turned into a carbenio ion 1.00c (Scheme 2.23).

Scheme 2.23 Glycosylation by SN1 mechanism.

Such intermediate is preferentially attacked from the α-axial face, due to stereoelectronical factors. Then, from a kinetic point of view we can observe the preferential formation of α- product 2.36. However, due to the anomeric effect, the product 2.36 is also the most thermodynamically stable, so the balance is totally moved to its formation. The solvent also enhances the stereoselectivity of the glycosylation. Thus, in this case we used a quite polar solvent such as dichloromethane, to provide the complete solubility of compounds and enhance the α-stereoselectivity.

The fully-O-protected disaccharide 2.36 had to be completely deprotected, in order to obtain the desired pseudomannobioside 2.09. In this case, the two portions of the disaccharide were protected with orthogonal groups, which could be removed under different conditions. The mannosidic portion and the C(3) position of carba moiety present all the hydroxyl groups protected respectively as benzoyl derivative and acetyl group, which are sensitive to basic conditions. The benzyl group on C(6) carba portion is, instead, stable to saponification and could be removed under reducing conditions.

Scheme 2.24 Possible sequence of deprotection for pseudodisaccharide 2.09.

Thus, we had to choose the sequence of deprotection in order to obtain the product with satisfactory yield and high purity degree (Scheme 2.24). Due to the complex synthetic pathway, we disposed of a quite poor amount of pseudodisaccharide 2.36, so the necessity to choose the sequence which provided more satisfactory results became essential.

Debenzylation of benzyl ether by catalytic hydrogenation was a crucial step because lead to formation of a primary amino group, which turned out to be difficult to purify, due to its basic features.

However, the product of reduction seemed to be stable under saponification conditions so we decided to follow the deprotection sequence shown in scheme 2.24. The glycosylation product 2.36 was dissolved in a 4:1 MeOH/DMF mixture: in this case MeOH was the solvent of the reaction and DMF was added in order to get better into solution the substrate. The catalyst Pd-C 10% was added and the reaction mixture was submitted to hydrogen satured atmosphere and was stirred at room temperature overnight, until the disappearance of the starting material. Dilution with MeOH and filtration through a Celite® pad afforded a crude reaction product, which was crystallized from diisopropyl ether to afford a pale yellow solid (70% yield) constituted by the partially-O-deprotected pseudomannobioside

2.37.

Unfortunately the very small amount of product (only 7 mg) doesn’t make possible to realize at the moment the last step of the synthesis, consisting in the saponification of the benzoyl protective groups to obtain the final product 2.09. In recent times of my thesis, I repeat again all the synthetic sequence, and at present only few steps are necessary to finish our project and to obtain the final pseudomannobioside 2.09 fully deprotected with the required sulfonyl amino portion on C(4) position and an α-ethoxy amino chain on the pseudoanomeric C(1) carbon.

In conclusion, we found the right pathway towards the synthesis of the new attractive pseudodisaccharide 2.09 as possible DC-SIGN antagonist with the main features represented by the presence of a mannose unit able to coordinate calcium ion by the equatorial hydroxyl groups on C(3) and C(4) position and a real mimic of D-mannose with tosyl sulfonamide on C(4) position able to establish favourable lipophilic interactions between the aromatic ring and the additional binding site of DC-SIGN.

The new pseudodisaccharide 2.09 will be send to Professor Frank Fieschi at Institute de Biologie Structurale in Grenoble in order to evaluate the affinity with DC-SIGN and the activity.