WO2003040163A2 - Immunomodulating saponins, a method for their preparation and their use for vaccines - Google Patents

Abstract

The present invention relates to new saponins for increasing the immune response in mammals and finds application in the field of immunology, supplying in particular new adjuvants, obtained by hemi-synthesis, starting from terpene sources. It also embraces the methods for the use of the new saponins as adjuvants to increase the immune response to an antigen in a mammal. The saponins of the present invention are suitable for veterinary and human pharmaceutical compositions which include one or more antigens, in particular those of synthetic origin, and one or more diluents, in pharmaceutically acceptable vehicles. These compositions can be used as immunopotenciators and/or immunomodulators in animals and human beings. The present invention further constitutes a vaccination method that includes the administration of one or more antigens and one or optionally more than one hemi-synthetic saponin of the invention.

Description

DESCRIPTION

"IMMUNOMODULATING AGENTS, A METHOD FOR THEIR PREPARATION AND THEIR USE FOR VACCINES"

FIELD OF THE INVENTION

This invention relates to adjuvants and more particularly to immunostimulating adjuvants.

The present invention relates to new saponins for increasing the immune response in mammals. The present invention finds application in the field of immunology, supplying in particular new adjuvants, obtained by hemi- synthesis, starting from terpene sources, which are commercially available and/or isolated pure and transformed by derivatization with appropriate chains of sugars to produce new saponins which modulate or stimulate the immunological response to an antigen. This invention also embraces methods for the use of the new saponins as adjuvants to increase the immune response to an antigen in a mammal.

The saponins of the present invention are suitable for veterinary and human pharmaceutical compositions which include one or more antigens, in particular those of synthetic origin, and one or more diluents, in pharmaceutically acceptable vehicles. These compositions can be used as immunopotenciators and/or immunomodulators in animals and human beings .

The present invention further constitutes a vaccination method that includes the administration of one or more antigens and one or optionally more than one hemi-synthetic saponin of the invention. Additionally, this invention also provides new non toxic saponins for use as oral adjuvants, in mammals, including human beings.

BACKGROUND OF THE INVENTION

A great variety of antigens stimulates the production of antibodies in animals and grant protection against subsequent infection. However, some antigens are unable to stimulate an effective immune response. The immunogenicity of those relatively weak antigens is frequently increased with the simultaneous administration of an adjuvant with the antigen. Adjuvants are substances that are not immunogenic when administered alone but they induce a state of systemic immunity or in the mucous when combined with the antigen.

Vaccination represents the only prophylactic way commonly used for the control and prevention of many infectious diseases or for their treatment. The vaccines exploit the natural mechanism of defence, the immune system, inducing resistance in the organism to the pathogenic agents responsible for the infectious diseases.

For the success of any vaccine it is indispensable to have knowledge of the nature of the pathogenic agent and the immunological mechanism that will generate the appropriate response for defence of the organism. In general, the humoral immune response acts against extracellular pathogenic microorganisms and toxins, and the control of the infections provoked by the intracellular pathogenic agents is performed by the cell immune response. Although the prevention of primary infections can be probably mediated by the humoral immune response (Seder and Hill, Nature, 2000, 406: 793-8) , the ideal immune response should generate antibodies and cytotoxic T-cells directed to the infectious agent.

Traditionally, the responses of the type Thl are interpreted as being associated with the production of cytokines INF-γ for the T-lymphocytes . Other cytokines such as IL-12 are not produced by T-cells and they can be directly associated with the responses of the type Th-1. On the contrary, the responses of the type Th2 are associated with the secretion of IL-4, IL-5, IL-6, IL-10 and with the tumour necrosis factor TNF-α (see, for example, Mosmann, T.R. and Coffman, R. L. (1989) "Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties, Annual Review of Immunology, 7, pl45-173) .

The advances in genetic engineering allow for the economic generation of pure antigens, but their application is limited by their weak immunogenic capacity, so that the co-administration of an immunological adjuvant is necessary to achieve the desired immune response. The vaccines prepared with this type of antigenic molecules present a series of advantages over conventional vaccines, principally that they are safer clinical products, as they reduce the chances of contamination of the final preparation by ^' undetected pathogens .

An immunological adjuvant is a substance or a group of substances that when administered together with an antigen, generates a immune response higher than the immune response generated by the antigen administered alone. The mechanism of action of the latest known adjuvants is not totally clear, which makes their classification difficult. The adjuvants for vaccines can act by several mechanisms: 1) formation of deposits in the administration site allowing the liberation of the antigen with time, increasing this way exposure of the immune system to the antigen; 2) increase of the antigen presentation; 3) induction of the secretion of i munomodulating substances, namely cytokines (Biopharmaceuticals : Biochemistry and Biotechnology, Gary Walsh, John Wiley & Sounds, Chichester, 1998) .

Immunological adjuvants are available such as Freund's complete (FCA) and incomplete (IFA) adjuvant, bacterial endotoxins and derivatives thereof, mineral salts, adjuvant Formulation Syntex (FANS) , RAS, saponins of Quillaja saponaria Molina. However, only the salts of aluminium, hydroxide gel or phosphate gel, have been accepted by FDA for use in human beings (Allison, 1999, Methods, 19: 87-93), and in the year of 1997 the MF59 adjuvant was licensed in Italy, an oil-in-water emulsion, wherein the oleous phase is constituted by squalene which is included in the Formulation of a vaccine for influenza (Flual^®, Quiron Pharmaceuticals) . The usefulness of the aluminium salts is limited to the type of immunological response that it can generate, it only increases the humoral immune response (Th2, T helper type 2) against bacteria or other antigens, it is not effective in the presence of antigens such as, for example, the conjugated tetanus toxin. Besides, the antigens adsorbed to the aluminium salts frequently promote a higher production of antibodies of the isotype E (Ig E) than in the absence of the aforementioned salts, being able to produce hypersensitivity to the administered vaccine antigens. Although the aluminium salts are acceptable for their application in human beings, by having no accentuated secondary effects, they present the limitation of not being able to induce a cell mediated immunological reaction.

Another group of adjuvants comprises the immunomodulators/immunopotenciators, substances that not only increase the immunological response but also modify the type of immune response, e.g. they change the isotype profile of the antibodies. Here derivatives from cell walls are included such as the muramildipeptides (MDP) and muramiltripeptides

(MTP) and also lipopolysaccharides (LPS) and their derivatives, e.g. lipid A. Also included in this group are the products derived from plants such as the saponins (of

Quillaja saponaria) and basydiolipids (Basidiomycetes) . These compounds are relatively toxic, being able, for example, to produce, in function of the considered molecule: cytolysis

(saponins) ; pyrogenicity, arthritis and anterior uveitis

(LPS, MDP and MTP) .

In veterinary medicine, compounds considered very useful as vaccine vectors are the immunostimulant lipophilic complexes (ISCOMS) containing small particles consisting of phospholipids, cholesterol and the saponins extracted from the bark of Quillaja saponaria Molina, Quil A, (see, e.g.: Morein, B., Fossum, C, Lovgren, K. , and Hoglund, S., The ISCOM-THE modern approach to vaccines, Semin Virol . , 1:49, 1990; and several other studies on ISCOMS later documenting a great variety of local and systemic immune responses after oral administration with an antigen (e.g., Mowat, A. Mel, Maloy, K. J. , Smith, R. E., Donachie, A. M. , ISCOMS the mucosal vaccine vectors, Vaccine Design: The Role of Cytokine Networks, Ed. Gregoriadis et al . , Plenum Press, New York, 1997) .

The mechanism of action of the Quillaja saponaria Molina saponins as adjuvants and immunostimulants is new and still not very well-understood and its usefulness and commercial impact for human vaccination seem to be limited to a pure fraction of known molecular structure designated as QS21 (see for example: Cleland, J: L. , Kensil, C. R., Lim, A., Jacobsen, N. E., Basa, L., Spellman, M. , Wheeler, D. A., Wu, J. Y., Powell, M. F., Isomerization and Formulation stability of the vaccine adjuvant QS-21, J. Pharm. Sci . , 85:1, 22-8, 1996) . In the last decade growing interest occurred for the use of the Quillaja saponaria Molina saponins. More than a dozen Patents were published referring compositions of vaccines comprising Quillaja saponins. Examples are Patent application WO 93/05789 describing conjugates wherein low immunogenicity proteins are covalently linked to a fraction of purified Quillaja saponaria Molina saponin, through the carboxylic acid group of the 3-O-glucuronic acid, inducing more intense immune responses. In another patent application, e.g., WO 0117551, 15 March 2001, of SMITHKLINE BEECHAM BIOLOG and WETTENDORFF MARTINE ANNE CECECIL (BE), and also No. PCT/EP94/04246 Sec. 371, July 2, 1996, PCT Pub. No. WO95/17210 PCT Pub. June 29, 1995, QS21 is advantageously mixed with other adjuvants, compositions of vaccines based in oil-in-water emulsions optionally constituted by monophosphoryl-lipid A 3-desoxy-O-acilated and QS-21, being described, as potent inductors of a great variety of immune responses. The toxicity of the saponins as adjuvants depends on the amount administered at the concentrations which are biologically active. An important fact described in the US Patent 5,750,110 (May 12, 1998) is that QS-21 and MPL together exhibit adjuvant activity at concentrations where they are not individually active.

Presently, the need of a non toxic adjuvant that stimulates either the humoral immunity or the cellular immunity is evident, and both two types of immunity preferably are needed. Agents such as the compounds of the present invention, that are terpenoid glycosides, constitute an effective and accessible system of antigens presentation potentially similar to Quillaja saponins inducing advantageous immune responses. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides saponins of the general Formula I

and the pharmaceutically acceptable salts thereof, wherein

Ri is a group of the Formula:

R₂ is CH₂OH or COOH group;

R₃ is hydrogen or methyl;

R₄ is selected from the group consisting of a (C₄-C₀) straight or branched chain alkyl group, and a (C₄-C₂o) straight or branched chain alkenyl group; either of which is optionally substituted by one or more of hydroxy, (Cι-C₆) alkoxy or carboxy group;

R₅ is methyl, CH₂0H, CHO or COOH; and,

R₆ is hydrogen or hydroxyl;

R₇ is CH₃, COOH or COOR₈,-and,

R₈ is hydrogen or a (C₄-C₃₀) straight or branched chain alkyl group, or a (C-C₂o) straight or branched chain alkenyl group, either of which is optionally substituted by one to six hydroxy or carboxy groups.

One preferred group of compounds is represented by

Formula II

II

and the pharmaceutically acceptable salts thereof; wherein

Ri is a group of the Formula:

The invention also refers to a pharmaceutical composition wherein the compounds of the present invention are used, for their immunostimulating properties, as adjuvants of vaccines, and wherein the said pharmaceutical compositions are aimed to the prophylaxis and treatment of immunitary weakness situations, including the improvement of the immune response in the elderly and in the newborn, in a mammal, that comprises the administration to said mammal of a therapeutically effective amount of the compound of Formula I, or of a pharmaceutically acceptable salt, prodrug or resulting hydrate.

In the above definitions, the term "alkyl", such as here used, unless otherwise stated, means saturated monovalent hydrocarbon radicals containing straight, cyclic or branched moieties. Said "alkyl" groups can include a carbon-carbon single, double or triple bond, where said alkyl group comprises at least two carbon atoms. It is understood that for cyclic moieties at least three carbon atoms in said alkyl group are requested.

The term "alkenyl", such as here used, unless otherwise stated, means unsaturated hydrocarbon straight or branched chain.

The term "halogen", such as here used, unless otherwise stated, means fluoro, chloro, bromo or iodo. Preferred halogen groups are fluoro, chloro and bromo.

The term "oxy" , such as here used, unless otherwise stated, means O-alkyl groups wherein "alkyl" is as above defined.

The term "cycloalkyl" , such as here used, unless otherwise stated, means a all-carbon monocyclic ring. Examples, without limitation, of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The sentence "pharmaceutically acceptable salts" , such as here used, unless otherwise stated, includes salts of acid or basic groups that can be present in compounds of Formula I . The compounds of Formula I that can be basic in its nature are capable to form an extensive variety of salts with several inorganic and organic acids . The acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of Formula I are those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as hydrochloride, hydrobromide, phosphoaluminate, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, hydroxyaluminate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantotenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrate, formate, benzoate, glutamate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate, palmitoate, polyethyleneglycolate, and pamoate [i.e., 1, l'-methylene-bis- (2-hydroxy-3-naphtoate) ] salts, or other alkyl chains of pharmaceutically acceptable adequate lipophilic chain. The compounds of Formula I that have acid nature, are capable to form base salts with several pharmacologically acceptable inorganic or organic cations. Examples of such salts include the alkaline metals and metals alkaline-earth salts and particularly, the salts of aluminium.

The compounds of the present invention have asymmetric centres and so they exist in different enantiomeric and/or diastereoisomeric forms. This invention refers to the use of all pharmaceutically acceptable ' optical isomers and stereoisomers of the compounds of the present invention, and resulting mixtures, and to all pharmaceutical compositions and treatment methods that can use them or contain them.

The invention also refers to a method of preparation of the saponins of the invention, as described in claim 7.

A non limitative example of process for the preparation of the compounds of the present invention is illustrated in Scheme 1. The starting intermediate compound V of the Example illustrated in Scheme 1, and detailed in the Experimental Part, can be prepared by one or more procedures described and published in the literature for the construction of saccharidic chains and linking these to hydrocarbon chains, including triterpenes, being these able on its turn, to be obtained and derivatized from natural sources and, in particular, from the cork of Quercus suber L. (vide, e.g., Moiteiro, C, Justino, F., Tavares, R. , Marcelo-Curto, M. J. , Florencio, M.H., Nascimento, M.S.J., Pedro, M. , Cerqueira, F., and Pinto, M.M. M., J^". Nat . Prod. , 64, 1273-1277, 2001). Glu III Xyl IV

Scheme I In the 1st Step of Scheme 1, the starting compounds of Formula V can be obtained starting from glucose of Formula III and from xylose of Formula IV for treatment with appropriate protecting agents (vide T. W. Greene and P.G.M. Wuts, "Protective groups in Organic Synthesis", Third Edition, John Wiley & Sounds, New York, 1999) to obtain, e.g., a donor for glycosylation such as the derivative xylopyranosyl chloride or bromide protected with benzoyl groups, e.g., compound XIV and a derivative of glucose as the acceptor such as, for example, ethyl 4, 6-0-benzylidene-2- O-levulinoyl-1-thio-β-D-glucopyranoside XV. Starting from these intermediates it is possible to assemble the disaccharide V as starting material to further produce the other glycosides of this invention, saponin XIII in Scheme 1 is an example, using the shown or other known coupling methods. (for details see, e.g., Danishefsky, S. J. , Bilodeau, M. T., Angew. Chem. , Int . Ed. Engl . , 35, 1380, 1996,; Zhang, Z., Ollmann, I. R. , Ye, X-S., Wischnat, R. , Baasov, T. , Wong, C-H. , J. Am. Chem. Soc , 121, 734-753, 1999) . Thus, one of the possible methods for the synthesis of disaccharide V is, for example, using the activation system silver triflate/2 , 6-lutidine, at reduced temperatures to couple the aforementioned xylopyranosyl bromide XIV with the aforementioned thio-glycoside XV.

The synthesis of the saponin XIII is presented in Example 1 (Scheme 1) , wherein the coupling of a triterpene VI to obtain the compound VII is possible, with yields ~ 60%, using the same silver triflate /2 , 6-lutidine system, as above described for the synthesis of the intermediate disaccharide V. For the objectives of the present invention, the synthetic procedure requires that, before the 1st Step of Example 1 (Scheme 1) , an adequate protecting group is introduced in position 2 of the glucose ring, whose selection should keep in mind the advantages of introducing a group sufficiently rich in electrons to promote the participation in the next coupling reaction without undesirable rearrangement, and that can be selectively removed. The selective removal of the protecting group in position 2 in the 2nd Step, in the case of this being, for example, a levulinic ester, can be made with good yield by selectively hydrolysing with hydrazine hydrate 0.5M in pyridine and acetic acid buffer at 0°C.

In the 3rd Step, the subsequent galactosylation of VIII, for example with the galactosyl bromide donor IX, using the coupling conditions identical to those described for the 1st Step, leads to the protected terpene trisaccharide X in 81% yield. The hydrolysis of a previously introduced protecting group R"3 and R 4, for example, benzylidene, in compound X, in the presence of trifluoracetic acid, in the 4th Step, proceeds in 74% yield leaving free the primary alcohol of the glucose ring (compound XI) for selective oxidation to glucuronic acid, in the 5th Step, for example in the presence of TEMPO (see, for example: Garegg, P. J. , Oscarsson, S., Tedebark, U. , J^". Carbohydr. Chem. , 17, 587, 1998) affording XII, which is then deprotected in the 6th Step, providing the desired saponin XIII in 73% yield over the two steps.

Other examples are given following alternative procedures (e.g., Schmidt, R.R.; Kinzy, W., Adv. Carbohydr. Chem. Biochem. , 50, 21, 1994). Namely, in the synthesis of glycyrrhetinic acid and friedelinolyl trisaccharides (Example 2) the synthetic route is achieved by first attaching the glucoside residue to the triterpene and then extending the sugar chain sequentially. The alternative glycosylation procedures are known to those skilled in glycoside assembling strategies (e.g., K. Toshima, K. Tatsuta, Chem. Rev. , 93, 1503, 1993; Garegg, P. J. , J^". Adv. Carbohydr. Chem. Biochem. , 52, 179, 1997; Zi-Hua Jiang, Dissertation, 'Studies in glycolipid synthesis: total synthesis and configurational assignment of plant growth regulator calonyctin A; a resin glycoside and synthetic studies on sulfated steroidal saponins of starfishes', Hartung-Gorre, onstanz, 1995; PhD Thesis of Jδrg Schimmel, Konstanz, 2001) where glycosylations are carried out on acetylated protected precursors activated through the use of appropriate protecting group patterns on the glucoside residue, which are chosen depending on the triterpene aglycone to be added. Removal of 4, 6-protecting groups followed by oxidation to the glucuronic acid and concomitant acetyl cleavage afforded the friedelanyl skeleton aglycone saponins, e.g., compound XXXIV (Scheme II), glycyrrhetinic acid methyl ester aglycone saponins, e.g., compound LIV (Scheme III) and cholestanol-type skeleton aglycone saponins, e.g., compound LXV (Scheme IV).

Scheme II (to be continued)

Scheme II (continuation)

XXXVII

p-TsOH (q.q.)

XXXVIII

Scheme III (to be continued)

Scheme III (continuation)

Scheme IV (to be continued)

Scheme IV (continuation)

The compounds of the present invention may have asymmetric carbon atoms . Such diastereoisomeric mixtures can be separated in their individual diastereoisomers based in their physical-chemistry properties by methods well-known to the skilled in the art, for example, by chromatography or fractionnated crystallization. Enantiomers can be separated by conversion of the enantiomeric mixtures in a diastereoisomeric mixture by reaction with an active appropriate compound (e.g., alcohol), separating the diastereoisomers and converting (e.g., by hydrolysing) the individual diastereoisomers in the corresponding pure enantiomers. All such isomers, including the diastereoisomeric mixtures and pure enantiomers are considered as part of the invention.

The compounds of general Formula I, that are acids by nature, are capable to form a great variety of different salts with several inorganic and organic bases. Although such salts should be pharmaceutically acceptable for administration to animals, it can be desirable in the practice to initially isolate the compound of Formula I of the reaction mixture as a pharmaceutically unacceptable salt and next to simply convert it in the free acid compound or in a salt of pharmaceutically acceptable base addition salt by treatment with an alkaline reactant . The base addition salts of the base compounds of this invention are readily prepared substantially by treatment of the acid compound with an equivalent amount of the chosen mineral or organic base, in an aqueous solvent medium or in an appropriate organic solvent, such as dimethylsulphoxide. After careful evaporation of the solvent, the wanted solid salt is readily obtained. The wanted salt can also be precipitated from a solution of the free acid in an adequate solvent by addition to the solution of an appropriate mineral or organic base and next evaporating the resulting solution to dryness, preferably at reduced pressure. In any case, stoichiometric amounts of reactants are preferably used in order to assure that the reaction is complete and that the maximum yields of the final wanted product are obtained.

For a better understanding of the invention, a few among the possible experimental examples are presented next, without limitative character, with the aid of enclosed figures representing the following data:

Figure 1. Secretion of IL-6 by the macrophages of the cell line J744A.1 after incubation with the saponin molecules T3T and T4T.

Figure 2. Secretion of IL-6 by the peritoneal macrophages after incubation with the saponin molecules T3T and T4T. Figure 3. Cytotoxicity of the saponin molecules T3T and T4T in peritoneal macrophages.

Figure 4. Cytotoxicity of the saponin molecules T3T and T4T and the purified saponin of Quillaja Saponaria QS-21 in the cells of the line J744A.1.

Figure 5. Cytotoxicity of the purified fractions of Quillaja saponaria (QH-A, QH-B, QH-C and Spikoside) in macrophages of the cell line J744A.1.

Figure 6. Effect of several Formulations where some of them included T3T and T4T saponins of general Formula I in the title of the sera of the immunized animals. The serum was collected up 2 weeks after the last immunisation and an equivalent volume of each collected serum of each group was used. The values correspond to the highest average value (title) of 3 replicas for each Formulation. The values are expressed as the Log₁₀ of the title of 4 animals in each group, this test corresponds to test 1. Figure 7. Comparison between the average values of the titles of the test 1 and test 2. The values are expressed as the LoglO of the title of 4 animals sera mixture in each group. Figure 8. Averages of the two above referred (Fig.6 and 7) in vivo tests. The values are expressed as the Logχ₀ of the title of the sera mixture of 4 animals in each group.

EXPERIMENTAL PART

I .General Procedures

All reactions were performed under inert atmosphere at room temperature, except when mentioned. The solvents used in the reactions were previously dried, by distillation on sodium-benzophenone (diethyl ether and tetra- tetrahydofuran (THF) ) or calcium hydride (dichloromethane (DCM) , toluene, dimethylformamide, methanol and acetonitrile) . The other reactants and solvents were used just as supplied or purified by reference procedures, if necessary. Unless otherwise specified, the aqueous solutions are saturated solutions.

The thin layer chromatography analysis (TLC) was performed using silica-gel glass plates (Merck Kieselgel 60 F₂₅) , and visualized under UV radiation or using ammonium molybdate (IV) in acid medium, as chemical developers. In the purification step by column chromatography (flash) Merck Kieselgel silica-gel was used. The petroleum ether used corresponds to the fraction with boiling points 40-60°C.

The ^Η- R and ¹³C-NMR spectra were registered using CDC1₃ as solvent (unless otherwise indicated) , in a Brucker AM-400 or Brucker DRX-600 spectrometer. The signal of residual proton of the CHC1₃ (δH = 7.26 ppm) and the carbon resonance of CDC1₃ (δC = 77.0 ppm), respectively, were normally used as internal references. The results of ¹H-NMR are described in the following way: chemical shift δ(ppm), (proton number, multiplicity, coupling constant J (Hz) , attribution) . The attribution of the signals in these spectra were also based on the analysis of the 2D-NMR spectra of COSY, TOCSY, HMQC (Heteronuclear Mul tiple Quantum Correlation) and HMBC (Heteronuclear Mul tiple Bond Correlation) . In the carbon assignment of the first synthetic approach (C*) refers to a quaternary carbon, (CH) to a carbon with one or three protons attached and (CH₃) to a carbon with three protons attached. In the proton assignment of the first synthetic approach A denotes a proton from the xylose residue, B denotes a proton from the gluc-residue, and C denotes a proton from the galactosyl residue .

The Infra-red spectra were obtained in Perkin Elmer FTIR 1620 instrument, with a film between disks of sodium chloride, or in chloroform solution.

The mass spectra were recorded in an Kratos Q-Tof or Quattro LC, or Bruker Esquire-LC spectrometer using either the ionization technique by Electrospray (ESI) or the technique of Chemical Ionization at Atmospheric Pressure (APCI) .

EXAMPLE 1- β-AMYRIN SAPONIN XIII (T4T)

1.1. Synthesis of the disaccharide V

Ethyl 4, 6-O-benzylidene-3-O-triethylsilyl-1-thio-β-D glucopyranoside XV

CH-CN (81%)

III Hid XV

Ethyl 4 , 6 -O-benzylidene-l -thio- β-D-glucopyranoside Hid (Verduyn, R . , Douwes , M . , Klein, P . A. M . van der, Moesinger, E.M., Marel, G.A.van der, Boom, J. H. van, Tetrahedron, 49 (33), 7301-7316, 1993) (8.9 g, 28.5 mmol) was mixed with imidazole (2.3 g, 34.2 mmol) and dissolved in THF (90 mL) . Chlorotriethylsilane (5.3 mL, 31.3 mmol) was added dropwise and the mixture was stirred for 40 minutes at room temperature. The mixture was then diluted with DCM and washed with water. The organic layer was dried with MgS0 , filtered and concentrated. The product ethyl 4, 6-0-benzylidene-3-0- triethylsilyl-1-thio-β-D-glucopyranoside XV (Rf= 0.3, diethyl ether/petroleum ether, 1:3), was isolated by flash chromatography (diethyl ether/petroleum ether, gradient 1:9- 1:4) as a colourless oil (7.2 g, 59%) as well as 3.5 g of mixed fractions containing the other regiosomer. υ_max (film) /cm^"1 3499 (OH), 2955, 2875, 1455 and 1384; δ_H (600 MHz) 7.47 (2H, d, J=6.0 Hz, Ph) , 7.37-7.35 (3H, m, Ph) , 5.50 (1H, s, HCPh) , 4.46 (1H, d, J=10.2, H-l) , 4.32 (1H, dd, J=10.8, 4.2 Hz, H-6) , 3.80-3.70 (2H, m, H-3 and H-6) , 3.51-3.43 (3H, m, H-2, H-4 and H-5) , 2.79-2.72 (2H, m, CH₂S) , 2.45 (1H, s, HO), 1.32 (3H, t, J 7.8 Hz, CH₃CH₂S) , 0.91 (9H, t, J=7.8 Hz, CN₃CH₂Si) , 0.61 (6H, q, J=7.8 Hz, CH₂Si) ; δ_c(150 MHz) [137.2, 129.0, 128.1, 126.2 (Ph) ] , 101.8 (CPh) , 86.5 (C-l) , 81.1 (C- 4), 75.8 (C-3), 74.1 (C-2) , 70.9 (C-5) , 68.6 (C-6) , 24.6 (CH₂S) , 15.2 (CH₃CH₂S) , 6.7 (CH₃CH₂Si) , 5.0 (CH₂Si) ; m/z Found: (MNa)⁺, 449.1182. Ca-H^OsNaSiS requires M, 449.1794.

Ethyl 4, 6-0-benzylidene-2-Q-levulinoyl-l-thio-β-D- glucopyranoside XVI

Et = Ethyl

Ph = Phenyl

Lev = Levulinoyl XVI Ethyl 4, 6-0-benzylidene-3-0-triethylsilyl-l-thio-β-D- glucopyranoside XV (1.0 g, 2.3 mmol) was dissolved in DCM (10 mL) , combined with DMAP (0.4 g, 3.5 mmol), levulinic anhydride was added (0 . 6 mL, 3.0 mmol) and the resulting solution was stirred for 15 minutes at room temperature. Next the mixture was diluted with DCM and washed with water, dried with MgS0 , filtered and concentrated. The residue was filtered through pad of silica-gel, using ether as eluent (Rf = 0.4, ether/toluene, 1:9). The crude residue was dissolved in THF (3 ml) and a stock solution of HF.pyridine in pyridine/THF (7 mL; 15.2 mL of HF.pyridine (Aldrich), 56 mL pyridine, 160 mL THF) was added. The reaction was stirred for 1 h after which time the mixture was diluted with DCM, washed twice with HCl 0.3 M and once with NaHC0₃, dried with MgS0₄ and concentrated to dryness. The product ethyl 4,6-0- benzylidene-2-O-levulinoyl-l-thio-β-D-glucopyranoside XVI (Rf=0.05, diethyl ether/petroleum ether, 1:1), was obtained with quantitative yield (1.09 g) without need of further purification.v_max (film) /cm^"1 3389 (OH), 2932, 2867, [1738, 1703 (C=0)], 1372 and 1156; δ_H(600 MHz) 7.51-7.32 (5H, m, Ph) , 5.55 (1H, s, HCPh), 4.97 (1H, t, J=9.5 Hz, H-2) , 4.51 (1H, d, J=9.5 Hz, H-l) , 4.36 (1H, dd, J=10.4 Hz, 5.0, H-6), 3.95 (1H, t, J=9.5 Hz, H-3), 3.77 (1H, t, J=10.4 Hz, H-6), 3.60 (1H, t, J 9.5 Hz, H-4) , 3.53-3.46 (1H, m, H-5) , 3.09 (1H, s, OH), 2.90-2.43 (6H, m, CH₂-lev and CH₂S) , 2.18 (3H, S, CH₃CO) , 1.26 (3H, t, J=7.5 Hz, CH₃CH₂S) ; δ_c(150 MHz) 207.1 (COCH₃) , 171.9 (C0₂CH₂) , [136.9, 129.2, 128.3, 126.3 (Ph) ] , 101.8 (CPh), 83.7 (C-l) , 80.4 (C-4) , 73.4 (C-3) , 72.9 (C-2) , 70.6 (C-5) , 68.5 (C-6) , 38.3 (CH₂CO) , 29.7 (CH₃CO), 28.2 (CH₂C0₂) , 24.0 (CH₂S) , 14.8 (CH₃CH₂S) ; m/z Found: (MNa)⁺, 433.1299. C₂₀H₂sO₇NaS requires M, 433.1297. Ethyl 4, 6-Q-benzylidene-2-0-levulinoyl-l-thio-3-0- (2,3,4-tri-O-benzoyl-β-D-xylopyranosyl) -β-D-glucopyranoside XVII

^XVI1 Bz=Benzoyl

The compound XVI (1.0 g, 2.4 mmol) and tri-O-benzoyl- - D-xylopyranosyl bromide XIV (1.66 g, 3.2 mmol) were combined, dissolved in toluene, concentrated and dried under vacuum. Activated molecular sieves were added (4A, beads) and the mixture was stirred in DCM (15 mL) for 3 hours, and later cooled to -35 °C. In parallel silver triflate (0.88 g, 3.4 mmol) was dried by azeotropic distillation in toluene and subsequently stored under vacuum. The silver triflate was then suspended in toluene (5 mL) and DCM (10 mL) and 2,6- lutidine (0.2 mL, 1.7 mmol) were added under argon. This mixture was added dropwise to the cooled solution of sugars (about 10 minutes for complete addition) , which was then stirred for a further 20 minutes at -35 °C. The reaction was quenched by addition of a few drops of triethylamine, then diluted with DCM and washed with Na₂S₂0₃ and 0.3M HCl . The organic layer was dried with MgS0, concentrated and purified by flash column chromatography (ethyl acetate/petroleum ether, 1:3), affording ethyl 4 , 6-0-benzylidene-2-0- levulinoyl-l-thio-3-O- (2 , 3 , 4-tri-O-benzoyl-β-D- xylopiranosyl) -β-D-glucopyranoside XVII (Rf=0.4, (ethyl acetate/petroleum ether, 1:1) as an amorphous solid (1.89 g, 92%) . ϋ_max (film) /cm^"1 3064, 2967, 2869, 1731 (C=0) , 1602, 1452, 1262, 1098; δ_H(600 MHz) 8.07-7.22 (20H, m, Ph) , 5.55- 5.61 (2H, m, H-3A and HCPh) , 5.22 (IH, d, J=2.7 Hz, H-1A) , 5.17 (IH, t, J=2.7 Hz, H-2A) , 5.15-5.07 (2H, m, H-4A and H- 2B) , 4.63 (IH, dd, J=13.1, 2.7 Hz, H-5A) , 4.48 (IH, d, J=10.0, H-IB) , 4.40 (IH, dd, J=4.9, 10.6 Hz, H-6B) , 4.16 (IH, t, J=9.1 Hz, H-3B) , 3.80 (IH, t, J=10.2 Hz, H-6B) , 3.72 (IH, t, J=9.4Hz, H-4B) , 3.60-3.54 (2H, m, H-5A and H-5B) , 2.78- 2.60 (4H, m, (CH₂) ) , 2.57 (2H, t, J=6.8 Hz, CH₂S) , 2.08 (3H, s, CH₃C0) , 1.24 (3H, t, J=6.8 Hz, CH₃CH₂S) ; δ_c(150 MHz) 206.2

(COCH₃) , 171.4 (C0₂CH₂) , [165.5, 165.2, 164.7 (PhCO) ] , [136.8, 129.5, 128.4, 125.9 (Ph) ] , 101.8 (PhCH0₂) , 97.9 (C-1A) , 84.4

(C-1B) , 79.6 (C-4B) , 77.0 (C-3B) , 72.3 (C-2B) , 70.9 (C-5B) , 68.9 (C-2A) , 68.7 (C-5A) , 67.9 (C-4A) , 67.7 (C-3A) , 59.4 (C- 6B) , 37.9 (CH₂C0C) , 29.6 (CH₃CO) , 28.1 (CH₂C0₂) , 24.2 (CH₂S) , 14.8 (CH₃CH₂S) ; m/z Found: (MNa)⁺, 877.2506, C₄₆H₄₆0₁₄NaS requires M, 877.2506.

1.2. Synthesis of Glycoside VII

Preparation of 4, 6-0-benzylidene-2-0-levulinoyl-3-0- (2,3,4-tri-O-benzoyl-β-D-xylopyranosy ) -β-D-glucopyranosyl chloride XVIII

XVII XVIII The donor chloride for the glycosylation was freshly prepared from the disaccharide XVII by dissolving it in a saturated solution of Cl₂ in CC1₄ and stirring for a maximum of 10 minutes. The solution was diluted with DCM and washed with Na₂S₂0₃ solution, dried with MgS0₄, filtered and concentrated, affording disaccharide XVII, without need for furher purification and immediately used in the coupling reaction.

Glycoside VII, R-OH = iS-amyrin

VII

Freshly prepared XVIII, prepared by treating XVII (0.30 g, 352 μmol) with a saturated solution of Cl₂ in CC1₄ (5 ml) in the manner described above was mixed with beta-amyrin (100 mg, 235 μmol) , dissolved in toluene and the solvent removed under reduced pressure. The mixture was redissolved in a mixture of CC1/DCM (1:1 1.5 ml) and beaded molecular sieves (4A, 0.5 g) were added and the mixture stirred for 3h. The reaction mixture was then cooled to -20°C and a suspension of freshly dried AgOTf (96 mg, 376 μmol), 2,6- lutidine (21 μL, 188 μmol) in CC1₄/toluene (1 mL, 3:2) was added dropwise over 5 minutes. The reaction mixture was stirred for a total of 30 minutes at -20 °C before being neutralized by addition of Et₃N (0.1 mL) , diluted with DCM, decanted, washed with Na₂S₂0₃, dried (MgS0₄) and concentrated.

Purification of the residue by flash column chromatography

(gradient of EtOAc/petroleum ether 1:9-1.5) afforded glycoside VII (171 mg, 60%) as a white amorphous solid. υ_max (film) /cm^"1 2948, 1727, 1602, 1452, 1380, 1262, 1098,

1027, 711; δ_H(600 MHz) 8.04 (4H, d, J=8.0 Hz, H-Ar) , 7.89

(2H, d, J=7.4 Hz, H-Ar), 7.59-7.22 (14H, m, H-Ar), 5.59-5.56

(2H, m, H-3A, PhCH0₂) , 5.20-5.14 (4H, m, H-1A, 2A, 2B, CH=C) ,

5.13-5.08 (IH, m, H-4A) , 4.63 (IH, dd, J=10.7, 2.0 Hz, H-

5Aa) , 4.56 (IH, d, J=7.8 Hz, H-IB) , 4.36 (IH, dd, J=10.2, 4.8

Hz, H-6Ba) , 4.12 (IH, t, J=9.3 Hz, H-3B) , 3.83 (IH, t, J=10.2

Hz, H-6Bb) , 3.75 (IH, t, J=9.3 Hz, H-4B) , 3.57-3.50 (2H, m,

H-5A, 5B) , 3.10 (IH, dd, J=ll.l, 4.7 Hz, H-CHO-glu) , 2.75-

2.55 (4H, m, CH₂-lev) , 2.08 (3H, s, CH₃C0) , 2.04-0.66 (47H, m, H-amyrin) ; δ_c(100 MHz) 206.5 (CH₃C0) , 171.1 (CH₂C0₂) ,

[165.5, 165.2, 164.8 (PhC0₂) ] , [145.2, 136.9 (C*)], [132.2,

130.0, 129.9 (CH)] , 129.6 (C*) , 129.5 (CH) , 129.4 (C*) ,

[129.3, 128.4, 128.3, 126.0 (CH) ] , 121.7 (CH=C) , 103.5 (C-

1B) , 101.8 (PhCH0₂) , 97.8 (C-1A) , 90.1 (CHO-glu) , 79.6 (C-

4B) , 76.4 (C-3B) , 74.2 (C-2B) , 68.8 (C-2A, 6B) , 67.8 (C-3A,

4A) _. 66.3 (C-5B) , 60.4 (C-5A) , [55.5, 47.6, 47.2 (CH) ] , 46.8

(CH₂) , [41.7, 39.8, 38.9 (C*)] , [38.6, 37.9, 37.1 (CH₂) ] ,

36.6 (C*) , 34.7 (CH₂) , 33.3 (CH) , 32.6 (CH₂) , [32.5, 31.0

(C*)] , [29.7, 28.4 (CH)] , 28.0 (CH₂) , 27.8 (CH) , [26.9, 26.1

(CH₂)] , 26.0 (CH) , 25.9 (CH₂) , 23.7 (CH) , [23.5, 18.2 (CH₂) ] ,

[16.8, 16.3, 15.5 (CH) ] ; m/z Found: (MNa⁺) 1241.6172,

C₇₄H₉o0₁₅Na requires M, 1241.6172. 1.3. Synthesis of the trisaccharide X

i) 3-β-O-amyrinyl- [4,6-0-benzylidene-3-0- (2, 3,4-tri-O- benzoyl-β-D-xylopyranosyl) -β-D-glucopyranoside] VIII

VII (171 mg, 140 μmol) was dissolved in a cooled solution of N₂H₄.H₂0 0.5M in pyridine/acetic acid 4:1 (5 ml) and stirred for 6 hours at 0°C. The mixture was diluted with DCM, washed three times with 0.3M HCl and once with NaHC0₃ solution, dried with MgS0₄ and concentrated to give crude VIII that was purified by flash chromatography (gradient of EtOAc/Petroleum ether 1:9-1:5) affording VIII (117 mg, 75 as a white amorphous solid. υ_max (film) /cm^"1 2948, 1727, 1602, 1452, 1380, 1262, 1098, 1027, 710; δ_H(600 MHz) 8.05 (2H, d, J=5.0 Hz, H-Ar), 8.03 (2H, d, J=5.1 Hz, H-Ar), 7.98 (2H, d, J=7.3 Hz, H-Ar), 7.57-7.26 (14H, m, H-Ar), 5.63 (IH, T, J=5.1 Hz, H-3a) , 5.56 (IH, s, PhCH0₂) , 5.45 (IH, d, J=3.0 Hz, H- 1A) , 5.34 (IH, t, J=4.7 Hz, H-2A) , 5.22-5.15 (2H, m, H-4A, CH=C) , 4.65 (IH, dd, J=12.9, 3.1 Hz, H-5Aa) , 4.44 (IH, d, J=7.8 Hz, H-IB), 4.34 (IH, dd, J=10.5, 4.9 Hz, H-6Ba) , 4.00 (IH, t, J=10.3 Hz, H-3B) , 3.80 (IH, t, J=10.5 Hz, H-6Bb) , 3.67-3.62 (3H, m, H-2B, 4B, 5Ab) , 3.49-3.42 (IH, m, H-5B) , 3.18 (IH, dd, J=11.6, 4.6 Hz, CHO-glu) , 2.45 (IH, d, J=2.7 Hz, H-OH), 2.02-0.73 (47H, m, H-amyrin) ; δ_c(100 MHz) [165.6, 165.4, 164.9 (PhC0₂) ] , [145.2, 137.1 (C*) ] , [133.4, 133.2, 129.9, 129.8 (CH) ] , [129.5, 129.4 (C*) ] , [129.0, 128.4, 128.3, 125.9 (CH) ] , 121.7 (CH=C), 105.3 (C-1B) , 101.5 (PhCH0₂) , 98.6 (C-IA) , 90.1 (CHO-glu) , 79.3 (C-2B) , 77.3 (C- 3B) , 76.1 (C-4B) , 69.2 (C-2A) , 68.9 (C-6B) , 68.3 (C-3A) , 68.2 (C-4A) , 66.5 (C-5B) , 59.6 (C-5A) , [47.6, 47.2 (CH) ] , 46.8 (CH₂) , [41.7, 41.3, 39.8 (C*)] , [38.5, 37.1 (CH) ] , 36.6 (C*) , 34.7 (CH₂) , 33.3 (CH) , 32.6 (CH₂) , [32.5, 31.1 (C*) ] , [28.4, 28.2 (CH) ] , [26.9, 26.1 (CH₂) ] , 26.0 (CH) , 25.9 (CH₂) , 23.7 (CH) , [23.5, 18.2 (CH₂) ] , [16.8, 16.6, 15.4 (CH) ] ; m/z Found (MNa⁺) 1143.5745, C₆₉H₈₄Oι ₅Na requires M, 1143.5803.

ii) 3-β-O-amyrinyl- [4, 6-0-benzylidene-2-Q- (2, 3, 4, 6- tetra-O-acetyl-β-D-galactopyranosyl) -3-0- (2,3,4-tri-O- benzoyl-β-D-xylopyranosyl) -β-D-glucopyranoside] X

X

Ac=Acetyl Bz=Benzyl Ph=Phenyl Glycoside VIII (32 mg, 29 μmol) and galactosyl bromide IX (40 mg, 98 μmol) were first dried by azeotropic removal of water by toluene and storage under vacuum. Activated beaded molecular sieves (4A, 0.2 g) and DCM (0.5 ml) were then added to the residue and the mixture stirred for 3h. After which time the reaction was cooled to -35 °C and a mixture of freshly dried AgOTf (26 mg, 103 μmol) and 2, 6-lutidine (6 μL, 49 μmol) in DCM/toluene (0.75 mL, 2:1) was added dropwise over 5 minutes. The reaction mixture was stirred for a total of 30 minutes and then neutralized by addition of Et₃N (0.1 mL) , diluted with DCM, decanted, washed with Na₂S₂0₃ solution, dried (MgS0₄) and concentrated. Subsequent purification of the residue by flash column chromatography (gradient of EtOAc/petroleum ether 1:5-1:3) produced X (34 mg, 81%) as an amorphous white solid; υ_max (film) /cm^"1 2948, 1755, 1725, 1600, 1453, 1371, 1251, 1218, 1098, 1038, 706;δ_H(600 MHz) 8.13 (2H, d, J=7.4 Hz, H-Ar), 8.05 (2H, d, J=7.4 Hz, H-Ar), 7.92 (2H, d, J=7.5 Hz, H-Ar) ,7.64-7.14 (14H, m, H-Ar), 5.53 (IH, s, PhCH0₂) , 5.51 (IH, brs, H-3A) , 5.46 (IH, d, J=3.1 Hz, H-4C) , 5.40 (IH, s, H-2A) , 5.30-5.29 (2H, m, H-1A, 3C) , 5.18 (IH, brs, CH=C) , 5.09 (IH, t, J=8.0 Hz, H-2C) , 5.04 (IH, d, J=8.0 Hz, H-1C) , 5.03-5.02 (IH, m, H-4A) , 4.75 (IH, d, J=11.7 Hz, H-5Aa) , 4.47 (IH, d, J=7.6 Hz, H-IB) , 4.35 (IH, dd, J=10.5, 4.9 Hz, H-6Ba) , 4.16-4.11 (3H, m, H-3B, 6C) , 4.02-3.98 (2H, m, H-2B, 5C) , 3.80 (IH, t, J=10.5 Hz, H-6Bb) , 3.68 (IH, t, J=9.5 Hz, H-4B) , 3.54(1H, d, J=11.7 Hz, H-5Ab) , 3.49-3.45 (IH, m, H-5B) , 3.10 (IH, dd, J=11.5, 4.5 Hz, CHO-glu), 2.11 (3H, s, CH₃C0₂) , 2.07-0.73 (56H, 3 x CH₃C0₂, H-amyrin); δ_c(l00 MHz) [170.3, 170.2, 169.7, 169.1 (CH₃CO) ] , [165.5, 165.1, 164.6 (PhC0₂)], [145.2, 136.9 (C*) ] , [133.6, 133.4, 133.3, 130.3, 129.9 (CH)], [129.8, 129.5 (C*) ] , 129.4 (CH) , 128.9 (C*) , [128.4, 128.3, 128.2, 126.0 (CH) ] , 121.7 (CH=C) , 104.5 (C-1B) , 102.0 (PhC0₂) , 99.1 (C-1C) , 96.8 (C-IA) , 91.1 (CHO- glu) , 79.3 (C-4B) , 78.2 (C-2B) , 76.8 (C-3B) , 71.0 (C-3C) , 70.4 (C-5C) , 69.5 (C-2C) , 69.0 (C-6B) , 67.2 (C-2A) , 67.1 (C- 4A, 4C) , 67.0 (C-3A) , 66.0 (C-5B) , 60.7 (C-6C) , 58.5 (C-5A) ,

[55.6, 47.6, 47.2 (CH) ] , 46.8 (CH₂) , [41.7, 39.8, 39.3 (C*)] , 38.7 (CH₂) , [37.1, 36.6 (C*)] , 34.7 (CH₂) , 33.3 (CH) . 32.6

(CH₂) , [32.5, 31.1 (C*)], [29.3, 28.4, 27.6 (CH) ] , [26.9, 26.1 (CH₂)], [25.9, 16.8, 16.2, 15.5 (CH) ] ; m/z Found (MNa⁺) 1473.8131, C₈₃H₁₀₂O₂₂Na requires M, 1473.6761.

iii) 3-β-O-amyrinyl- [2-0- (2, 3, 4, 6-tetra-O-acetyl-β-D- galactopyranosyl) -3-0- (2, 3,4-tri-O-benzoyl-β-D- xylopyranosyl) -β-D-glucopyranoside] XI

XI

Glycoside X (105 mg, 72 μmol) was dissolved in DCM (3 ml) and a solution of TFA/H₂0 1:1 (200 μL) was added. After stirring the mixture for 6 h at room temperature, the reaction was neutralized with triethylamine and concentrated. The residue was purifed by flash column chromatography (gradient of EtOAc/petroleum ether 1:5-1:1), affording XI (73 mg, 74%) as a white amorphous solid. υ_max (film) /cm^"1 3582,

2948, 1741, 1732, 1453, 1369, 1252, 1071, 712; δ_H(600 MHz)

7.98 (4H, d, J=8.0 Hz, H-Ar), 7.92 (2H, d, J=7.5 Hz, H-Ar),

7.57-7.33 (9H, m, H-Ar), 5.83 (IH, t, J=8.0 Hz, H-3A) , 5.56

(IH, dd, J=8.1, 5.9 Hz, H-2A) , 5.40-5.36 (IH, m, H-4A) , 5.17

(IH, s, CH=C) , 5.08-5.04 (2H, m, H-1A, 2C) , 5.00 (IH, d,

J=3.2 Hz, H-4C) , 4.82 (IH, dd, J=10.5, 3.3 Hz, H-3C) , 4.65

(IH, d, J=7.9 Hz, H-1C) , 4.62 (IH, dd, J=12.3, 4.8 Hz, H-

5Aa) , 4.35 (IH, d, J=7.5 Hz, H-IB) , 3.92-3.84 (4H, m, H-6C,

6B) , 3.80-3.68 (3H, m, H-2B, 3B, 5Ab) , 3.57 (IH, t, J=9.2 Hz,

H-4B) , 3.34-3.32 (IH, m, H-5B) , 3.06 (IH, dd, J=11.3, 4.9 Hz,

CHO-glu), 2.58 (IH, t, J=6.7 Hz, H-5C) , 2.11-0.71 (59H, 4 x

CH₃CO₂ , H-amyrin) ; δ_c (150 MHz) [170 . 1 , 170 . 0 , 169 . 8 , 169 . 0

(CH₃CO) ] , [165 . 5 , 165 . 4 , 164 . 9 (PhC0₂) ] _. 145 . 1 (CH=C) ,

[133.7, 133.7, 129.9, 129.7 (CH) ] , 129.0 (C*) , 128.9 (CH) ,

128.7 (C*) , 128.5 (CH) , 121.8 (CH=C) , 103.7 (C-1B) , 100.2 (C-

1C) , 99.2 (C-IA) , 90.8 (CHO-glu), 85.8 (C-2B) , 76.1 (C-3B) ,

74.8 (C-5B) , 70.9 (C-3C) , 70.7 (C-2A) , 70.4 (C-3A) , [69.8,

69.7 (C-5C, 2C, 4B) ] , 69.2 (C-4A) , 66.8 (C-4C) , 63.0 (C-6C) ,

62.3 (C-5A) , 60 . 6 (C-6B) , 55.5 (CH) , 47.6 (C*) , [47.6, 47.2 (CH)], 46.8 (CH₂) , [41.7, 39.8, 39.2 (C*) ] , [38.6, 37.1 (CH₂)], 36.6 (C*) , 33.3 (CH₂) , 32.6 (CH) , 32.5 (CH₂) , [31.1,

28.4 (C*)], [27.7, 26.9 (CH) ] , [26.3, 26.1, 26.0 (CH₂) ] , 23.7 (CH) , 23.5 (CH₂) , [20.9, 20.7, 20.6, 20.5 (CH) ] , 18.2 (CH₂) , [16.8, 16.3, 15.4 (CH) ] ; m/z Found (MNa⁺) 1385.744,

C₇₆H₉₈0₂₂Na requires M, 1385 . 6648 . iv) 3-β-O-amyrinyl- [2-0- (2, 3, 4, 6-tetra-O-acety -β-D- galactopyranosyl) -3-0- (2, 3,4-tri-O-benzoyl-β-D- xylopyranosyl) -β-D-glucuronic acid] XII

XII Alcohol XI (35 mg, 26 μmol) and TEMPO (6 mg) were dissolved in THF (0.8 mL) . Water (0.2 mL) containing NaBr (6 mg) was added and the mixture cooled to 0°C, stirring vigorously. A mixture of NaOCl (4-5% Cl₂ content, 0.18 mL) and aqueous saturated NaHC0₃ (0.12 mL) was added dropwise and the reaction stirred for 25 minutes. The reaction was immediately diluted with DCM (10 mL) containing MeOH (0.2 mL) and the organic layer separated. The aqueous layer was extracted repeatedly with DCM (2 x 10 mL) . The combined organic washings were then washed with a 0.1 M HCl solution in brine and concentrated. The residue was purifed by flash column chromatography (gradient DCM-DCM:Acetone (85 : 15) -DCM: eOH (95:5-4:1)) to afford the partially purified XII (27 mg) contaminated by trace products arising from acetolysis; m/z Found (MNa⁺) 1399.4. This material was used directly in the next reaction. 1.4. Deprotection of 3-β-O-amyrinyl- [2-0- (2, 3, 4, 6- tetra-O-acetyl-β-D-galactopyranosyl) -3-0- (2,3,4-tri-O- benzoyl-β-D-xylopyranosyl) -β-D-glucuronic acid] XII

XIII

The partially protected glycoside XII (43 mg) was dissolved in MeOH/THF (3.5 mL, 6:1) and a spatula tip of K₂C0₃ was added. After 3 hours the reaction was neutralized by addition of Amberlite IR 120 resin, and the reaction mixture was filtered and diluted with more MeOH (30 mL) . The reaction mixture was then washed repeatedly with n-hexane and the methanol layer was separated and concentrated to XIII (28 mg) still contaminated by methyl benzoate (approx. 5 wt%).δ_H(600 MHz, CDCI₃: MeOD 2:1): 5.00 (IH, S, CH=C) , 4.62 (IH, d, J=7.6 Hz, H-1C) , 4.35 (IH, d, J=6.9 Hz, H-1A) , 4.31 (IH, d, J=7.7 Hz, H-IB) , 3.77 (IH, dd, J=11.2, 5.2 Hz, H-5Aa) , 3.67-3.60

(4H, m, H-2B, 4B, 4C, 6Ca) , 3.51-3.46 (3H, m, H-3B, 5B, 6Cb) , 3.41-3.39 (IH, m, H-4A) , 3.35-3.28 (3H, m, H-2C, 3C, 5C) , 3.21-3.13 (2H, m, H-2A, 3A) , 3.09 (IH, t, J=11.2 Hz, H-5Ab) , 3.00 (IH, dd, J=11.8, 4.3 Hz, CHO) , 1.84-0.56 (47H, m, H- amyrin) ; δ_c(150 MHz, CDC1₃ : MeOD 2:1): 144.9 (C=CH) , 121.5

(C=CH) , 104.2 (C-1B) , 103.5 (C-IA) , 102.5 (C-1C) , 91.0 (CHO- glu), 85.9 (C-5B) , 77.3 (C-2B) , 76.7 (C-2A or 3A) , 75.2 (C- 5C) , 74.6 (C-4B) , 73.5 (C-3C) , 73.4 (C-2A or 3A) , 72.2 (C- 2C) , 69.9 (C-3B) , 69.1 (C-4A) , 68.8 (C-4C) , 65.7 (C-5A) , 63.1 (CHC=CH), 61.3 (C-6C) , 55.4 (CH) , [47.4, 47.0 (CHO] , 36.4 (C*) , 34.5 (CH₂) , 33.0 (CH) , 32.4 (CH₂) , [32.2, 30.8 (C*)], [28.0, 27.2 (CH) ] , [26.7, 25.9, 25.7, 23.3, 17.9 (CH₂) ] , [16.5, 15.7, 15.2 (CH) ] ; m/z Found (MNa⁺) 919.5048, C₄₇H_7S0₁₆Na requires M, 919.5030.

EXAMPLE 2- FRIEDELIN SAPONIN XXXIV

2.1. 3-β-O-Friedelinolyl 2-Q-acetyl-3,4, 6-tri-O-benzyl- β-D-glucopyranoside XXII

To a solution of imidate XX (Scheme II) (521 mg, 0,82 mmol) and 3-?-0-friedelinol XXI (Moiteiro, C, Justino, F., Tavares, R., Marcelo-Curto, M. J. , Florδncio, M.H. , Nascimento, M.S.J., Pedro, M. , Cerqueira, F. , and Pinto, M.M. M., J. Nat. Prod., 64, 1273-1277, 2001) (176 mg, 0.41 mmol) in dry CH₂C1 (2 mL) was added TMSOTf (9 μL, 0.041 mmol). The mixture was stirred for 10 min at room temperature and then quenched by addition of Et₃N and the solvent evaporated. Purification of resulting residue by flash chromatography (petroleum ether/ethyl acetate, 10:1) gave XXII (190 mg, 51%) . MALDI-MS: m/z 926 [M+Na] ⁺, 943 [M+K]⁺. ^XH NMR (600 MHz, CDCI₃) 7.33-7.21 (m, 15H, 3xOBn) , 5.03 (t, J = 7.9 Hz, IH, 2'-H) , 4.79, 4.67 (2d, J = 11.5 Hz, 2H, 2xOCH₂Ph) , 4.62, 4.58 (2d, J = 12.4 Hz, 2H, 2xOCH₂Ph) , 4.79, 4.58 (2d, J = 11.2 Hz, 2H, 2xOCH₂Ph) , 4.30 (d, J = 7.9 Hz, IH, I'-H), 3.72 (d, J = 10.8 Hz, IH, 6'-H), 3.64 (m, IH, 3',4'-H), 3.65 (d, IH, 6'- H) , 3.52 (s, IH, 3-H) , 3.44 (m, IH, 5'-H), 2.2 (d, J = 11.8 Hz, IH, 2-H) , 1.96 (s, 3H, COCH₃) , 1.69 (d, IH, J = 12.5 Hz, CH₂) , 1.16 (s, 3H, CH₃) , 1.00, 0.98 (s, 12H, 4xCH₃) , 0.94 (s, 3H, 3xCH₂) , 0.84, 0.82 (s, 12H, 4xCH₃) . ¹³C NMR (150.8 MHz, CDCI3) 169.2 (IC, COMe) , 138.3, 137.97 (3C, ipso-Ph) , 128.4- 127.5 (15C, Ph) , 103.1 (IC, l'-C) , 83.2 (IC, 3',4'-C) , 83.1 (IC, 3-C) , 78.2 (IC, 3',4'-C) , 75.0 (3C, 2xOCH₂Ph, 5'-C) , 73.4 (2C, OCH₂Ph, 2'-C) , 69.0 (IC, 6'-C) , 34.2 (IC, 2'-C) , 21.0 (IC, COMe) , 34.2 (IC, 4'-C) . Anal. Calcd for C₅₉H₈₂θ₇ : C, 78.45; H, 9.15. Found: C, 78.49; H, 9.45.

2.2. 3-β-O-Friedelinolyl 3,4, 6-tri-O-benzyl-β-D- glucopyranoside XXIII

XXIII To a solution of 3-β-O-friedelinolyl 2-0-acetyl-3 , 4 , 6- tri-O-benzyl-β-D-glucopyranoside XXII (120 mg, 0.13 mmol), in MeOH/CH₂CL₂ (117 mL, 2:1) was added NaOMe (catalytic quantity) . The mixture was stirred at room temp, for 18 h, neutralized by addition of ion exchange resin (Amberlite IR- 120, H⁺ form) and then filtered. The filtrate was concentrated in vacuo and the resulting residue purified by flash chromatography (petroleum ether/ethyl acetate, 13:1) to afford XXIII (96 mg, 86%). MALDI-MS: m/z 884 [M+Na]⁺, 900

[M+K]⁺. ^XH NMR (600 MHz, CDC1₃) 7.38-7.20 (m, 15H, 3xOBn) , 4.98, 4.81 (2d, J = 11.2 Hz, 2H, 2xOCH₂Ph) , 4.83, 4.59 (2d, J = 9.5 Hz, 2H, 2xOCH₂Ph) , 4.85, 4.56 (2d, J = 10.9 Hz, 2H, 2xOCH₂Ph) , 4.22 (d, J = 7.0 Hz, IH, l'-H), 3.72 (d, J = 10.5 Hz, IH, 6'-H), 3.66 (d, J = 5.1 Hz, IH, 6'-H), 3.63 (s, IH, 3-H) , 3.50 (m, IH, 3'-H), 3.59 (d, J = 7.0 Hz, IH, 2'-H), 3.55 (m, IH, 4'-H), 3.46 (m, IH, 5'-H), 2.22 (d, J = 11.7 Hz, IH, 2-H) , 1.73 (d, J = 12.6 Hz, CH₂) , 1.18 (s, 3H, CH₃) , 1.00

(s, 9H, 3xCH₃) , 0.95 (s, 3H, 3xCH₃) , 0.86 (s, 3H, CH₃) . ¹³C NMR (150.8 MHz, CDCl₃) 138.8, 138.4, 138.2 (3C, ipso-Ph), 128.4-127.5 (15C, Ph) , 105.0 (IC, l'-C), 84.6 (IC, 3'-C), 83.1 (IC, 3-C), 77.6 (IC, 4'-C), 75.8 (IC, 2'-C), 75.1 (3C, 2xOCH₂Ph, 5'-C), 73.4 (IC, OCH₂Ph) , 69.1 (IC, 6'-C), 61.4

(IC, CH₂) , 34.3 (IC, 2'-C), 35.0, 32.8, 32.3, 20.1, 18.6, 18.3, 16.3, 11.9 (8C, 8xCH₃) . Anal. Calcd for C₅₇H₈₀O₆ : C, 79.49; H, 9.35. Found: C, 78.83; H, 9.47. 2.3. 3-β-O-Friedelinolyl [ (2,3,4, 6-tetra-O-acetyl-β-D- galactopyranosyl) - (1—>2) ] -3,4, 6-O-benzyl-β-D-glucopyranoside XXV

To a solution of saponin XXIII (44 mg, 0.05 mmol) in dry

CH₂C1₂ (2 mL) and the imidate XXIV (50 mg, 0.102 mmol) was added a solution of TMSOTf (1.1 μL, 0.005 mmol) in CH₂C1₂

(0.7 mL) . The mixture was stirred for 10 min at room temp. and then quenched by addition of Et₃N and the solvent evaporated. Purification of resulting residue by flash chromatography (petroleum ether/ethyl acetate, 8:1, 5:1, 3:1) gave XXV (41 mg, 69%). MALDI-MS: m/z 1215 [M+Na]⁺. ^XH NMR

(600 MHz, CDC1₃) 7.34-7.10 (m, 15H, 3xOBn) , 5.37 (d, J = 2.6

Hz, IH, 4"-H), 5.25 (t, J = 2.6 Hz, IH, 2 " -H) , 4.99 (m, 2H,

3",1'-H), 4.85 (d, J = 7.9 Hz, IH, 1"-H), 4.86-4.44 (6d,

6H, 3xOCH₂Ph) , 4.11 (m, IH, 6"-H), 3.91 (m, IH, 5'-H), 3.90

(m, IH, 5''-H), 3.85 (t, J = 9.4 Hz, IH, 3 ' -H) , 3.72, 3.60

(m, 2H, 6'-H), 3.71 (d, IH, 2 ' -H) , 3.62 (m, IH, 3-H) , 3.61

(m, IH, 4'-H), 2.14, 2.05, 1.97, 1.75 (4s, 12H, C0CH₃) . ¹³C

NMR (150.8 MHz, CDCl₃) 170.2, (3C, ipso-Ph), 168.8 (4C,

COMe), 128.5-127.6 (15C, Ph) , 101.9 (IC, 1"-C), 95.9 (IC,

I'-C), 81.3 (IC, 3'-C), 80.0 (IC, 2'-C), 78.0 (IC, 4'-C),

77.9 (IC, 3-C), 75.0, 73.5, (3C, 3xOCH₂Ph) , 71.2 (IC, 3''-C), 70.7 (IC, 5''-C) , 70.4 (IC, 5'-C), 69.0 (IC, 2"-C), 68.6 (IC, 6'-C), 67.3 (IC, 4"-C), 61.6 (IC, 6"-C) . Anal. Calcd for C₅₇H₈o0₆: C, 79.49; H, 9.35. Found: C, 78.83; H, 9.47. Anal. Calcd for C₇ιH₉₈0ι₅ : C, 71.57; H, 8.29. Found: C, 71.11; H, 8.37.

2.4. 3-β-O-Friedelinolyl [ (2,3,4, 6-tetra-O-acetyl-β-D- galactopyranosyl) - (l- 2) ] -β-D-glucopyranoside XXVI

To a solution of saponin XXV (312 mg, 0.26 mmol) in MeOH/CH₂Cl₂ (22 mL, 2:1) was added Pd/C (100 mg) . The mixture was stirred at room temperature under hydrogen atmosphere for 10 min., filtered off and evaporated. The resulting residue was purified by flash chromatography (petroleum ether) to afford XXVI (127 mg, 53%). MALDI-MS: m/z MALDI-MS: m/z 945 - [M+Na]⁺, 961 [M+K]⁺.

2.5. 3-β-O-Friedelinolyl [ (2 " , 3 ' ' ,4' ' , 6"-tetra-0- acetyl-β-D-galactopyranosyl) - (l->2)] -3 -O-benzyl-4' ,6' -O- benzylidene-β-D-glucopyranoside XXVII

Using the general benzylidene protection procedure oulined in Scheme II, XXVII was obtained from XXVI in 92% yield. MALDI-MS: m/z 1032 [M+Na]⁺, 1048 [M+K]⁺. ^XE NMR (600 MHz, CDCI₃) 7.49-7.26 (5H, Ph) , 5.51 (IH, CHPh) , 5.36 (IH, 4"-H), 5.16 (IH, 2"-H), 5.11 (IH, 1"-H), 5.02 (IH, 3"-H), 4.36 (IH, l'-H), 4.30 (IH, 6'-H), 4.12 (IH, 6"-H), 3.89 (IH, 5"-H), 3.80 (2H, 2", 3'-H), 3.75 (IH, 6'-H), 3.56 (IH, 3-H), 3.51 (IH, 4'-H), 3.35 (IH, 5'-H) . ¹³C NMR (150.8 MHz, CDCI₃) 104.9 (IC, l'-C) , 102.2 (IC, CHPh), 100.4 (IC, 1"-C), 84.8 (IC, 3-C), 80.9 (IC, 4 -C), 78.4 (IC, 2 ' or 3'-C), 75.6 (IC, 2' or 3'-C), 71.4 (IC, 3"-C), 70.7 (IC, 5"-C), 70.1. (IC, 2"-C), 69.1. (IC, 6'-C), 67.5. (IC, 4"-C), 65.9. (IC, 5'-C), 61.5. (IC, 6^""-C) . Anal. Calcd for C₅₇H₈4θι₅ : C, 67.83; H, 8.39. Found: C, 67.60; H, 9.27. 2.6. 3-β-Q-Friedelinolyl (2' ' ' , 3' ' ' ,4' ' ' -tri-O-acetyl-β- D-xylopyranosyl) - (l->3) - [ (2' ' ,3' ' ,4' ' ,6' ' -tetra-O-acetyl-β-D- galactopyranosyl) - (1— >2) ] -4' , 6' -O-benzylidene-β-D- glucopyranoside XXX

The procedure was the same as above described in § 2.2. where compound XXIII was replaced by XXVII, reacting with imidate XXIX to yield XXX in 75% yield. MALDI-MS: m/z 1289

[M+Na]⁺, 1306 [M+K]⁺. ^XE NMR (600 MHz, CDC1₃) 7.45-7.26 (5H, Ph) , 5.46 (IH, CHPh), 5.36 (IH, 4"-H), 5.25 (IH, 3"-H), 5.11 (IH, 2""-Ε), 4.99 (IH, 3"'-H), 4.97 (IH, 2"'-H), 4.93

(IH, 1"-H) , 4.89 (IH, 1"'-H), 4.81 (IH, 4"'-H), 4.31 (IH, l'-H), 4.28 (IH, 6'-H), 4.14 (IH, 6"-H), 4.11 (IH, 5"'-H), 3.95 (2H, 2',3^{^}-H), 3.94 (IH, 5"-H), 3.75 (IH, 6'-H), 3.73

(IH, 4'"-H) , 3.51 (IH, 3-H) , 3.37 (IH, 5"-H) , 3.06 (IH, 5"'-H) . ¹³C NMR (150.8 MHz, CDC1₃) 104.9 (IC, l'-C) , 102.2

(IC, CHPh), 99.0 (IC, 1"-C), 98.4 (IC, l'"-C), 85.1 (IC, 3- C) , 80.2 (IC, 2" or 3'-C) , 79.4 (IC, 4"^*-C) , 76.8 (IC, 2" or 3"-C) , 70.6 (2C, 3"^*,5"-C) , 70.4 (2C, 2"',3"'~C), 70.2 (IC, 2"'-C) , 69.3 (IC, 6'-C) , 68.0. (IC, 4"'-C) , 67.8. (IC, 4" - C) , 65.9. (IC, 5"-C) , 61.1. (IC, 6"-C) , 60.9 (IC, 5"-C) . Anal. Calcd for C₆8H₉s0₂₂ : C, 63.39; H, 7.51. Found: C, 64.06; H, 8.36. 2.7. 3-β-O-Friedelinolyl (2 ' ' ' , 3 ' ' ' ,4' ' ' -tri-O-acetyl-β- D-xylopyranosyl) - (l->3) - [ (2 " , 3 ' ' ,4' ' , 6 ' ' -tetra-O-acetyl-β-D- galactopyranosyl) - (l->2) ] -β-D-glucopyranoside XXXI

As outlined in Scheme II, XXXI was obtained from XXX by reaction with EtSH in dichloromethane, in the presence of p- TsOH, in 80% yield. MALDI-MS: m/z 1200 [M+Na]⁺. ²H NMR (600 MHz, CDCI₃) 5.34 (IH, 4"'-H), 5.17 (IH, 3"'-H), 5.13 (IH, 2"-H), 4.08 (IH, 3"'-H), 5.06 (IH, 3"-H), 4.99 (IH, 4'"- H) , 4.85 (IH, 1"'-H), 4.84 (IH, 1 " -K) , 4.25 (IH, 5""-H), 4.24 (IH, l'-H), 4.15, 4.09 (2H, 6"-H), 3.86 (IH, 6"-H), 3.84 (IH, 5"-H), 3.79 (IH, 2"-H), 3.72 (IH, 6'-H), 3.63 (IH, 3'-H), 3.50 (IH, 3-H), 3.49 (IH, 5""-H), 3.46 (IH, 4"-H)_. 3.26 (IH, 5"-H), 2.04 (2H,OH). ¹³C NMR (150.8 MHz, CDC1₃) 103.9 (IC, l'-C), 100.3 (IC, l^{^}'-C), 98.9 (IC, l'"-C), 87.1 (IC, 3'-C), 84.4 (IC, 3-C), 75.3 (IC, 2'-C), 74.7 (IC, 5"-C), 71.2 (2C, 2'",3'"-C), 70.7 (IC, 3'"-C), 70.4 (IC, 5"-C), 69.9 (IC, 2',2"-C), 69.0 (IC, 4"'-C), 67.1. (IC, 4"-C), 63.0. (IC, 6"_rC), 62.3. (IC, 5"'-C), 60.9 (IC, 6"-C). Anal. Calcd for C_SιH₉₂0₂₂ : C, 62.23; H, 7.88. Found: C, 61.83; H, 8.21. 2.8. Friedelinolyl 3-β-O- (β-D-xylopyranosyl) - (1→3) - [ (β- D-galactopyranosyl) - (1—>2) ] -β-D-glucopyranoside XXXII

Alcaline hydrolysis of XXXI with NaOMe in methanol yielded the deprotected XXXII according to Scheme II. MALDI- MS: m/z 909 [M+Na]⁺. ^XH NMR (600 MHz, CDC1₃ + d₄-MeOH) 4.60 (IH, 1"-H), 4.48 (IH, l'-H), 4.44 (IH, 1"'-H), 3.90 (IH, 5"'-H), 3.87 (IH, 4"-H), 3.82 (IH, 6"-H), 3.74 (IH, 6"- H) , 3.65 (IH, 6'-H), 3.64 (IH, 3-H), 3.58 (IH, 2 -H), 3.54 (IH, 3'-H), 3.52 (IH, 4"'-H), 3.50 (IH, 2"-H), 3.47 (IH, 5"-H), 3.44 (IH, 3"-H), 3.37 (IH, 4'-H), 3.29 (IH, 3""-H), 3.26 (IH, 2""-H), 3.23 (IH, 5'-H), 3.21 (IH, 5"'-H). ¹³C NMR (150.8 MHz, CDC1₃) 104.4 (IC, l'"-C), 103.9 (IC, 1"-C), 100.9 (IC, l'-C), 86.2 (IC, l'-C), 80.3 (IC, 2'-C), 79.7 (IC, 3-C), 77.3 (IC, 3"'-C), 76.1 (IC, 5'-C), 75.3 (IC, 5"-C), 73.8 (IC, 2"'-C), 73.5 (IC, 3"-C), 72.5 (IC, 2"-C), 69.7 (IC, 4 -C) , 69.0 (IC, 4'-C), 66.1 (IC, 5"^"*-C), 62.1 (IC, 6'-C) , 61.2 (IC, 6"-C) . Anal. Calcd for C₄₇H₇₈0₁₅ ^■ 6H₂0 : C, 56.95; H, 9.15. Found: C, 57.14; H, 8.83. 2.9. 3-β-O-Friedelinolyl (2 ' " , 3 ' ' ' ,4" ^• -tri-O-acetyl-β-D- xylopyranosyl) - (l-3) - [ (2 ' ' , 3 ' ' ,4' ' , 6' ' -tetra-O-acetyl-β-D- galactopyranosyl) - (1→2) ] -6-β-D-glucuronic acid XXXIII

To a mixture of compound XXXI (14 mg, 0.012 mmol), NaBr (0.20 mg, 0.002 mmol), TBABr (0.21 mg, 0.0006 mmol) and TEMPO (0.16 mg, 0.001 mmol) in CH₂C1₂/H₂0 6:1 (0.25 mL) cooled to 0°C was added a mixture of NaOCl (0.05 mL) , H₂0 (0.04 mL) , NaHC0₃ (0.07 mL) . After stirring for 20 min was added MeOH (0.08 mL) . The compound was extracted in CH₂C1₂ and the solvent evaporated to give a residue, which was purified by flash chromatography (CH₂Cl₂/MeOH, 9.5:0.5) to afford XXXIII (11 mg, 76%) . MALDI-MS: m/z 1218 [M+Na]⁺. ^XH NMR (600 MHz, d₄- MeOH) 5.30 (IH, 4"-H), 5.09 (IH, 3 " -H) , 5.04 (2H, 2", 3"'-H), 4.99 (IH, 2"'-H), 4.92 (IH, l'"-H), 4.87 (IH, 4'''-H), 4.83 (IH, 1"-H), 4.34 (IH, 5"'-H), 4.22 (IH, I ' ll) , A . 6 (m, IH, 6"-H), 3.87 (IH, 5"-H), 3.82 (IH, 2 ' -H) , 3.74 (IH, 4'-H), 3.67 (2H, 3',5'-H), 3.46 (IH, 3-H). 3.43 (IH, 5"'-H). ¹³C NMR (150.8 MHz, CDC1₃) 103.9 (IC, l'-C), 98.5 (IC, 1"-C), 98.4 (IC, 1"'-C), 84.6 (IC, 3-C), 83.2 (IC, 3'-C), 75.7 (IC, 2'-C), 73.9 (IC, 5'-C), 70.4 (IC, 3"- C) , 70.3 (IC, 5"-C), 69.9 (3C, 2 " , 2 " ' , 3 " ' -C) , 69.8 (IC, 4'-C), 68.0 (IC, 4'''-C), 67.2 (IC, 4"-C), 60.7 (2C, . 5"',6"-C) . Anal. Calcd for C₆₁H₉₀O₂₃ C, 61.50; H, 7.61. Found: C, 63.21; H, 7.71.

2.10. Friedelinolyl 3-β-O- (β-D-xylopyranosyl) - (l->3) - [ (β-D-galactopyranosyl) - (l->2) ] -6-β-D-glucuronic acid XXXIV

Compound XXXIII (11 mg, 0.009 mmol) in MeOH (5 mL) and NaOMe (few drops of IN sodium solution in MeOH) was stirred for 12 h at room temperature. Quenched by Amberlyte addition, filtered off and the solvent evaporated. Purification by column chromatography (CH₂Cl₂/MeOH 2:1, 1:1, 1:2) afforded the compound XXXIV (8 mg, q.q.). MALDI-MS: m/z 921 [M+Na]⁺. ¹H NMR (600 MHz, CDCl₃ + d₄-MeOH) 4.86 (IH, 1"-H) , 4.60 (IH, 1""-H), 4.38 (IH, l'-H), 3.93 (IH, 5"'-H), 3.88 (IH, 2'-H), 3.85 (IH, 4""-H), 3.72 (2H, 3',6""-H), 3.69 (IH, 6"-H), 3.74

(IH, 6"-H), 3.72 (IH, 2'-H), 3.60 (IH, 5 -H), 3.59 (IH, 4'- H) , 3.53 (IH, 4"'-H), 3.51 (IH, 2"-H), 3.50 (IH, 5"-H), 3.48 (IH, 3'"-H), 3.34 (IH, 3""'-H), 3.28 (IH, 2"'-H), 3.26

(IH, 5"'-H) . ¹³C NMR (150.8 MHz, CDC1₃) 105.1 (IC, l'-C) , 104.1 (IC, 1"'-C) , 103.1 (IC, 1"-C) , 86.2 (IC, 3'-C) , 84.4

(IC, 5'-C) , 78.1 (IC, 2'-C), 77.4 (IC, 3""-C) , 76.0 (IC, 5"-C), 74.5 (IC, 2"'-C) , 74.3 (IC, 3"-C) , 73.0 (IC, 2"- C) , 70.4 (IC, 4'-C) , 70.2 (IC, 4^{' •}C) , 69.4 (IC, 4"-C) , 66.6 (IC, 5""-C) , 61.4 (IC, 6"'-C)

EXAMPLE 3- GLYCYRRHET NIC ACID SAPONINS

3.1. Glycyrrhetinic acid 30-methyl ester 3-β-Q-(2^#-Q- acetyl-3' -O-benzyl-4' ,6' -O-benzylidene-β-D-glucopyranoside) XLIII

XLIII

Using the reaction conditions outlined in Scheme III, based in the imidate intermediate XLI, and the above referred standard TMSOTf glycosidation procedure, XLIII was obtained in 67% yield from glycyrrhetinic acid XLII. MALDI-MS: m/z 890 [M+Na]⁺. 906 [M+K]⁺. ^XH NMR (600 MHz, CDC1₃) 7.50-7.26 (m, 10H, 2xPh) , 5.66 (s, IH, 12-H) , 5.57 (s, IH, CHPh), 5.06 (t, J = 8.7 Hz, IH, 2'-H), 4.87, 4.68 (2d, J = 12.2 Hz, 4H, 2xOCH₂Ph) , 4.50 (d, J = 7.9 Hz, IH, l'-H), 4.33 (m, J = 5.0 Hz, IH, 6'-H), 3.83 (t, 10.3 Hz, IH, 6'-H), 3.77 (t, J = 9.3 Hz, IH, 4'-H), 3.70 (m, IH, 3'-H), 3.68 (s, 3H, 0CH₃) , 3.40 (m, IH, 5'-H), 3.09 (m, J = 5.3 Hz, IH, 3-H) , 2.78 (d, J = 13.6 Hz, IH, 1-H) , 2.30 (s, 3H, COCH₃) , 1.84-1.76 (m, 2H, 2- H) , 1.34, 1.14 (2s, 6H, 2xCH₃) , 1.12 (s, 6H, 2xCH₃) , 0.91, 0.80, 0.77 (3s, 9H, 3xCH₃) . ¹³C NMR (150.8 MHz, CDC1₃) 200.1 (IC, CHCO) , 176.9 (IC, COOMe) , 169.1 (IC, COMe) , 138.3, 137.2 (2C, ipso-Ph) , 129.0-126.0 (10C, Ph) , 103.6 (IC, l'-C) , 101.2 (IC, CHPh), 89.9 (IC, 3-C) , 81.5 (IC, 4'-C) , 78.5 (IC, 3'-C) , 73.9 (IC, OCH₂Ph) , 73.2 (IC, 2'-C) , 68.8 (IC, 6'-C) , 66.1 (IC, 5'-C), 51.8 (IC, OCH₃) , 39.0 (IC, 1-C) , 28.5, 28.3, 27.6 (3C, 3xCH₃) , 26.5 (IC, 2-C) , 23.3, 18.6 (2C, 2xCH₃) , 18.3 (3C, 2xCH₃, CH₂) . Anal. Calcd for C₅₃H₇₀O₁₀ : C, 73.41; H, 8.14. Found: C, 73.07; H, 8.28.

3.2. Glycyrrhetinic acid 30-methyl ester 3-β-Q-(3^,-Q- benzyl-4' ,6' -O-benzylidene-β-D-glucopyranoside) XLIV

Standard hydrolysis procedure of XLIII with NaOMe/MeOH according to Scheme III yielded XLIV. MALDI-MS: m/z 849 [M+Na]⁺, 866 [M+K]⁺. ^XH NMR (600 MHz, CDC1₃) 7.50-7.26 (m, 10H, 2xPh) , 5.66 (s, IH, 12-H) , 5.57 (s, IH, CHPh) , 4.96, 4.80 (2d, J = 11.7 Hz, 4H, 2xOCH₂Ph) , 4.46 (d, J = 7.3 Hz, IH, l'-H), 4.32 (m, J = 4.9 Hz, IH, 6'-H) , 3.81 (t, 10.3 Hz, IH, 6'-H), 3.71 (m, IH, 4'-H) , 3.68 (s, 3H, OCH₃) , 3.66 (m, IH, 3'-H) , 3.63 (m, IH, 2 ' -H) , 3.43 (m, IH, 5'-H) , 3.20 (m, J = 7.6 Hz, IH, 3-H) , 2.80 (d, J = 13.6 Hz, IH, 1-H) , 1.35, 1.15 (2s, 6H, 2xCH₃) , 1.12 (s, 6H, 2xCH₃) , 1.05, 0.86, 0.81 (3s, 9H, 3xCH₃) . ¹³C NMR (150.8 MHz, CDC1₃) 200.1 (IC, CHCO) , 176.9 (IC, COOMe) , 169.1 (IC, COMe), 138.5 (2C, ipso-Ph), 128.9-126.0 (10C, Ph) , 105.4 (IC, l'-C) , 101.2 (IC, CHPh) , 89.8 (IC, 3-C) , 81.3 (IC, 4'-C) , 80.5 (IC, 3'-C) , 75.0 (IC, 2'-C), 74.6 (IC, OCH₂Ph) , 68.8 (IC, 6'-C) , 66.4 (IC, 5'-C) , 51.8 (IC, OCH₃) . Anal. Calcd for C₅ιH₆₈0₉ : C, 74.24; H, 8.31. Found: C, 73.91; H, 8.40.

3.3. Glycyrrhetinic acid 30 -methyl ester 3-β-O- (2' ' ,3' ' ,4' ' ,6' '-tetra-O-acetyl-β-D-galactopyranosyl) - (l->2) - 3' -Q-benzyl-4^/ , 6' -O-benzylidene-β-D-glucopyranoside XLV

Using the same conditions as described above in § 2.3 (but using the inverse glycosilation procedure) by replacing XXIII for XLIV and the same imidate intermediate XXIV, the disaccharide glycoside XLV was obtained (Scheme III) in 85% yield. MALDI-MS: m/z 1180 [M+NaJ⁺. ^XH NMR (600 MHz, CDC1₃) 7.47-7.31 (m, 10H, 2xPh) , 5.66 (s, IH, 12-H) , 5.55 (s, IH, CHPh), 5.32 (d, J = 2.9 Hz, IH, 4"-H), 5.18 (m, IH, " -H) , 5.08 (d, J = 8.0 Hz, IH, 1"-H) , 4.95 (d, J = 3.5 Hz, IH, 3"-H), 4.93, 4.65 (2d, J = 10.5 Hz, 2H, 2xOCH₂Ph) , 4.46 (d, J = 7.6 Hz, IH, l'-H) , 4.32 (m, J = 5.0 Hz, IH, 6'-H) , 4.10- 4.04 (m, IH, 6"-H) , 3.86 (t, J = 8.0 Hz, IH, 2 ' -H) , 3.79 (m, IH, 6'-H) , 3.78 (m, IH, 5"-H) , 3.70 (m, IH, 4'-H), 3.69 (s, 3H, OMe) , 3.40 (m, IH, 5'-H) , 3.13 (m, J = 5.5 Hz, IH, 3-H) , 2.78 (IH, J = 13.5 Hz, 1-H) , 2.13, 2.06, 2.01, 1.98 (4s, 12H, COMe) . ¹³C NMR (150.8 MHz, CDC1₃) 200.1 (IC, CHCO), 176.9 (IC, COOMe) , 170.3-169.1 (4C, COMe) , 137.9, 137.2 (2C, ipso-Ph) , 129.0-126.0 (10C, Ph) , 104.4 (IC, l'-C) , 101.1 (IC, 1"-C) , 101.2 (IC, CHPh) , 90.6 (IC, 3-C) , 82.8 (IC, 3'-C), 81.5 (IC, 4'-C) , 77.2 (IC, 2'-C) , 75.4 (IC, CH₂Ph) , 71.1 (IC, 3"-C) , 70.3 (IC, 5"-C) , 69.6 (IC, 2"-C), 68.8 (IC, 6'-C) , 67.3 (IC, 4"-C), 65.9 (IC, 5'-C), 61.1 (IC, 6' ' -C) , 51.8 (1C,0CH₃) . Anal. Calcd for CssHsoOis : C, 65.40; H, 7.57. Found: C, 65.52; H, 7.77.

3.4. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2, 3,4, 6- tetra-O-acetyl-β-D-galactopyranosyl) - (1— >2) -β-D- glucopyranoside XLVI

To a solution of saponin XLV (119 mg, 0.103 mmol) in MeOH/CH₂CL₂ (8.8. mL, 2:1) was added Pd/C (68 mg) . The mixture was stirred at room temperature under hydrogen atmosphere for 2 h, filtered off and concentrated in vacuo. The resulting residue was purified by flash chromatography (petroleum ether) to afford XLVI (101 mg, quantitative yield) . MALDI-MS: m/z 1000 [M+Na]⁺, 1016 [M+K]⁺. ^XH NMR (600 MHz, CDC1₃) 5.67

(s, IH, 12-H) , 5.37 (d, J = 2.7 Hz, IH, 4"-H), 5.23 (m, IH, 2"-H), 5.01 (m, IH, 3 ' ' -H) , 4.99 (d, J = 7.9 Hz, IH, 1"-H), 4.45 (d, J = 5.6 Hz, IH, l'-H), 4.13 ( , IH, 6"-H), 3.92 (t, IH, 5"-H), 3.88-3.79 (m, IH, 6'-H), 3.69 (s, 3H, OMe) , 3.56

(m, 3H, 2',3',4'-H), 3.33 (m, IH, 5"-H) , 3.16 (m, IH, 3-H) . ¹³C NMR (150.8 MHz, CDCl₃) 103.8 (IC, l'-C), 100.7 (IC, 1"- C) , 89.7 (IC, 3-C), 80.2, 76.6, 70.5 (4C, 2 ' , 3 ' , 4 ' , 5 " -C) , 74.7 (IC, 5'-C) , 70.8 (IC, 3"-C) , 70.2 (1C,2"-C) , 66.7 (IC, 4"-C), 62.6 (IC, 6'-C) , 60.6 (IC, 6"-C) , 51.9 (1C,0CH₃) . Anal. Calcd for C₅ιH_7sO_X8: C, 62.69; H, 7.84. Found: C, 61.81; H, 8.10.

3.5. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2, 3,4, 6- tetra-O-acetyl-β-D-galactopyranosyl) - (l->2) -4, 6-Q- benzylidene- β-D-glucopyranoside XL VII

To a solution of saponin XLVI (80 mg, 0.082 mmol) in CH₃CN (5 mL) and C₆H₅CH(OMe)₂ (14 μL, 0.098 mmol) was added p- toluenosulfonic acid (catalytic quantity) . After stirring at room temp, for 1.5 h, the mixture was quenched by addition of Ξt₃N and concentrated in vacuo . The resulting residue was purified by flash chromatography (petroleum ether/ethyl acetate, 2:1) to afford XLVII (77 mg, 88%). MALDI-MS: m/z 1088 [M+Na]⁺, 1104 [M+K]⁺. ^Η NMR (600 MHz, CDC1₃) 7.49, 7.37 (m, 5H, Ph) , 5.66 (s, IH, 12-H) , 5.51 (s, IH, CHPh), 5.36 (d, J = 2.7 Hz, IH, 4"-H), 5.18 (m, IH, 2"-H), 5.03 (m, IH, 3"-H), 5.02 (d, J = 3.2 Hz, IH, 1"-H), 4.49 (d, J = 7.4 Hz, IH, l'-H), 4.31 (m, J = 5.0 Hz, IH, 6'-H), 4.12 (d, J = 6.7 Hz, IH, 6"-H), 3.90 (t, J = 6.7 Hz, IH, 5"-H), 3.81 (m, IH, 3'-H), 3.76 (m, IH, 6'-H), 3.75 (m, IH, 2 ' -H) , 3.69 (s, 3H, OMe) , 3.51 (t, J = 6.7 Hz, IH, 4'-H), 3.38 (m, IH, 5'-H), 3.15 (m, IH, 3-H), 2.78 (IH, J = 13.5 Hz, 1-H) , 2.13, 2.06, 2.04, 1.98 (4s, 12H, COMe). ¹³C NMR (150.8 MHz, CDC1₃) 200.2 (IC, CHCO), 176.9 (IC, COOMe) , 170.4, 170.1, 169.8, 169.2 (4C, COMe), 136.9, (IC, ipso-Ph), 129.4-126.2 (5C, Ph) , 104.1 (IC, l'-C), 102.0 (IC, CHPh), 100.8 (IC, 1"-C), 90.2 (IC, 3- C) , 80.5 (IC, 4'-C), 79.7 (IC, 2 ' -C) , 74.8 (IC, 3 ' -C) , 71.1 (IC, 3"-C), 70.5 (IC, 5"-C), 69.9 (IC, 2"-C), 68.9 (IC, 6'-C), 67.1 (IC, 4"-C), 65.8 (IC, 5'-C), 61.1 (IC, 6"-C), 51.8 (1C,0CH₃) . Anal. Calcd for C₅₈H₈₀Oι₈ : C, 65.40; H, 7.57. Found: C, 65.52; H, 7.77.

3.6. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2, 3,4- tri-O-acetyl-β-D-xylopyranosyl) - (1—>3) - [ (2,3,4, 6-tetra-O- acetyl-β-D-galactopyranosyl) - (1—>2) ] -4, 6-O-benzylidene-β-D- glucopyranoside XLIX

To a solution of saponin XLVIII (60 mg, 0.06 mmol) in dry CH₂C1₂ (1.7 mL) and the imidate XXIX (28 mg, 0.07 mmol) was added a solution of TMSOTf (0.65 μL, 0.003 mmol) in CH₂C1₂ (0.5 mL) . The mixture was stirred for 10 min at room temp, and then quenched by addition of Et₃N and the solvent evaporated, Purification of resulting residue by flash chromatography (petroleum ether/ethyl acetate, 2:1, 1:1) gave XLIX (68 mg, 86%). MALDI-MS: m/z 1346 [M+Na]⁺. ¹H NMR (600 MHz, CDC1₃) 7.44, 7.35 (m, 5H, Ph) , 5.66 (s, IH, 12-H), 5.46

(s, IH, CHPh), 5.35 (d, J = 3.1 Hz, IH, 4"-H), 5.25 (dd, J = 3.5 Hz, J = 10.4 Hz, IH, 3''-H), 5.09 (m, IH, 2"-H), 4.97

(m, 2H, 2"',3"'-H), 4.95 (m, IH, 1"'-H), 4.93 (m, IH, 1"- H) , 4.78 (m, IH, " ' -H) , 4.41 (d, J = 7.1 Hz, IH, l'-H), 4.31 (m, J = 4.9 Hz, IH, 6'-H), 4.18 (dd, J = 3.8 Hz, J = 12.3, IH, 5"'-H), 4.12 (d, J = 6.9 Hz, IH, 6"-H), 3.97 (m, IH, 3'-H), 3.96 (m, IH, 5"-H), 3.95 (m, IH, 2'-H), 3.75 (m, IH, 6'-H), 3.67 (s, 3H, OMe) , 3.63 (t, J = 9.1 Hz, IH, 4'-H), 3.38 (m, IH, 5'-H), 3.13 (m, IH, 5"'-H), 3.09 (m, IH, 3-H) , 2.77 (IH, J = 13.6 Hz, 1-H) , 2.13, 2.08, 2.04, 2.00, 1.95 (5s, 12H, COMe). ¹³C NMR (150.8 MHz, CDC1₃) 200.2 (IC, CHCO), 176.9 (IC, COOMe), 170.4-169.1 (7C, COMe), 137.1, (IC, ipso- Ph), 129.3-126.0 (5C, Ph) , 104.2 (IC, l'-C), 101.8 (IC, CHPh), 99.0 (IC, 1"-C), 97.9 (IC, 1"'-C), 90.8 (IC, 3-C), 79.4 (IC, 4'-C), 78.6 (IC, 3'-C), 77.6 (IC, 2'-C), 70.5 (IC, 5''-C), 70.4 (IC, 3''-C), 70.2 (IC, 2"-C), 69.5 (2C, 3"',2'"-C), 69.1 (IC, 6'-C), 67.5 (IC, 4"-C), 67.4 (IC, 4"'-C), 65.8 (IC, 5'-C), 61.0 (IC, 6"-C), 60.1 (IC, 5'"- C) , 51.8 (IC, OCH₃) . Anal. Calcd for C_S9H₉₄0₂₅ : C, 62.62; H, 7.16. Found: C, 62.17; H, 7.22.

3.7. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2 ',3 ,4¹ -tri-Q-acetyl-β-D-xylopyranosyl)- (l-3) ■ [ (2^X * , 3 * * , 4 ^τ , 6 * -tetra-Q-acetyl-β-D-galactopyranosyl) (l-»2) ] -β-D-glucopyranoside L

Benzylidene deprotection of XLIX with EtSH in dichloromethane in the presence of p-TsOH yielded L in 83% yield (Scheme III). MALDI-MS: m/z 1258 [M+Na]⁺, 1274 [M+K]⁺. ^XH NMR (600 MHz, CDCl₃) 5.66 (s, IH, 12-H), 5.35 (d, J = 2.6 Hz, IH, 4"-H) , 5.16 (m, IH, 3 " ' -H) , 5.15 (m, IH, 2''-H) , 5.07 (m, IH, 2"'-H) , 5.06 (m, IH, 3''-H) , 4.99 (m, IH, 4'"- H) , 4.84 (m, IH, l'"-H) , 4.82 (m, IH, 1"-H) , 4.37 (d, J = 7.7 Hz, IH, I'-H) , 4.26 (dd, J = 4.7 Hz, J = 12.2, IH, 5'"- H) , 4.12 (m, IH, 6"-H), 3.88 (m, IH, 6'-H), 3.85 (m, IH, 5"-H), 3.79 (t, J = 8.5 Hz, IH, 2 ' -H) , 3.74 (m, IH, 6'-H) , 3.68 (s, 3H, OMe) , 3.64 (m, IH, 3 ' -H) , 3.48 ( , 2H, 5'"-H, 4'-H) , 3.30 (m, IH, 5'-H) , 3.09 (dd, J = 4.6 Hz, J = 11.6 Hz, IH, 3-H) , 2.14, 2.10, 2.05, 2.04, 1.97 (5s, 12H, COMe) . ¹³C NMR (150.8 MHz, CDC1₃) 200.1 (IC, CHCO) , 176.9 (IC, COOMe) , 170.3-169.0 (7C, COMe) , 103.5 (IC, l'-C) , 100.0 (IC, 1"'-C) , 99.3 (IC, 1"-C), 90.5 (IC, 3-C), 86.0 (IC, 3'-C) , 76.0 (IC, 2'-C), 74.7 (IC, 5'-C), 70.9 (2C, 3"-C, 2"'-C), 70.5 (IC, 5"-C) , 70.6 (IC, 3"-C) , 69.8 (IC, 2"-C) , 69.6 (IC, 4'-C), 68.6 (IC, 4"'-C) , 67.0 (IC, 4"-C) , 62.8 (IC, 6'-C) , 62.1 (IC, 5"'-C) , 60.8 (IC, 6"-C) , 51.8 (IC, OCH₃) . Anal. Calcd for C_s2H₉₀O_2Ξ: C, 60.27; H, 7.34. Found: C, 60.41; H, 7.38.

3.8. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2" ' ,3' " ,4" ' -tri-O-acetyl-β-D-xylopyranosyl) - (l-»3) - [ (2' ^• ,3' ' ,4' ' , 6' ' -tetra-O-acetyl-β-D-galactopyranosyl) (1→2)] -β-D-glucopyranoside LI

To a solution of saponin L (58 mg, 0.047 mmol) in MeOH (2.5 mL) was added a 1M solution of NaOMe in MeOH (0.4 mL) . The mixture was stirred at room temp, for 24 h, neutralized by addition of ion exchange resin (Amberlite IR-120, H⁺ form) and then filtered. The filtrate was concentrated in vacuo to afford LI (44 mg, q.q.). MALDI-MS: m/z 964 [M+Na]⁺. ^λE NMR (600 MHz, d4-MeOH) 5.56 (s, IH, 12-H), 4.84 (br, IH, 1"-H), 4.58 (br, IH, l'"-H), 4.45 (br, IH, l'-H), 3.90 (br, IH, 5"'-H), 3.71 (br, IH, 2 ' -H) , 3.68 (s, 3H, OMe) , 3.51 (br, IH, 4"'-H), 3.25 (br, IH, 5"'-H), 3.24 (br, IH, 2 " ' -H) , 3.19 (m, IH, 3-H) . ¹³C NMR (150.8 MHz, CDC1₃) 202.6 (IC, CHCO), 178.6 (IC, COOMe), 105.8 (IC, 1"-C), 104.9 (IC, _. ' ^{• •} - C) , 104.9 (IC, 1"-C), 90.8 (IC, 3-C), 87.0 (IC, 3'-C), 78.6 (IC, 2'-C), 74.6 (IC, 2"'-C), 72.8 (IC, 2"-C), 66.3 (IC, 5"'-C). Anal. Calcd for C₄₇H₇₄Oι₈ ^• 5H₂0 : C, 61.26; H, 8.14. Found: C, 55.68; H, 7.60.

3.9. Glycyrrhetinic acid 3-β-O- (β-D-xylopyranosyl) - (l-»3) - [ (O-β-D-galactopyranosyl) - (1—>2) ] -β-D-glucopyranoside LII

Hydrolysis of methyl ester group of LI using LiOH 3% in MeOH (Scheme III) yielded LII in 54% yield. MALDI-MS: m/z 950 [M+Na]⁺. ^XH NMR (600 MHz, d₄-MeOH) 5.57 (s, IH, 12-H), 4.85 (IH, 1"-H), 4.58 (IH, 1"'-H), 4.46 (IH, l'-H), 3.91 (IH, 5"'-H), 3.84 (IH, 6'-H), 3.80 (IH, 4 "H) , 3.75 (IH, 6"-H), 3.71 (IH, 2'-H), 3.66 (IH, 3'-H), 3.65 (IH, 6'-H) , 3.62 (IH, 6 " -E) , 3.51 (IH, 4"'-H), 3.48 (IH, 2"-H), 3.45 (IH, 5"- H) , 3.44 (IH, 3"-H), 3.33 (IH, 4'-H), 3.30 (IH, 3"'-H), 3.27 (IH, 5'-H) , 3.25 (IH, 5""-H), 3.24 (IH, 2"'-H), 3.21 (IH, 3-H) . ¹³C NMR (150.8 MHz, CDC1₃) 105.2 (IC, l'-C), 104.7 (IC, 1"'-C), 103.8 (IC, 1"-C), 91.5 (IC, 3-C), 87.6 (IC, 3'-C), 78.9 (IC, 2'-C), 77.8 (IC, 3"'-C), 77.3 (IC, 5'-C), 76.8 (IC, 5"-C), 75.0 (IC, 2"'-C), 74.8 (IC, 3"-C), 73.3 (IC, 2"-C), 70.8 (IC, 4'"-C), 70.0 (IC, 4"-C), 66.9 (IC, 5"'-C), 62.4 (2C, 6',6"-C). Anal. Calcd for C₄₇H₇₄0₁₈ • 7H₂0 : C, 53.60; H, 8.42. Found: C, 53.42; H, 8.29.

3.10. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2 ' ' ^• , 3 ' ' ' , 4 ' ' ' -tri-O-acetyl-β-D-xylopyranosyl) - (l->3) - [(2' ' , 3 ' ' ,4 ' , 6 ' '-tetra-O-acetyl-β-D-galactopyranosyl) - (l->2) ] -β-D-glucopyranoside LII

To a mixture of compound L (53 mg, 0.043 mmol), NaBr

(0.75 mg, 7.5 mol), TBABr (0.75 mg, 2.34 mol) and TEMPO (0.60 mg, 3.86 μmol) in CH₂C1₂/H₂0 6:1 (0.94 mL) cooled to 0°C was added a mixture of NaOCl (0.18 mL) , H₂0 (0.15 mL) , NaHC0₃

(0.26 mg) . After stirring for 15 min was added MeOH. The compound was extracted in CH₂C1₂ and the solvent evaporated to give a residue, which was purified by flash chromatography

(CH₂Cl₂/MeOH, 9.5:0.5) to afford LIII (40 mg, 75%). MALDI-MS: m/z 1272 [M+Na]⁺, 1288 [M+K] ⁺ . ^Η NMR (600 MHz, d4-MeOH) 5.57

(s, IH, 12-H), 5.35 (s, IH, 4''-H), 5.17 (m, IH, 3"-H), 5.13

(m, IH, 2"-H), 5.08 (m, IH, 1"'-H), 5.05 (m, IH, 3 " ' -H) ,

5.01 (m, IH, 2"'-H), 4.94 (d, J = 7.5 Hz, IH, 1"-H), 4.88

(m, IH, 4"'-H), 4.45 (m, IH, 1 ' -H) , 4.49 (m, IH, 5'"-H),

4.16, 4.11 (m, IH, 6"-H), 4.02 (m, IH, 5"-H), 3.80 (m, IH,

3'-H), 3.78 (m, IH, 2'-H), 3.69 (s, 3H, OMe) , 3.68 (m, IH,

5'-H), 3.63 (m, IH, 4'-H), 3.69 (s, 3H, OMe), 3.51 (m, IH,

5'''-H) , 3.14 (m, IH, 3-H) . ¹³C NMR (150.8 MHz, CDC1₃) 202.4

(IC, CHCO) , 178.6 (IC, COOMe) , 172.3-171.0 (7C, COMe) , 104.5

(IC, l'-C), 100.2 (IC, 1"-C) , 99.4 (IC, 1"'-C) , 91.1 (IC,

3-C), 83.0 (IC, 3'-C), 78.4 (IC, 2'-C) , 76.1 (IC, 5'-C), 71.8

(IC, 3''-C) , 71.5 (2C, 4'-C,5''-C), 71.1 (IC, 2"-C) , 70.9

(IC, 3'''-C) , 70.7 (IC, 2"'-C) , 69.0 (IC, 4"'-C) , 68.4 (IC,

4"-C) , 62.0 (IC, 6"-C) , 61.4 (IC, 5" '-C), 52.1 (IC, 0CH₃) .

Anal. Calcd for C₆₂H₈₈0₂₆ ^• 1H₂0 : C, 58.75; H, 7.16. Found: C,

58.73; H, 7.29. 3.11. Glycyrrhetinic acid 30-methyl ester 3-β-O- (2, 3,4- tri-O-acetyl-β-D-xylopyranosyl) - (l-»3) - [ (2,3,4,6-tetra-O- acetyl-β-D-galactopyranosyl) - (1—>2) ] -β-D-glucopyranoside LIV

To a solution of saponin LIII (24 mg, 0.019 mmol) in MeOH (1.0 mL) was added a IM solution of NaOMe in MeOH (0.2 mL) . The mixture was stirred at room temp, for 16 h, neutralized by addition of ion exchange resin (Amberlite IR- 120, H⁺ form) and then filtered. The filtrate was concentrated in vacuo to afford LIV (18 mg, q.q.). MALDI-MS: m/z 978 [M+Na]⁺, 993 [M+K]⁺. ^XH NMR (600 MHz, d4-MeOH) 5.56

(s, IH, 12-H), 4.84 (d, IH, l''-H), 4.62 (d, J = 7.6 Hz, IH, 1"'-H), 4.49 (d, J = 7.5 Hz, IH, l'-H), 3.92 (m, IH, 5"'- H) , 3.81 (m, IH, 4"-H), 3.77 (m, IH, 2'-H), 3.74 (m, IH, 6"-H), 3.72 (m, H, 3'-H), 3.69 (m, IH, 5'-H), 3.68 (s, 3H, OMe), 3.63 (m, IH, 6"-H), 3.58 (m, IH, 4'-H), 3.51 (m, IH, 4"'-H), 3.50 (m, IH, 2''-H), 3.46 (m, IH, 5"-H), 3.45 (m, IH, 3"-H), 3.31 (m, IH, 3"'-H), 3.26 (m, IH, 2"'-H), 3.19

(m, IH, 3-H) . ¹³C NMR (150.8 MHz, CDC1₃) 202.6 (IC, CHCO),

178.7 (IC, COOMe) , 105.3 (IC, l'-C) , 104.6 (IC, l'''-C),

103.8 (IC, 1"-C), 91.3 (IC, 3-C) , 86.6 (IC, 3'-C) , 78.9 (IC, 2'-C) , 77.9 (IC, 3"'-C) , 76.9 (IC, 5'-C) , 76.8 (IC, 5' '-C), 74.9 (IC, 3",2'"-C) , 73.3 (IC, 2"-C) , 71.5 (IC, 4'-C), 70.7 (IC, 4"'-C) , 70.0 (IC, 4''-C) , 66.9 (IC, 5"'-C) , 62.4 (IC, 6"-C) , 52.1 (IC, O.CH₃) . Anal. Calcd for C₄₈H₇₄0₁₉ ^• 5H₂0 : C, 55.16; H, 8.10. Found: C, 55.00; H, 8.50.

EXAMPLE 4- CHOLESTANOL SAPONINS

4.1. Cholestenyl 3-β-O- (β-D-xylopyranosyl) - (l->3) - [(β-D- galactopyranosyl) - (l-»2) ] -6-β-D-glucuronic acid LXIV

The compound LXIII was used as starting material to prepare LXIV, being intermediate LXIII made in the same way as that described in Example 3 (3.1 to 3.8) and the compound LXIII here used instead of LI in above referred Example 3.9.

To a mixture of compound LXIII (Scheme IV) (39 mg, 0.034 mmol), NaBr (0.61 mg, 0.006 mmol), TBABr (0.61 mg, 0.002 mmol) and TEMPO (0.48 mg, 0.003 mmol) in CH₂C1₂/H₂0 6:1 (0.76 mL) cooled to 0 °C was added a mixture of NaOCl (0.15 mL) , H₂0 (0.12 mL) , NaHC0₃ (0.21 mL) . After stirring for 45 min. was added MeOH (0.2 mL) . The compound was extracted in CH₂C1₂ and the solvent evaporated to give a residue, which was purified by flash chromatography (CH₂Cl₂/MeOH, 9.5.-0.5) to afford LXIV (13 mg, 57% + 16 mg of recovered LXIII) . MALDI-MS: m/z 1177 [M+Na]⁺. ^XH NMR (600 MHz, CDC1₃ + d₄-MeOH) 5.32 (IH, 4"-H), 5.06 (3H, 2 " " , 3 " " , 3 ~-H) , 4.96 (IH, 1'"- H) , 4.88 (IH, 4'"-H), 4.89 (IH, 2""-H), 4.50 (IH, l'-H) , 4.31 (IH, 5"'-H), 4.12, 4.09 (2H, 6"-H), 3.91 (IH, 5"-H), 3.76 (2H, 4',5'-H), 3.60, 3.59 (2H, 2',3'-H), 3.58 (IH, 3-H), 3.39 (IH, 5-""-H). ¹³C NMR (150.8 MHz, CDCl₃) 99.5 (IC, l'-C), 99.0 (IC, 1"-C) , 98.5 (IC, l'"^*-C), 81.1 (2C,.4 " , 5'-C) , 79.2 (IC, 3-C), 74.2 (IC, 2'or 3"-C), 70.4 (IC, 5"-C) , 70.3 (4C, 3""',3-"^',2'-",2^'"-C) , 70.1 (IC, 3' or 2'-C), 68.1 (IC, 4'"^' "^'- C) , 66.8 (IC, 4"-C), 60.9 (2C, 5'",6"-C).

4.2. Cholestenyl 3-β-O- (β-p-xylopyranosyl) - (l->3) - [ (β-p- galactopyranosyl) - (l-»2)] -6-β-D-glucuronic acid LXV (T3T)

Deprotection of LXIV was carried out using NaOMe and MeOH (scheme IV). MALDI-MS: m/z 880 [M+Na] ⁺ . E NMR (600 MHz, CDCI₃ + d₄-MeOH) 4.68 (IH, 1"-H) , 4.62 (IH, 1""-H) , 4.54 (IH, X-E) , 3.91 (IH, 5""-H), 3.88 (IH, 4""-H), 3.72 (2H, δ'-'-H), 3.70 (IH, 3-H), 3.69 (IH, 2",3'-H), 3.61 (IH, 5'-H), 3.54, 3.53, 3.52, 3.51, 3.50 (5H, 4", 2 ", 3 " , 3 " ", 4 " " '-H) ,

3.35 (IH, 3""-H), 3.28 (2H, 2 -H) , 3.24 (IH, 5^' -H) 13.

NMR (150.8 MHz, CDCl₃) 103.8 (IC, 1"'-C), 103.6 (IC, 1"-C), 100.1 (IC, l'-C), 84.1 (IC, 3-C), 79.9 (2C, 2",3"-C), 76.6 (IC, 3"'-C), 76.4 (IC, 5'-C) , 76.8 (IC, 5"-C), 73.8 (IC, 2"'-C), 73.6, 72.5, 71.0, 69.9 (4C, 4 " , " , 3 ' " , 4 " "-C) , 69.1 (IC, 4"-C), 66.0 (IC, 5"-C), 61.3 (IH, 6"-H). Anal. Calcd for C₄₄H₇₀Oι₆: C, 54.87; H, 8.58. Found: C, 54.74; H, 8.66.

4.3. Cholesteryl 3-β-O- (β-D-xylopyranosyl) - (l->3) - [ (β-D- galactopyranosyl) - (l->2) ] -β-D-glucopyranoside LXVI

Deprotection of LXIII with NaOMe in MeOH yielded LXVI (Scheme IN). MALDI-MS: m/z 869 [M+Νa]⁺. 1H NMR (600 MHz, CDC1₃ + d₄-MeOH) 4.77 (IH, 1 "-H), 4.43 (IH, 1"'-H), 4.27 (IH, l '-H), 3.89 (IH, 5"'-H), 3.81 (IH, 2'-H), 3.79 (IH, 6'-H), 3.76 (IH, 2'-H), 3.73 (IH, 6"-H), 3.65 (IH, 6'-H), 3.64 (IH, 2"-H), 3.54 (IH, 3,3 '-H), 3.51 (IH, 4"'-H), 3.46 (IH, 2"-H), 3.43 (IH, 5"-H), 3.39 (IH, 5"-H), 3.36 (IH, 4'-H), 3.27, 3.26 (2H, 2'", 3"'-H), 3.20 (IH, 5"'-H), 3.19 (IH, 5'-H). ¹³C NMR (150.8 MHz, CDC1₃) 104.1 (IC, l '-C), 103.4 (IC, 1 "'-C), 102.0 (IC, 1"-C), 86.8, 83,3 (2C, 3, 3'-C), 77.1 (IC, 2'-C), 76.7 (IC, 2'" or 3"'-C), 75.1 (IC, 5'-C), 74.5 (IC, 5"-C), 73.2 (IC, 3"-C), 73.1 (IC, 2'" or 3"'-C), 68.8 (2C, 4',4'"-C), 68.5 (IC, 4"-C), 65.3 (IC, 5"'-C), 62.1 ( IC, 6 '-C), 6 1.0 ( 1C, 6 "-C). Anal. Calcd for C₄₄H₇₄Oι₅-5H₂0: C, 57.75; H, 9.03. Found: C, 57.54; H, 8.87. II. Tests of biological activity

The compositions of the present invention have proved to be useful as immunological adjuvants to generate immune active response in mammals and preferably humans.

The immunological adjuvants are substances that when administered to an individual or tested in vi tro allow to observe an increment in the immune response to an antigen or an increase in the activity of the cells that belong to the immune system. The function of the immunological adjuvants is to increase the immune response in the presence of an antigen, giving to the antigen more immunogenicity and/or a smaller amount of necessary antigen to generate the appropriate response. This function is fundamental because some antigens have little immunogenicity or still because at the concentrations that generate the appropriate immune response they are toxic.

The in vi tro immunoadjuvant activity of the compounds of the present invention can be evaluated by well-known methods (Behboudi, S., Morein, B. and Villacres-Eriksson; Clin . Exp. Immunol . , 1996, 105: 26). The secretion of cell mediators in the culture medium after stimulation of antigen presenting cells with the molecules of the invention can be used to assess their adjuvant activity. The method needs incubation of the adjuvant, in the antigen absence, with the cells for 24 hours, after what the supernatant is removed for the quantification of the pro-inflammatory cell mediators, mainly the cytokines, IL-6 and IL-1 (interleukine) , TNF-α (tumour necrosis factor) and nitrous oxide.

The in vivo adjuvant activity can be evaluated equally by well-known methods (Cleland, J.L, Kensil, C.R.; Lim, N. ; Jacobsen, N.E.; Basa, L. ; Spellman, M; Wheeler, D.S.; Wu, J.Y. and Powell, M.F., J. Pharm. Sci . , 1996, 85: 22). The titles of antibodies can be assessed in the serum of the Balb/c mice immunized with subcutaneously administered ovoalbumine together with the hemi-synthesis molecules of the present invention. The increments of the titles can be used to evaluate the immunoadjuvant activity of the saponins of the present invention.

Evaluation in vitro of the immunoadjuvant activity

The saponins of general formula I of the present invention can be used as the sole adjuvant or together with other adjuvants like, for example, oil in water emulsions, liposomes and aluminium salts; or immunomodulators like MPL (monophosphoryl lipid A) , pure substances, QS-21, or pure fractions of the bark extract of Quillaja saponaria Molina, and cytokines like IL-2, IL-12 and GM-CSF.

The saponins of the present invention have immunoadjuvant activity in a wide interval of concentrations. The new compounds of the present invention can be administered by parentherical route (endovenous, endoarterial, subcutaneous, intramuscular, intracutaneous , intraperitoneal), orally or nasal route. For the oral administration of the molecules of the present invention they can be included in solid or liquid forms (solutions, emulsions, suspensions) . For the parenthetical administration, they will be in sterile liquid forms as solutions, emulsions or suspensions, being the carrier an inert material, like the saline phosphate buffer for example.

The administration of the saponins of the invention with the antigen or together with another adjuvant or immunomodulador, may need one or more successive administrations at appropriate intervals. The dose to be administered will depend on the weight and the individual ' s age to be immunized.

The amount and relationship between the adjuvant of the present invention and the antigen will depend on the nature and antigen type. The controls used were several purified bark extracts of Quillaja saponaria M. and LPS, liposaccharide from E. coli,

The QS-21 sample used as reference (SmithKline Beecham Biolog BE) , purified fraction of the bark extract of the Quillaja saponaria M. tree, presents a great cytotoxic effect when evaluated in the cells of the line J-744A.1. The observed cytotoxicity is dependent on the concentration of the molecule in the medium when the concentrations are higher than 0.63 μg/mL. Besides, it should be underlined that at the concentrations of 20 and 40 μg/mL there is no cell survival, being possible to consider that at these concentrations the cytotoxicity is total .

The macrophages when stimulated, produce and release regulator molecules such as cytokines, chemokines and nitrous oxide. Among these releasable substances, the interleukine 6 (IL-6) , the tumour necrosis factor (TNF-α) and also the nitrous oxide (NO) were quantified. Besides the stimulation the cytotoxicity of the saponins that can be supplied in this invention was also evaluated.

The controls used were several purified extracts of bark of Quillaja saponaria and LPS, liposaccharide originated from

E. coli .

The cytotoxicity of molecules of this invention was evaluated. Tests of macrophage stimulation

The macrophages were stimulated by incubation of the substances in study, in well-known concentrations, the molecules segregated in the supernatant after 24 hours to 37°C and wet environment being quantified. The cell concentration used in all tests was 1 x 10⁶ cells/mL.

Example 1

Macrophages Stimulation for the test saponins

A immunoadjuvant potential activity can be evaluated in vitro using antigen presenting cells, namely macrophages and monocytes. These tests are based on the knowledge that an increase of the immune response is related to the secretion of the antigen presenting cells of pro-inflammatory cell mediators (Nathan, C.F., J. Clin . Invest . , 1987, 79: 319).

The stimulating activity of the antigen presenting cells was evaluated by incubation of growing concentrations of saponins provided by the process of this invention with antigen presenting cells of different origin, such as macrophages extracted from the mouse Balb/c peritoneum with buffer phosphate at pH 7.4 and cellular monocyte macrophages lines: the line J744A.1, coming from mice ancestry that respond to the action of the cellular wall of microorganisms Gram negative (lipopolysaccharide LPS) and the cellular line DMBM-2 coming from mice that do not respond to LPS.

For the accomplishment of the tests, the cells were incubated for 24 hours in a stove of C0₂ (5%) with temperature (37 °C) and moist controlled atmosphere. The complete culture means (glutamine, penicillin, streptomycin and fetal bovine serum, Gibco) were used for the quantification of the cell mediators segregated during the incubation, namely IL-6, TNF-α and nitrous oxide as nitride.

The quantification of TNF-α and IL-6 was performed by the reference technique known as "ELISA sandwich" using the reactants supplied in the MiniKit^® of Endogen. The nitrous oxide was quantified as nitride with the Griess-Illosvay method.

Table 1 demonstrates the stimulation in the production of 11-6 and NO in cells of the line DMBM-2 by one of the molecules of general Formula I provided by the present invention, the saponin T4T.

Table 1. Levels of IL-6 and N0₂ ^" in the supernatant of the DMBM-2 cell culture after incubation with T4T.

Concentration IL-6 N0₂ ^" (μM) of T4T (ng/mL) (average + (μg/mL) (average ± s.d.) s.d. )

10 36 . 5±5 . 0 3 . 84+0 . 2

20 23 .4±2 . 6 6 . 3+0 . 5

In figures 1 and 2 the obtained result is presented demonstrating the stimulation of the secretion of IL-6 in antigen presenting cells of different origin for two saponins T3T and T4T of general Formula I of the present invention. Besides, the levels of IL-6 detected in the supernatants were still more important. Figure 1 shows that T4T molecule induces the production of this interleukine at concentrations higher than 1.25 μg/mL. In the case of the peritoneal macrophages the stimulation is produced in all tested concentrations of the compounds of the present invention.

Example 2

Comparative cytotoxicity of adjuvant molecules

The main problem for the acceptance, on the part of the sanitary authorities, of the different immunological adjuvants, under study or under patent until today, is their toxicity. The tests of cytotoxicity of the new adjuvant of the present invention were performed in vi tro either on the cell lines mentioned in example 1 or on the peritoneal macrophages .

The cytotoxicity of the molecules of this invention here exemplified for the saponins T3T and T4T was evaluated by the MTT method (Mosman, T., J. Immunol . Method. , 1983, 65: 55), that allows to assess the cellular viability in function of the activity of the cell mitocondrial dehydrogenase. As reference substances the purified fractions, QH-A, QH-B, QH-C and Spikoside, of the extract of bark of Quillaja Saponaria Molina (supplied by Doctor K. Lovgren-Bengtson and Prof. Morein, Uppsalla) and the pure saponin, QS-21, also the extract of cork of Quillaja Saponaria Molina (supplied by SmithKline Beecham, Rixensat, Belgium) were used. These fractions of marketed natural saponins presents recognized immunoadjuvant and immunomodulating activity, but their application is limited by their toxicity both in vi tro and in vivo.

After the incubation of the cells with the new synthetic saponins or the control substances for 24 hours, as mentioned in example 1, the MTT solution is added in buffer saline phosphate at pH 7.4 and the incubation process continues for 4 hours more . To read the colour developed in a spectrophotometer UV/VIS, a solution is added to the 11% of SDS in HC1 0. OlN/isopropanol (1:1).

The results of MTT indicate that the T3T and T4T saponins inhibited the synthesis of the dehydrogenase to concentrations higher than 5 mcg/mL (Figure 3 and Figure 4) in the peritoneal macrophages . On the contrary the molecules used as reference, the purified fractions, QH-B, QH-C and Spikoside, and the pure saponin, QS-21, of Quillaja saponaria showed to be cytotoxic in the cell line J744A.1 at concentrations higher than 1.25 mcg/mL (Figure 4 and Figure 5) . These facts demonstrate that in particular the saponin T4T of general formula I of this invention inhibits the dehydrogenase at much higher concentrations and therefore it is less cytotoxic than the control molecules.

Biological tests in vivo

A well established model was used to determine if the Formulations of a new immunostimulating saponin together with aluminium hydroxide could work as optimised immunological adjuvant. Briefly, experiences to compare these Formulations of the saponin were performed and the reference immunostimulating saponin QS-21 was used separately. The new immunostimulating saponin that was selected to serve as an adjuvant was saponin of β-amyrin (T4T) and cholestanol saponin (T3T) of general Formula I . The reference saponin that was selected to serve as an adjuvant was QS-21.

An experience to determine if T4T and T3T of general Formula I could potentiate the production of specific antibodies to antigens, and/or to influence the isotype profile of the response of specific antibodies to antigens and of the response of functional antibodies to a subunitary antigen, ovalbumine (OVA) was performed.

A second experience to determine if T4T and T3T could serve as adjuvants for a subunitary vaccine, ovalbumine (OVA) , in mice, in the induction of responses CTL and/or responses to antibodies was performed. In this experience, two immune adjuvants were tested consisting of T4T and T3T of general Formula I with suboptimal doses of QS-21 (< 2.5 μg) to determine if T4T can affect the adjuvant effect of QS-21 at these lower doses .

DETERMINATIONS OF THE GLOBAL RESPONSE TO ANTIBODIES

Mice

Male BALB/C mice 8 weeks old were used, obtained from the Instituto Gulbenkian de Ciencia. 4 mice were used by variation.

Antigen:

As antigen, Ovalbumine of chicken egg (Sigma A2512) , prepared at the concentration of 1 mg/ml in PBS, 25 μg per mouse was used.

Adjuvants

The used adjuvants were the Inject Alum (Pierce 77161 constituted by 3-5% (p/v) of aluminium hydroxide = Al (OH) ₃ in water and by Freund's Incomplete Adjuvant (Sigma F-5505) .

Immunomodulators :

The following compounds were used:

QS21: Quillaja, Fraction 21 (purified from GSK)¹'; concentration: 2 mg/mL in H₂0.

F3 : T3T, 50 μg/mL in PBS.

F4: T4T, 50 μg/mL in PBS. Immunisations

Groups of 4 mice, having been immunized with intraperitoneal injections, with 125 μl of the Formulations described in the following table were used:

Note: values in microlitres

Dates of immunisations and blood collection:

The immunisations were performed at intervals of 15 days and the blood collection was done 3 days before each immunisation. Collection of blood was performed starting from a cut in the terminal zone of the tail being collected from 200 to 500 μl of blood from each animal.

Preparation of the serum

The collected blood was maintained for 2 hours at 4°C, and next centrifuged at 12,000 g for 10 minutes at 4°C. The collected serum was kept at -20 °C for later analyses. ELISA IMMUNOTEST

The chosen immunotest was the ELISA (Enzyme-Link Immunoassay) test . The test was performed in the following way:

A mixture in equal volumes of serum samples (combined) for each (same) variation was prepared.

ELISA tests were performed to verify the production of antibodies after the administration of vaccines containing the saponins of the invention, in particular the T3T and T4T saponins .

The conditions of the test were the following ones:

Polystyrene plates of 96 wells were used in which a volume of 50 μl of the antigen at the concentration of 10 μg/mL was applied in the plate. In all the variations and dilutions of the serum it was always used a well with a control in which the antigen was not applied. The incubation was made for 1 hour at 37 °C. The plate was washed with TBS buffer (10 mM Tris-HCl and 125 mM NaCl, pH=7.5). The plate was next blocked with 100 μl of gelatin (BIORAD 170-6537) at the concentration of 10 mg/ml, for 1 hour at room temperature (20 °C) . Next the gelatin was removed and 50 μl of the combined serum of 4 mice by variation was added. The following dilutions in TBS with gelatin at 1 mg/ml of series 2 were made (first dilution: 1/100, last dilution: 1/204,800). The plates were incubated for 1 hour at 30°C. After that time the plates were washed 3 times with TBS-T, that corresponds to TBS containing Tween-20 (Sigma P-1379) in the final concentration of 0.05% (v/v) . Next 50 μl of a mixture of 3 serums conjugated with alkaline phosphatase was added: anti-IgA (Sigma A-4937) , anti-IgG (Sigma A-3438) and anti-IgM (Sigma A-9688) in the final dilution of 1/10,000 each one. The plates were next incubated at 30 °C, for 1 hour. After this incubation the plates were washed 3 times with TBS-T and 1 time with distilled water. Next the p- nitrophenylphosphate substratum was added (JT Baker 3351) at the concentration of 1 mg/ml in the following buffer: diethanolamine 10 mM (Merck 16205), 100 mg/1 of zinc chloride and 100 mg/1 (Merck 116763) of magnesium chloride hexa- hydrated (Merck 1.05833).

The plates were placed at 4°C and the spectrophotometric readings were performed with the 405 nm filter.

Test

The serum collected 2 weeks after the last immunisation was used.

To determine the title of the antibodies 1 plate of 96 wells was used by each variation and where the dilutions of series 2 were made. For each dilution 3 replicas were made, each replica has control without antigen (Ovalbumine) and the test was 2 times repeated.

A result of an absorbance value 3 times higher than the absorbance value of the control (wells without antigen) for the same variation and dilution of the serum is considered as positive. The values are expressed as the Logχ₀ of the title of the mixture of serum of 4 animals in each group.

Formulation 1, just with PBS

Formulation 2: with aluminium hydroxide as the sole adjuvant

Formulation 3 and 4: with QS 21/SKB at the concentrations of

1.25 and 2.5 μg/mL

Formulation 5 and 6: F3 = T3T at the concentrations of 1.25 and 2.5 μg/mL

Formulation 7 and 8: F4 = T4T at the concentrations of 1.25 and 2.5 μg/mL

Formulation 9 and 10: positive controls. Obtained results:

The graphs of Figures 6, 7 and 8 present the higher values for the titles observed by variation. From these figures the following conclusions can be withdrawn:

■=> For the same concentrations, it is obtained a higher title with the F4=T4T fractions than for QS 21. ^ The F3 and F4 fractions at the concentration of 2.5 μg/mL are comparable with the Freund's incomplete adjuvant and QS21 at 10 μg/mL. *=> The titles obtained for the F4 fraction are higher than for F3 and also higher than for QS 21 in terms of induction of a immunological response to the antigen. ■= In average the tests with the F4 fraction (samples 7 and

8) present higher values than the QS 21 for the same concentration (3 and 4) . ■= The test using PBS buffer only, presents in average the lowest value . ■=> Sample 2 includes the aluminium hydroxide adjuvant

(common to samples 3 to 8, except 7 with Freund's

Adjuvant) . Any stimulation effect would have to be always higher than the value of sample 2, what is confirmed. ==> For the 2 adjuvants Freund and QS 21 at 10 μg/ml

(positive controls, samples 9 and 10) the largest average values are obtained. ■= By the obtained results, compounds T3T and T4T of general Formula I present immunostimulating capacity.

Claims

1 . Saponins Formula I

and the pharmaceutically acceptable salts thereof, wherein

Ri is a group of the Formula:

R₂ is CH₂OH or COOH group; R₃ is hydrogen or methyl; R₄ is selected from the group consisting of a (C₄- C₂₀) straight or branched chain alkyl group, and a (C₄- C₂₀) straight or branched chain alkenyl group; either of which is optionally substituted by one or more of hydroxy, (Cι-C₆) alkoxy or carboxy group;

R₅ is methyl, CH₂0H, CHO or COOH; and,

R₆ is hydrogen or hydroxyl;

R₇ is CH_3/ COOH or COOR₈; and,

R_a is hydrogen or a (C₄-C₃₀) straight or branched chain alkyl group, or a (C₄-C₂₀) straight or branched chain alkenyl group, either of which is optionally substituted by one to six hydroxy or carboxy groups .

2. A compound according to claim 1 represented by Formula II

and the pharmaceutically acceptable salts thereof; wherein

Rx is a group of the Formula:

3. A process for the preparation of a compound according to claims 1 and 2 characterized by performing a disaccharide donor's conjugation with a selected aglycone moiety followed by extension of the sugar unit .

4. A process for the preparation of a compound according to claims 1 and 2 where glycosylations are successively carried out on acetylated protected precursors activated through the use of appropriate protecting group patterns on the glucoside residue, which are chosen depending on the aglycone to be added.

5. The use of a compound according to claims 1 to 4 characterized by applying it in the preparation of a vaccine for the stimulation of the immune response in an antigen of a mammal.

6. A pharmaceutical composition with immunoadjuvant activity characterized by incorporating a compound according to claims 1 to 4.