NO311004B1 - Use of benzaldehyde derivatives for the preparation of a therapeutic agent for the treatment of microbial infections - Google Patents

Description

This invention relates to the use of benzaldehyde derivatives for the production of therapeutic agents for the prevention and/or treatment of infections by viruses, protozoa, fungi and other microorganisms by means of modulation of the immune system.

Aldehydes react with a number of O-, S- or N-containing nucleophilic groups such as hydroxyl groups, sulfhydryl groups and amino groups to form carbonyl-containing condensation products such as acetals, mercaptals, aminals etc. With primary amines, the reaction will normally lead to an addition compound in the form of a Schiff base (an imine). It is well known that in vivo Schiff base formation is involved in key biochemical processes such as transamination, decarboxylation and other amino acid modifying reactions catalyzed by pyridoxal phosphate, the action of aldolase on fructose diphosphate in glycolysis and the condensation of retinal with rhodopsin in the vision process. It is also known that signaling through membranes involves carbonyl condensation reactions, e.g. when a response from the immune system is to be initiated.

The formation of imines occurs by a two-step mechanism. By adding an amino nucleophile to the carbonyl group, the intermediate carbinolamine (aminohydrin) is formed, then a dehydration step follows in the formation of the double bond C=N. Both steps are reversible, but are favored at different pH values. Therefore, the reaction follows a characteristic bell-shaped pH/rate profile where the highest overall reaction rate occurs at moderate pH.

However, it is known that Schiff bases are easily formed also under physiological conditions, and many carbonyl condensation reactions are well known in vivo (E.Schauenstein et. al., Aldehydes in Biological Systems, London, Pion Ltd., 1977).

The Schiff base is often itself a reactive molecule, and often participates in further reactions that lead to the addition of nucleophilic units to the double bond. For certain sulphur-containing amines, especially the amino acids cysteine and methionine and for glutathione, the Schiff base that is first formed will be able to undergo reversible internal ring formation where the sulfhydryl group is added to the imine to form thiazolidine carboxylate (M.Friedman, The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins, Oxford, Pergamon Press, 1973).

Indications of reactions between carbonyl compounds and free amino groups in proteins to form reversible Schiff bases were reported by G.E.Means and R.E.Feeney (Chemical Modification of Proteins, pp. 125-138, San Francisco, Holden-Day, 1971). Aromatic aldehydes are generally more reactive than saturated aliphatic aldehydes, and can form Schiff bases even without removing water during the reaction. (R.W.Layer, Chem. Rev. 63(1963), 489-510). This is important when considering the formation of Schiff bases under physiological conditions. With hemoglobin as a source of amino groups, Zaugg et. eel. (J. Biol. Chem. 252

(1977), 8542 - 8548) showed that aromatic aldehydes are two to three times as reactive as aliphatic aldehydes for the formation of Schiff bases. An explanation for the limited reactivity of the alkanal channels could be that in water solution at neutral pH a very large excess of free aldehyde is required to shift the equilibrium in the direction of Schiff base formation (E.Schauenstein et. al., Aldehydes in Biological Systems, London , Pion Ltd., 1977). Benzaldehyde and salicylaldehyde readily form Schiff base imines with membrane amino groups, and high equilibrium constants have been measured for the reaction of benzaldehyde with amines (J.J. Pesek and J.H.Frost, Org. Magnet. Res. 8 (1976), 173 -176, J.N.Williams Jr . and R. M. Jacobs, Biochim. Biophys. Acta., 754 (1968), 323-331). With salicylaldehyde, the imine can be further stabilized by hydrogen bonding between the lone pair of electrons in the imine nitrogen and the ortho-hydroxyl group (G.E.Means and R.E.Feeney, Chemical Modification of Proteins, pp. 125 -138, San Francisco, Holden-Day, 1971, J.M. Dornish and E.O.Pettersen , Biochem. Pharmac. 33(1990), 309-318).

We have previously shown by photographing reactions between radioactively labeled reagents that the benzaldehyde does not enter the cells, but binds to the cell membrane (Dornish, J.M. and Pettersen, E.O., Cancer Letters 29(1985), 235-243). This agrees with a previous study showing that the benzaldehyde reacted with the membrane proteins of E. coli (K.Sakaguchi et. al., Agric. Biol. Chem. 43 (1979), 1775-1777). It was also found that both pyridoxal and pyridoxal-5-phosphate protect the cells against the cytotoxic anticancer drug cis-DDP. Cis-DDP acts on the nucleus inside the cell. Although pyridoxal could in principle penetrate the lipophilic cell membrane, this is impossible for pyridoxal-5-phosphate due to the ionic phosphate group. The pyridoxal-5-phosphate must therefore exert its protective effect outside the cell membrane. A simultaneous observation of a shift in spectrographic absorbance for pyridoxal-5-phosphate to shorter wavelengths is consistent with the formation of Schiff base addition compounds between the aldehyde and amino groups in the cell membranes (J.M. Dornish and E.O. Pettersen, Cancer Lett. 29, (1985), 235 - 243).

These findings suggest that aldehydes bind to amines and other nucleophilic units on the cell membrane by forming Schiff bases and other condensation products. It is known that cells are stimulated to grow by a cascade of events that act from outside the cell membrane. In the same way, the derivatives in the present patent application can act by forming addition compounds with ligands on the cell membrane and giving rise to impulses inside the cell that have an impact on cell growth parameters such as protein synthesis and mitosis, and on the expression of immune response and tumor suppressor genes. Since the condensation reactions are reversible, the cell effects can be modulated by an equilibrium shift involving the molecules that are bound together. That there are dynamic equilibria at the chemical level is consistent with the reversible and non-toxic mode of action of the benzaldehyde derivatives.

The immune system is carefully designed to identify and eliminate all substances that are perceived as foreign, whether they originate from a bacterial, viral or protozoan infection or from abnormal cells such as cancer cells. In order to be able to provide a specific response to the large range of biological variation that the invading substances represent, the immune system must be very versatile. However, overstimulation of this fine-tuned system can lead to various allergic and inflammatory reactions and to autoimmune diseases. Modulating the immune system, either by up- or down-regulating a specific response, is therefore a major therapeutic challenge.

In an immunological recognition process, a fragment of a foreign protein is trapped within the cleft of an MHC class II protein on the surface of an antigen-presenting cell (APC). The receptor for a T-helper cell is also bound to this MHC-antibody complex. To activate a T-helper cell, at least two signals are needed. The primary signal is given by the antigen itself, via the MHC class II complex and is amplified by the CD4 co-receptors. The second signal can be obtained from a specific signaling molecule that is bound in the plasma membrane on the surface of the APC cell. A corresponding coreceptor protein is located on the surface of the T helper cell. Both signals are necessary to activate the T cells. When they are activated, they will stimulate their own reproduction by secreting interleukin-based growth factors and synthesizing corresponding receptors on the surface. The binding of the interleukin to these receptors then directly stimulates the T cells to multiply.

In the 1980s, it was known that a cyclodextrin-benzaldehyde-based inclusion complex could stimulate the immune system by enhancing the lymphokine-activated killer cells in a mouse model (Y. Kuroki et al., J. Cancer Res. Clin. Oncol. 117, (1991), 109- 114). In wrro studies have subsequently revealed the nature of the chemical reactions at the APC donor/T cell receptor interaction sites responsible for the second stimulatory signal, and that these reactions take the form of carbonyl-amino condensations (Schiff base formation). Furthermore, these interactions can be imitated by synthetic chemical entities. These discoveries open up new therapeutic possibilities for artificial modulation of the immune system. WO 94/07479 claims a patent for the use of certain aldehydes and ketones which form Schiff bases and hydrazones with amino groups on the T-cell surface. In EP 0609606 A2, the preferred immunostimulatory substance is 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (Tucaresol), a compound originally designed to cure sickle cell anemia. This substance is given orally and is systemically bioavailable. The potential of Tucaresol to cure a number of diseases such as bacterial, viral and protozoan infections, autoimmune diseases and cancer are now being investigated (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504) and combination strategies where Tucaresol is given together with a vaccine to cure chronic hepatitis B, HIV and malignant melanoma are under development.

Measurement of immune parameters in vitro and studies of effects in vivo resulted in a bell-shaped dose/response profile (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504). This dose/response relationship, which is otherwise somewhat unusual, can be justified by assuming that a high concentration of the aldehyde drug leads to the co-stimulatory ligands needed for effective binding of the APC to the T cell being saturated with drug molecules so that the effect is weakened. A dose sufficient to form a dynamic equilibrium conducive to stimulation without blocking the binding between cells appears to be optimal.

In general, aldehydes themselves are unstable to oxidation. 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (Tucaresol) presented in EP-0609606 is significantly more active in vivo than in vitro. The reason for this may be that the drug is oxidized in aqueous solution in vitro (H.Chen and J.Rhodes, J. Mol. Med. (1996) 74:497-504). Many aldehydes are too reactive to be given as aldehydes, and although benzaldehyde has been shown to be an active anticancer drug in vitro, it is highly irritating and unsuitable for direct use in vivo. In a biological system, the carbonyl group in the aldehyde will react rapidly with nucleophilic groups that are present in large quantities in all body fluids. These unwanted side reactions can lead to rapid metabolism of the drug and difficulties in controlling the serum concentration of the active drug. Controlling the drug at the cellular level within a narrow concentration window is essential to achieve effective immunomodulation. Tucaresol is given orally as an unprotected aldehyde, and there may be reason to suspect that the drug may be subject to oxidation and that the pharmacokinetics may be difficult to control.

The benzaldehyde derivatives 4,6-benzylidene-D-glucose and the deuterated analogue (compounds 1 and 2) have been shown to have high bioavailability whether given intravenously or orally. The bioavailability for BALB mice measured as concentration in serum after oral administration of compound 2 was 93-99% (C.B. Dunsaed, J.M. Dornish and E.O. Pettersen, Cancer Chemother. Pharmacol. (1995) 35: 464-470). In addition, the glucose group can have an affinity for receptors on the cell surface so that the drug becomes more available at the cell level. The free aldehyde can be easily released by hydrolysis of the acetal so that the carbonyl group becomes available for Schiff base formation with the target ligands.

In the present patent application, the aldehydes are derivatized with biologically acceptable carbohydrates such as glucose, galactose and others and form acetals with them. The sugar group will thus contribute to increasing the stability and improving the bioavailability of the aldehyde function for the target cells. This surprisingly leads to the carbonyl condensation reactions becoming more efficient and the pharmacokinetics easier to control when using our compounds compared to previously known compounds.

To compare compound 2 with Tucaresol, protein synthesis and inactivation of cells were measured at equal concentrations of the two drugs. As can be seen from fig. 1, it was demonstrated that compound 2 was more effective than Tucaresol with respect to both measured parameters.

The immunostimulating effect of the compounds according to the invention can also be used in the treatment of certain viral diseases in combination with other treatment against viruses such as e.g. antiviral drugs or vaccines. Many types of virus will, after the first infection, incorporate themselves into the cell nucleus and are inactive for a long time. Oncogenic viruses such as hepatitis B and C, certain retroviruses and certain papillomaviruses can lead to the development of cancer. During these latent periods, it is very difficult to cure the viral infection. But such viruses will often be activated by the immune response so that they enter the blood, and in this step it is possible to get rid of the viral infection. The benzaldehyde derivatives' ability to trigger the immune response can be used in combination with antiviral agents or vaccines to develop a treatment for these diseases.

It is a main purpose of the invention to provide compounds for the prevention and/or treatment of diseases related to the immune system.

Another object of the invention is to provide compounds which are able to enhance the immune response to make it possible to fight infectious diseases caused by viruses, bacteria, fungi and other microorganisms.

A third object of the invention is to provide compounds for the prevention or treatment of diseases related to immune problems, without the compounds having toxic side effects.

These and other objects of the invention are achieved by the attached patent claims.

The compounds used according to the present invention have a general formula (I):

where L is H or D;

Ar is phenyl or mono- or di-substituted phenyl, where the substituent (e) is selected from the group consisting of alkyl with 1-6 carbon atoms, CF3, fluorine, chlorine, nitro, cyano, OR<1>, SR<1>, N(R<1>)2 or COOR<1> where R<1> is H, CF3 or alkyl with 1-6 carbon atoms, or OC(0)R<2>, CA(OR<2>)2, CA [OC(0)R<2>]2, NHC(0)R<2> or N[C(0)R<2>]2 where A is H or D and R2 is CF3 or alkyl with 1-6 carbon atoms , or C(O)R3 where R3 is H, D, CF3 or alkyl of 1-6 carbon atoms;

Y is selected from the atoms or group consisting of H, D, fluorine, chlorine, nitro, OR<1>, OC(0)R<2>, N(R<1>)2, NHC(0)R<2> or N[C(0)R<2>]2 where R1 and R2 are as defined above;

R is H, CF3 or alkyl of 1-6 carbon atoms;

or a pharmaceutically acceptable salt thereof.

It is understood that any stereoisomer according to formula (I) is encompassed by the present invention.

Detailed description of the invention

The invention is further described below with examples and attached figures.

Description of the figures

Fia. 1: The rate of protein synthesis relative to untreated control group of NHIK 3025 cells treated with compound 2 (■) or Tucaresol (A) for 1 hour at 37°C. The rate of protein synthesis was measured as the amount of [<3>H]-valine incorporated during the first hour after the start of drug treatment. The rate of protein synthesis was measured relative to the total amount of protein in the cells. The data are representative of an experiment performed in quadruplicate. The standard deviations are shown if they are larger than the symbols. Fig. 2: Peripheral blood mononuclear cells and superantigen in Ex Vivo 10 medium were exposed to either benzaldehyde, deuterated benzaldehyde, compound 2 or zilascorb(<2>H). The proliferation of peripheral blood mononuclear cells was measured as incorporation of tritiated thymidine at the different drug concentrations. Fig. 3: NMRI mice were infected intraperitoneally with spleen-invading Friend erythroleukemia virus. Infected and uninfected mice were treated intraperitoneally daily with 5 mg/kg of either compound 2 or compound 5. After 19 days of treatment, the spleen was dissected out and weighed.

Manufacturing

As is known, aldehydes undergo acid-catalyzed condensation reactions with alcohols to form acetals. At the same time, water is obtained as a by-product. The reaction is reversible, and in solution an equilibrium mixture of aldehyde/alcohol and acetal/water is formed. The equilibrium position is mainly determined by the reactivity and concentration of each molecular species. To complete the reaction, one of the products (acetal or water) is removed from the reaction mixture.

In the present patent application, various sugars, deoxysugars and aminosugars are condensed with aldehydes or aldehyde equivalents to sugar acetals. Particularly preferred is a reacetalization strategy where the aldehyde is introduced protected as the dimethyl acetal instead of the aldehyde itself. Methanol is thus formed as a by-product. The reaction mixture is moderately heated under reduced pressure to remove the methanol as soon as it is formed. In most cases, these reaction conditions will easily shift the equilibrium in favor of the acetal.

Acetalization of sugars will normally lead to the formation of mixtures of regio- and stereoisomers. Ring contractions can also occur which lead to mixtures of pyranoses and furanoses and in some cases di-acetalisation products are formed. If you do not use protection strategies, you will therefore often get extremely complex reaction mixtures. However, surprisingly pure product fractions were produced after suitable work-up, especially by liquid chromatography. The identification of the products was done by GC-MS spectroscopy and various NMR techniques.

The specific reaction conditions and which solvents and catalysts are used will depend in each individual case on the solubility and reactivity of the reactants and on the properties of the product. The catalyst can be a mineral acid, e.g. sulfuric acid, an organic acid, e.g. paratoluenesulfonic acid, an acidic ion exchange agent, e.g. Amberlyst 15, an electrophilic mineral clay, e.g. montmorillonite K-10 or a resin-supported superacid, e.g. Nafion NR 50. The reaction can preferably be carried out in a dipolar, aprotic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, dimethoxyethane or the like. Paratoluenesulfonic acid in dimethylformamide was the preferred and most commonly used reaction medium.

The compounds of formula (I) where L is deuterium can be prepared as described above, but starting from the dimethyl acetal of an aldehyde which is deuterated in the formyl position. Production of deutero-benzaldehyde can be carried out with a modified Rosenmund reduction with D2 gas in a deuterated solvent, as described in EP 0 283 139 B1. Deuterated benzaldehyde derivatives with substituents in the phenyl ring can be prepared according to the examples given in EP 0 493 883 A1 and EP 0 552 880 A1.

The following examples illustrate how the compounds of the present invention can be prepared.

Compound 1:4. 6- O- benzvlidene- D- qlukopvranose

This known compound was prepared as described for compound 2, starting from undeuterated benzaldehyde dimethyl acetal. The identity was confirmed by <1>H NMR spectroscopy in DMSO-d6: 5 rel. to TMS: 7.58-7.29 (5H, m, Ar-H), 6.83 (0.4H, d, OH-1-p), 6.60 (0.6H, d, OH-1 -a), 5.61 (1H, s+s, acetal-H), 5.25 (0.4H, d, OH_-3-p), 5.21 (0.4H, d, OH-2- P), 5.62 (0.6H, d, OH-3-ct), 5.00 (0.6H, H-1-cc), 4.82 (0.6H, d, OH-2-cc ), 4.49 (0.4H, t, H-1-P), 4.18-4.02 (1H, m, H-6'-a+<p>), 3.89-3.77 ( 0.6H, m, H-5-cc), 3.75-3.57 (1.6H, m, H-6"-a+p and ]H-3-a), 3.45-3, 27 (2.5H, m, H-3-p, H-4-a+p, H-5-P and H-2-a) and 3.11-3.00 (0.4H, m, H -2-p).

Compound 2: 4. 6- O-( benzvlidene-di)- D- alucopvranose

Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1. The preparation of 4,6-0-(benzylidene-di)-D-glucopyranose is also described in EP 0 283 139 B1, but this time the compound was prepared according to an alternative method where high purity was prioritized: D(+)-glucose ( 706 g, 3.92 mol), benzaldehyde dimethyl acetal-di (571 g, 3.73 mol), dry DMF (1.68 kg) and paratoluenesulfonic acid (4.5 g, 24 mmol) were mixed in a dry distillation apparatus connected to a vacuum pump through a cold flux condenser. The mechanically stirred mixture was heated to max. 69°C at 30 torr to distill off methanol and after 2 hours 235 g had been collected. The reflux condenser was then shut off and the temperature increased to max. 73°C to distil off DMF. After a further 2 hours a further 1385 g had been collected and the distillation was stopped. The distillation residue was cooled to 40°C and ice water (2.9 L) was added before 5 min had elapsed. The temperature fell below 0°C and a precipitate formed, partly as large lumps. The mixture was transferred to a beaker and an additional 8-9 L of ice water was added to cause the lumps to fall apart and go into suspension. The suspension was filtered on two nutch filters and the two filter cakes were left overnight on the filters connected to a water jet pump and both filter cakes were supplied with N2 through an inverted funnel. The filter cakes were spread out on two plates and dried at 32°C for 20 hours in a vacuum oven. The vacuum was first set to 13 millibars and then regulated down to 1 millibar.

The crude product was recrystallized (to remove dibenzylidene acetals) and washed with water (to remove DMF and glucose) until these impurities were eliminated. Accordingly, the crude product (500 g) was dissolved in hot dioxane (800 mL) and the solution transferred to boiling chloroform (9 L) through a pleated filter. The solution was cooled, first to room temperature, then in an ice bath overnight. The precipitate was filtered off, dried for 2 hours on the filter (while supplying N2 as described above) and further dried overnight at 31 °C in vacuum on a rotavapor. The product (142 g) was suspended in ice water (1 L), filtered on a nutch (washing with 200 ml ice water) and dried on the filter overnight as described above. It was then ground, sieved (0.5 mm grid) and dried in vacuum for 5 hours at 31 °C on a rotavapor. The product (96 g) was resuspended in ice water (500 ml), filtered (washing with 150 ml ice water) and dried (7 hours under N 2 flow). It was finally ground in a mortar, sieved (0.5 mm) and dried in a vacuum oven.

The product was a white, finely divided powder of high purity according to the HPLC analysis. The yield was 95 g, 10% of theoretical yield. NMR in DMSO-d6 indicated an a:P anomer ratio of approximately 7:3.

<1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.55-7.28 (5.00H, m, Ar-H), 6.85 (0.27H, d, OH-1-<p>), 6.58 (0.71H, d, OH-1-a), 5.24 (0.27H, d, OH-3-P), 5.19 (0.28H, d, OH-2-P), 5.61 (0.71 H, d, OH-3-a), 4.99 (0.72H, H-1-a), 4.82 (0.71 H, d, OH-2-cc), 4.48 (0.29H, t, H- 1-p), 4.20-4.04 (1.04H, m, H-6'-a+P), 3.88-3.73 (0.78H, m, H-5-a), 3.73-3.56 (1.72H, m, H-6"-a+p and H-3-a), 3.46-3.21 (2.61 H, m, H-3-P,

H-4-a+p, H-5-p and H-2-cc) and 3.09-2.99 (0.28H, m, H.-2-P); 137.881, 128.854, 128.042, 126.435 (Ar-C), 100.462 (acetal-C), 97.642 (C-1-P), 93.211 (C-1-cc), 81.729 (C-4-a), 80.897 (C -4-p), 75.796 (C-2-P), 72.906 (C-2-cc and C-3-P), 69.701 (C-3-a), 68.431 (C-6-a), 68.055 ( C-6-P), 65.810 (C-5-p) and 62.032 (C-5-cc).

Compound 3: 4, 6- O- benzvlidene- D- qalactopvranose

D(+)-galactose (15.0 g, 0.083 mol) and dry DMF (80 mL) were mixed with stirring at 50°C in a still. To the resulting suspension was added benzaldehyde dimethyl acetal (12.2 g, 0.083 mmol) and paratoluenesulfonic acid (0.14 g) and methanol/DMF was slowly distilled off using a water jet pump. After 3 hours, most of the galactose was used up and the remaining DMF was removed on a rotavapor connected to a vacuum pump. The distillation residue, a rather tough syrupy mass, was purified on a Lobar C RP-8 column with methanol/water (1:1) as eluent. The product fractions were freeze-dried.

Gas chromatography for the TMS derivatives showed that the product consisted mainly of two isomers. On the basis of <1>H, <13>C, COSY, DEPT and C-H correlation NMR spectra, the product was identified as the α and β anomers of the title compound.

<1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.49-7.27 (5H, m, Ar-H), 5.57 (1H, s, acetal-H), 5.22 (0.5H, d, H-1-a), 4.56 (0.5H, d, H-1-P), 4.23+4.18 (0.5H+0.5H, d+d, H-4-oc+P), 4.14- 3.98 (2H, m, H-6-cc+P), 3.94-3.79 (1.5H, m, H-2-ct, H-3-cc and H-5-a), 3.69-3.49 (1.5H, m, H-2-p, H-3-P and H-5-P); 137.422, 129.981 128.902 126.639 and 126.590 (Ar-C), 101.325 (acetal-C), 96.540 (C-1-P), 93.161 (C-1-a), 76.581 (C-4-a), 76.093 (C -4-P), 71.889 + 71.802 (C-2-P+C-3-P), 69.404 (C-6-a), 69.182 (C-6-P), 68.566 + 68.057 (C-2-cc + C-3-P), 66.579 (C-5-P) and 62.886 (C-5-cc).

Compound 4: metvl- 4. 6- 0- benzvlidene- a- D- mannopyranoside

Methylx-mannopyranoside (18.1 g, 0.093 mol), benzaldehyde dimethyl acetal (21.0 g, 0.138 mol) and dry DMF were mixed with stirring at 50-55°C in a still. Paratoluenesulfonic acid (approx. 0.1 g) was added and 10 min. later it was connected to a water jet pump to distill off methanol. After 4 hours, the reaction mixture was evaporated to a white solid. The evaporation residue was washed with dibutyl ether, filtered and the filter cake was dissolved in acetonitrile. A precipitate began to form and the mixture was refrigerated for 5 days. The precipitate was then filtered off and the filtrate evaporated. The evaporation residue was purified on a Lobar C RP-8 column with 30% acetonitrile in water as eluent. Product fractions from 4 separate syntheses were freeze-dried and combined.

Gas chromatographic analysis of the TMS derivatives indicated that the product consisted of 95 area percent monoacetals. The monoacetals for their part consisted of 4 peaks which could be integrated to 0.4, 3.2, 94.1 and 2.4 area-% respectively. On the basis of <1>H, <13>C, COSY, DEPT and C-H correlation NMR spectra together with gas chromatography and MS spectroscopy, the dominant molecular species was identified as the title compound.

<1>H and <13>C NMR (acetone-d6), 8 rel. to TMS: 7.54-7.30 (5H, m, Ar-H), 5.60 (1H, s, acetal-H), 4.71 (1H, s, H-1), 4.34 ( 1H, broad s, OH), 4.22-4.02 (2H, m+broad s, IH-6'+OH), 3.94-3.82 (3H, m, H-2, H-3 and H-4), 3.80-3.60 (2H, m, H-5+H-6") and 3.39 (3H, s, CH3); 139.264, 129.396, 128.662 and 127.211 (Ar-C ), 102.825 (C-1), 102.468 (acetal-C), 79.888 (C-4), 72.090 (C-3), 69.308 (C-6), 69.127 (C-2), 64.363 (C-5) and 54.921 (CH 3 ).

Connection 5:4. 6-( benzvlidene-di)- 2- deoxyv- D- alucopvranose

Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.

2-deoxy-D-glucose (10 g, 60.9 mmol), dry DMF (35 mL),

benzaldehyde dimethyl acetal-di (11.7 g, 76.4 mmol) and paratoluenesulfonic acid (70 mg, 0.37 mmol) were mixed under N 2 to a white slurry. On heating to 45-50°C, a colorless solution was formed within half an hour. A vacuum pump was connected to remove methanol through a cooled column (to prevent loss of benzaldehyde dimethyl acetal-di). The pressure was regulated stepwise down from 70 millibars to 20-30 millibars during 4.5 hours and the temperature was maintained at 40-45°C. The distillation was then interrupted, the apparatus rebuilt without the column and the DMF was removed by short path distillation at 50-55°C, max. vacuum. The rest was a pale yellow syrupy mass.

1/4 of the syrup mass was dissolved in weakly alkaline (NaHCOa) methanol/water 60/40 and purified on a Merck LiChroprep RP-8 reverse phase column with methanol/water 60/40 as eluent. Product fractions were concentrated to remove methanol and freeze-dried to a white, off-white solid. Products from four separate syntheses were combined in a yield of 3.5 g, 23% of theory.

Gas chromatographic analysis of the TMS derivatives and NMR spectroscopy showed that the product consisted of a 1:1 mixture of the α- and (3-anomers.

<1>H and <13>C NMR (DMSO-d6), 6 (ppm) rel. to TMS: 7.52-7.28 (m, 5H, Ar-H I + II), 6.9-6.65 (broad s, 1/2 H, OH-1 II), 6.55-6 .32 (broad s, 1/2 H, OH-1 I), 5.25-5.12 (m, 1 H, OH-3 II and H-1 I), 5.12-5.0 (d , 1/2 H, OH-3 II), 4.84-4.73 (dd, 1/2 H, H-1 II), 4.20-4.02 (m, 1H, H-6 l+ ll), 3.98-3.73 (m, 1H, H-3 I and H-5 I), 3.73-3.58 (m, 1.5H, H-6' 1+ll and H- 3 II), 3.42-3.18 (2.5 H, H-4 l+ll and H-5 II and hbO), 2.10-1.86 (m, 1H, H-2 l+ll ) and 1.62-1.34 (m, 1H, H-2' 1+11); 137.979, 137.926, 128.841, 128.036 and 126.432 (Ar-C l+ll), 101.5-100.0 (acetal-C l+ll), 94.057 and 91.424 (C-1 l+ll), 83.916 and 83.093 ( C-4 l+ll), 68.374 and 68.119 (C-6 l+ll), 66.889, 66.092, 64.174 and 62.604 (C-3 l+ll and C-5 l+ll) and 41.932 and 40.051 (C-2 l+ll).

Compound 6: 4, 6- Q-( 4- carbomethoxybenzvlidene)- D- qulcopvranose

Methyl-4-formylbenzoate (100 g, 0.609 mol), methanol (91.5 g, 2.86 mol), trimethyl orthoformate (71 g, 0.67 mol) and concentrated hydrochloric acid (165 µL) were mixed to a slurry in a 500 ml wooden flask. The porridge was converted to a slightly yellow solution in a few minutes and the temperature increased spontaneously from 15°C to 30°C. After stirring for 15 minutes, the reaction mixture was refluxed at 58°C for another 25 minutes and then cooled to 10°C (ice water). An alkaline solution was made by dissolving KOH (8.3 g) in methanol (53 mL) and 7 mL of the solution was added to the reaction mixture. After 25 minutes of stirring at 10°C, the reactor was rebuilt for short path distillation and all volatile substances removed in vacuum (water jet pump). Distillation was then continued with a vacuum pump until a colorless oil was left at 112-114°C/0.5 millibar. The oil turned into a colorless solid with a melting point of 32-33°C, and was identified by NMR as methyl-4-formylbenzoate dimethyl acetal. The yield was 108.75 g, 85% of the theoretical.

D(+)-glucose (8.0 g, 44.4 mmol), dry DMF (25 mL), methyl 4-formylbenzoate dimethyl acetal (10.4 g, 49.5 mmol) and paratoluenesulfonic acid were mixed at 50°C under N2 to a white suspension. The apparatus was connected to a vacuum pump through a vertical condenser and the evaporation of methanol started at 80-100 millibar, 55°C. The vacuum was gradually increased to 40 millibars while the temperature was maintained at 55-60°C. The reaction mixture gradually became clear and the apparatus was rebuilt for short path distillation of DMF. The distillation residue was a pale yellow syrupy mass.

The syrup mass was dissolved in a warm solution of 100 mg of NaHCO 3 in 20 ml of methanol and 8 ml of water and filtered by adding 100 ml of ethyl acetate. The precipitate was isolated from the mother liquor by filtration, washed with cold water (4 x 15-20 ml) and transferred to a rotayapore flask. The moisture was removed by adding ethyl acetate and evaporating twice. The product was finally dried under high vacuum. It was filtered off more sediment from the mother liquor and washed and dried for another product portion. The two portions were combined to give 1.94 g of pure product, 13% of the theoretical yield.

Gas chromatographic analysis of the TMS derivatives showed two isomers in the ratio 2:1.

<1>H- and <13>C NMR (DMSO-d6), 8 (ppm) rel. to TMS: 7.99 and 7.61 (dd, 2+2H, furfuryl-H), 6.87 (d, 0.67H OH-1 II), 6.59 (d, 0.28H, OH-1 I) , 5.68 (s+s, 1H, acetal-H l+ll), 5.29 (d, 0.68H, OH-3 II), 5.21 (d, 0.67H, OH-2 II), 5 .16 (d, 0.31 H, OH-3 I), 5.00 (t, 0.30H, H-1 I), 4.85 (d, 0.28H, OH-2 I), 4.48 (t, 0.73H, H-1 II), 4.25-4.08 (m, 1.14H, H-6), 3.95-3.77 (m, 3.43H, OCH3 and H-5 I) , 3.78-3.59 (m, 1.40H, H-3 I and H-6'), 3.49-3.23 (m, 3.59H, H-4 I and II, H-5 II , H-2 I and H-3 II), 3.10-2.98 (m, 0.72H, H-2 II); 165,978, 142.553, 142553, 129,959, 129,067, 126,763, 99,982, 99,812, 97,661, 93,238, 81,794, 81,794, 80,965.95.9.9.9.CLIBRATION, 75, 75, 75, 75, 75, 75, 75.

Compound 7: 4, 6- 0- benzvlidene- 2- deoxv- D- qlucopyranose

12-deoxy-D-glucose (10.0 g, 60.9 mmol), dry DMF (34 mL), benzaldehyde dimethyl acetal (11.6 g, 76.2 mmol) and paratoluenesulfonic acid (70 mg, 0.37 mmol) were mixed under N2 to a white mush. The reaction mixture was stirred at room temperature for 30 minutes and by heating to 45-50°C the solid substance was gradually dissolved. A vacuum pump was connected to remove methanol through a cooled column (to prevent loss of benzaldehyde dimethyl acetal) and the reaction continued for 4.5 hours. The distillation was then interrupted, the column removed and DMF distilled from by short path distillation at 50-55°C, maximum vacuum. The distillation residue was a pale yellow syrupy mass.

The syrup mass was dissolved in weakly alkaline (NaHCO 3 ) methanol/water 60/40 and purified on a Merck LiChroprep RP-8 reverse phase column with methanol/water 60/40 as eluent. The product fractions were concentrated to remove methanol and freeze-dried to a white, woolly solid. The products from four separate syntheses were combined to give 3.18 g, 21% of the theoretical yield.

Gas chromatographic analysis of the TMS derivatives and NMR spectroscopy showed that the product consisted of a 1:1 mixture of the α- and β-anomers.

<1>H and <13>C NMR (DMSO-d6), 8 (ppm) rel. to TMS: 7.52-7.30 (m, 5H, Ar-H I + II), 6.85-6.68 (broad s, 1/2 H, OH-1 II), 6.50-6 .35 (broad s, 1/2 H, OH-1 I), 5.61 (s+s, 1H, acetal-H l+ll), 5.23-5.12 (m, 1 H, OH- 3 II and H-1 I), 5.12-5.02 (d, 1/2 H, OH-3 II), 4.84-4.74 (dd, 1/2 H, H-1 II) , 4.20-4.04 (m, 1H, H-6 1+11), 3.98-3.74 (m, 1H, H-3 I and H-5 I), 3.74-3, 57 (m, 1.5H, H-6' 1+ll and H-3 II), 3.42-3.18 (2.5 H, H-4 1+ll and H-5 II and HaO), 2.08-1.88 (m, 1H, H-2 1+ll) and 1.62-1.32 (m, 1H, H-2' 1+ll).

Compound 8: 2- acetamido- 4, 6- 0- benzvlidene- di- 2- deoxv- D- qlucopyranose

Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.

Benzaldehyde dimethyl acetal-di (8.7 g, 56.8 mmol), N-acetyl-D-glucosamine (10.0 g, 45.2 mmol), dry DMF (30 mL) and paratoluenesulfonic acid (88 mg, 0.46 mmol ) was mixed under N2 to a white suspension. The reaction mixture was stirred at 50°C for 45 minutes, then a vacuum pump was connected through a vertical condenser and the reaction continued for 2 hours at 55°C/60-70 mbar. The apparatus was rebuilt to remove DMF by short path distillation and the distillation continued at 55-60°C, max. vacuum for 1 more hour. The distillation residue was a yellow-white, soft solid.

A solution was made by mixing NaHCO 3 (150 mg) in 30 ml methanol/water (3:2) and the distillation residue was neutralized by adding the solution. This resulted in a creamy slurry which was filtered, washed 2-3 times with a 1% NaHCO 3 solution and several times with ether. The product was found to be sufficiently pure by analysis (GC) and dried in vacuo. The yield was 8.8 g, 63% of the theoretical.

Gas chromatographic analysis of the TMS derivatives indicated a 1:1 isomeric mixture. NMR spectroscopy in DMSO-d6 solution identified the product as a 3:1 anomeric mixture.

<1>H and <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.83 (s+s, 1H, NH), 7.51-7.28 (m, 6H, Ar-H), 7.0-6.2 (broad s, 1H, OH-1) , 5.65-5.05 (broad s, 1 H, OH-3), 4.99 (d, 1H, H-1 I), 4.61 (d, 0.3H, H-1 II), 4.21-4.03, 3.92-3.67 and 3.51-3.22 (m, 6H, H-2, H-3, H-4, H-5 and H-6), and 1.85 (s+s, 3H, CH3); 169,452 (C = 0), 137,818, 128,869,128,035 and 126,438 (AR-C), 100,505 (Acetal-c), 96,056, 91,500, 82,471, 81,505, 70.10541, 68,11, 68,1, 67.91, 67.91, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67,300, 67.9. -C) and 23.123 and 22.674 (CH 3 ).

Compound 9: 2- acetamido- 2- deoxy- 4. 6- O-( 3- nitrobenzvlidene)- D- glucopvranose

3-Nitrobenzaldehyde (100 g, 0.66 mol), methanol (99 g, 3.1 mol), trimethyl orthoformate (77.3 g, 0.73 mol), and concentrated hydrochloric acid (165 µl) were mixed in a 500 mL three-necked flask to a yellow mush which turned into a solution in after 5 minutes. The reaction mixture was refluxed at ∼50°C for 15 min and cooled to 10°C with ice water. The reaction was quenched by adding 6.6 ml of a solution made by dissolving 2.5 g of KOH in 16 ml of methanol. Stirring was continued for 15 minutes and the reactor was converted for short path vacuum distillation. Volatile substances (CH3OH + HCOOCH3) were distilled off with a water jet pump, the distillation was interrupted and a vacuum pump connected. Distillation then continued and a yellow oil was distilled from at 93 - 97.5°C/20 mbar. By NMR, the oil was found to be 3-nitrobenzaldehyde dimethyl acetal of high purity. The yield was 127 g, 97.6% of the theoretical.

3-Nitrobenzaldehyde dimethyl acetal (5.5 g, 0.028 mol), N-acetyl-D-glucosamine (5.0 g, 0.023 mol), paratoluenesulfonic acid (50 mg, 0.263 mmol) and dry DMF (15 mL) were mixed with stirring at 50°C to a pale yellow suspension. After 1/2 hour a vacuum pump was connected and the reaction continued at 56°C/50 mbar for 11 hours. The reaction mixture was evaporated and the evaporation residue partitioned between a small volume of weakly alkaline (NaHCO 3 ) water and chloroform. The aqueous phase (which formed a cheesy suspension) was extracted twice with chloroform and filtered and washed several times with water and ether. The product was dried in vacuo to a slightly brownish powder. The yield was 570 mg, 7% of the theoretical.

Gas chromatographic analysis of the TMS derivatives indicated that the product consisted of two isomers in a 2:1 ratio. NMR spectroscopy identified the product as a mixture of α- and β-anomers. <1>H and 1<3>C NMR (DMSO-d6), 8 rel. to TMS: 8.4-8.1 (m, 2H, Ar-H), 8.05-7.79 (m, 2H, Ar-H), 7.79-7.60 (t, 1H, NH ), 6.80 (d, 1H, OH-1), 5.81 (s+s, 1H, acetal-H), 5.30 and 5.18 (s+s, 1/2 H + 1/2 H, H-1), 4.30-4.10, 3.93-3.3 (m, 6H, H-2 - H-6), and 1.85 (d, 3H, CH3); 169,331 (C=0), 147,490, 139,544, 133,018, 129,862, 123,732, 120,884 (Ar-C), 99,000, 98,806 (acetal-C), 95,924, 91,406, 82,433, 81,454, 70,320, 68,240, 67,907, 67,020, 65,574 , 61.833, 57.875, 54.609 (sugar-C) 23.014 and 22.557 (CH3).

Compound 10: 4, 6- O-( benzvlidene-di)- D- qalactopvranose

Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.

D(+)-galactose (15.0 g, 0.0833 mol) and dry DMF (80 mL) were stirred in a still at 45°C. Benzaldehyde dimethyl acetal-di (12.8 g, 0.0836 mol) and paratoluenesulfonic acid (0.14 g) were added and methanol and DMF slowly distilled off in vacuo (water jet). After 3 hours a vacuum pump was fitted and the remaining DMF was distilled off. Aliquots of the distillation residue were dissolved in methanol/water (1:1) containing NaHCO 3 (11 mg/ml) and purified on a Lobar C RP-8 column with methanol/water (1:1) as eluent. Product fractions from 7 individual syntheses were freeze-dried and pooled into a white, woolly product. The yield was 6.62 g, 30% of the theoretical.

Gas chromatographic and NMR analysis showed that the product consisted of a 1:1 anomeric mixture. <1>H and 13C NMR (DMSO-d6), 8 rel. to TMS: 7.52-7.30 (m, 5H, Ar-H), 6.62 (0.5H, d, OH-1-P), 6.32 (0.5H, d, OH-1- a), 5.05 (0.5H, t, H-1-a), 4.85+4.69+4.49 (1H+0.5H+0.5H, m+d+d, OH-2 +OH-3), 4.35 (0.5H, t, H-1-P), 4.12-3.92+3.81 -3.71 +3.69-3.59+3.49 -3.39+3.39-3.28 (3H+1H+0.5H+1H+2H, m+m+m+m+m, H-2-H-6+H2O); 138.753, 138.690, 128.607, 128.319, 127.913 and 126.3 (Ar-C), 99.345 (acetal-C), 97.307 and 93.178 (C-1), 76.738 and 76.158 (C-4), 72.101, 71.68, 68.97, 68.97 68.486 and 67.730 (C-2, C-3 and C-6) and 65.829 and 62.068 (C-5).

Compound 11: 4.6-0-(benzvlidene-di)-D-mannopvranose

Benzaldehyde di was prepared and converted to benzaldehyde dimethyl acetal di as described in EP 0 283 139 B1.

D(+)-mannose (15.0 g, 0.0833 mol) and dry DMF (70 mL) were stirred in a still at 40°C. Benzylidenedimethylacetal-di and paratoluenesulfonic acid (0.14 g) were added and a clear solution formed. A vacuum pump was connected and methanol and DMF were slowly distilled from at 70-20 mbar, 45-50°C. After 3 hours, the remaining DMF was distilled off at max. vacuum, so that a faint yellow syrupy mass remained.

The residue was washed several times with ether to remove lipophilic substances. Some portions of the crude product were dissolved in weakly alkaline (NaHCO 3 ) methanol/water (3:2) and purified on a Lobar C RP-8 column with methanol/water (3:2) as eluent. gas chromatography of the TMS derivatives indicated that the product consisted of 4 isomers in the ratio 10:3:1:4. It was re-eluted with methanol/water (1:4) to give a white, woolly product consisting of only two isomers in the ratio 70/30, according to gas chromatographic analysis. The yield was 1.42 g, 6.4% of the theoretical.

On the basis of 1H, <13>C, COSY, DEPT and C-H correlation NMR, the chemical structure was confirmed and the α- and β-anomers were found to reach an equilibrium at a 1:8 ratio.

<1>H and <13>C NMR (DMSO-d6) of the dominant isomer, 8 rel. to TMS: 7.5-7.28 (m, 5H, Ar-H), 6.56 (d, 1H, OH-1), 5.0-4.85 (m, 3H, OH-2 and OH -3), 4.10-4.02 (m, 1H, H-6), 3.63-3.40 (m, 5H, H-2, H-3, H-4, H-5 and H -6'); 138.045, 128.825, 128.029, 126.438 (Ar-C), 100.802 (Acetal-C), 95.233 (C-1), 78.967 (C-4), 72.032 (C-3), 68.317 (C-6), 67.252 ( C-2), 63.495 (C-5).

Compound 12: 2- acetamido- 4, 6- 0- benzvliclen- 2- cleoxv- a- D- qalactopvranose

Benzaldehyde dimethyl acetal (2.0 mL, 14 mmol) followed by paratoluenesulfonic acid monohydrate (15 mg) was added to a stirred suspension of N-acetyl-D-galactosamine (1.50 g, 6.77 mmol) in acetonitrile (37 mL) . The reaction mixture was then immersed in a hot (60°C) oil bath and stirred under nitrogen for 3 hours, while a thick white precipitate was observed to form. The reaction mixture was then filtered and the solid washed with cold dichloromethane (about 2 mL), then continued with suction filtration under nitrogen flow. The white powder was then placed in a preweighed glass and placed under vacuum (0.06 mbar) for 72 hours to give the pure desired product as only the α-isomer (1.74 g, 83%).

<1>H NMR 8H (300 MHz, d6-DMSO) 1.83 (3H, s, CH3), 3.80-4.17 (6H, m, H-2, H-3, H-4, H -5 and H-6), 4.65 (1H, d, OH-3), 5.06 (1H, t, H-1), 5.59 (1H, s, ArCH), 6.52 (1H , d, OH-1), 7.33-7.55 (5H, m, ArH) and 7.69 (1H, d, NH); 13C NMR 8C{<1>H} (75 MHz, d6-DMSO ) 23 (CH3), 50, 62, 65, 69 and 76 (C-2, C-3, C-4, C-5, C-6), 91 (C-1), 100 (ArCH), 126 , 128,128 and 138 (arom. C) and 170 (C=0).

Compound 13: 4. 6- O-(3- nitrobenzene)- D- alucopvranose

3-Nitrobenzaldehyde dimethyl acetal was prepared as described for compound 9.

3-nitrobenzaldehyde dimethyl acetal (21.9 g, 0.11 mol), D(+)-glucose (16.0 g, 0.09 mol), paratoluenesulfonic acid (100 mg, 0.5 mmol) and dry DMF (50 mL) was mixed under N2 and stirred at 58°C for 25 min. A vacuum pump was connected and methanol and DMF were slowly distilled off through a cooled column at 55-60°C, 30-40 mbar over 4 hours and 15 minutes. The apparatus was rebuilt to remove most of the DMF by short path distillation and the distillation continued for another 1.5 hours. The distillation residue was a pale yellow syrupy mass.

The syrup mass was dissolved in weakly alkaline (NaHCO 3 ) methanol/water 60:40 and purified on a Lobar C RP-8 column with methanol/water 60:40 as eluent. The product fractions were evaporated (to remove methanol), freeze-dried and combined to give 5 g of a white, woolly solid. The product was purified once more with methanol/water 60:40 as eluent to obtain the title compound sufficiently pure. The yield was 3.3 g, 12% of the theoretical. Gas chromatography indicated that the product consisted of two isomers in a 70:30 ratio.

<1>H NMR (DMSO-d6), 5 rel. to TMS: 8.33-7.63 (5H, m, Ar-H), 6.89+6.60 (1H, d+d, OH-1-l+ll), 5.78 (1H, s +s, acetal-H-i+ii), 5.34 (0.65H, d, OH-3-II), 5.57+5.71 (1.12H, d+d, OH-2-II + OH-3-I), 4.99 (0.56H, m, H-1-I), 4.88 (0.32H, OH-2-I), 4.49 (0.74H, m, H-1- II), 4.28-4.12 (1H, m, H-6'-1+11), 3.85-3.53 (2.27H, m, H-3-I, H-5-I and H-6"-l+ll), 3.49-3.32 (2.58H, m, H-2-I, H-3-II, H-4-I+II and H-5-II) and 3.12-2.98 (0.85H, m, H-2-II).

Compound 14: 4, 6- 0-( 2- hydroxybenzvlidene)- D- qulkopvranose

Salicylaldehyde (74.4 g, 0.609 mol), methanol (91.5 g, 2.86 mol), trimethyl orthoformate (71 g, 0.67 mol) and concentrated hydrochloric acid (165) are mixed in a 500 ml three-necked flask. The reaction mixture is stirred at room temperature for 15 minutes and refluxed for another 25 minutes. After cooling (ice water), an alkaline solution prepared by dissolving KOH (1.1 g) in methanol (7 ml) is added and stirring is continued for 25 minutes. All volatiles are removed ( water jet pump) and the formed salicylaldehyde dimethyl acetal is distilled in vacuo (vacuum pump). D(+)-glucose (8.0 g, 44.4 mmol), dry DMF (25 ml), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol begins. The temperature is maintained at 55-60°C and the vacuum is gradually regulated down to complete the reaction. The apparatus is converted for short path distillation to remove DMF. a syrupy residue is formed.

The residue is dissolved in weakly alkaline (NaHCOa) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.

Compound 15: 2-deoxy-4.6-0-(2-hydroxybenzvlidene)-D-qlucopvranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.

2-deoxy-D-glucose (7.3 g, 44.5 mmol), dry DMF (25 mL),

salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.

The residue is dissolved in weakly alkaline (NaHCC«3) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.

Compound 16: 2-acetamido-2-deoxy-4.6-Q-(2-hydroxybenzvlidene)-D- glucopvranose

Salicylaldehyde dimethyl acetal is prepared as described for compound 14.

Mix salicylaldehyde dimethyl acetal (9.5 g, 56.5 mmol), N-acetyl-D-glucosamine (10.0 g, 45.2 mmol), dry DMF (30 mL) and paratoluenesulfonic acid (88 mg, 0.46 mmol) under N2. The reaction mixture is stirred at 50°C under reduced pressure to drive the reaction to completion. The apparatus is rebuilt to remove. DMF by short path distillation and the distillation is continued at 55-60°C, max. vacuum.

The residue is neutralized by adding methanol/water containing a little NaHCO 3 and purified on a Lobar RP-8 column with methanol/water as eluent. The identity is confirmed by NMR spectroscopy.

Compound 17: 4, 6- O-( 2- hydroxybenzvlidene)- D- qalactopvranose

D(+)-gaiactose (8.0 g, 44.4 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.

The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.

Compound 18: 2-deoxy-4.6-0-(2-hydroxybenzvlidene)-D-galactopyranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.

2-deoxy-D-galactose (7.3 g, 44.5 mmol), dry DMF (25 mL),

salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.

The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.

Compound 19: 2- acetamido- 2- deoxy- 4. 6-( 2- hydroxybenzvlidene)-D- chalactopvranose

Salicylaldehyde dimethyl acetal is prepared as described for compound 14.

N-Acetyl-D-galactosamine (10.0 g, 45.2 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A distillation residue is formed.

A weakly alkaline solution is made by mixing NaHCO3 in methanol/water and the distillation residue is neutralized by adding this solution. The slurry that forms is filtered and washed with 1% NaHCO 3 solution and water. The product is analyzed by gas chromatography and recrystallized if necessary. The product is dried in a vacuum when it is sufficiently clean.

The identity is confirmed by NMR analysis.

Compound 20: 4. 6- 2-hydroxybenzvlidene)-D-mannopyranose Salicylaldehyde dimethyl acetal is prepared as described for compound 14.

D(+)-mannose (8.0 g, 44.4 mmol), dry DMF (25 mL), salicylaldehyde dimethyl acetal (8.33 g, 49.5 mmol) and paratoluenesulfonic acid are mixed under N 2 at 50°C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is kept at around 55-60°C and the vacuum is gradually regulated down to drive the reaction to completion. The apparatus is converted for short path distillation to remove DMF. A syrupy residue is formed.

The residue is dissolved in weakly alkaline (NaHCO 3 ) methanol/water and purified on a Lobar RP-8 column with methanol/water as eluent. The product fractions are freeze-dried and combined. The identity is confirmed by NMR analysis.

Biological experiments

Example 1

Protein synthesis

The rate of protein synthesis was calculated as previously described (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981). Briefly, the cell protein was labeled to saturation point by at least 2 days of preincubation with [<14>C]-valine of constant specific radioactivity (0.5 Ci/mol). To keep the specific radioactivity constant, a high concentration of valine was used in the medium (1.0 mM). At this valine concentration, the dilution of [<14>C]valine in the intracellular and the proteolytically generated valine will be negligible (Ronning, O.W., et al., Exp. Cell Res. 123: 63-72, 1979). The protein synthesis rate was calculated from the incorporation of [<3>H]valine relative to the total [<14>C] radioactivity in the protein at the beginning of the different measurement periods and expressed as a percentage per hour (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981).

It can be seen from fig. 1 that compound 2 leads to stronger inhibition of protein synthesis than Tucaresol.

Example 2

Experiments with compound 2 and compound 5 on NMRI mice infected with FRI END ervtroleukemia virus (FLV)

Virus: Eveline cells were provided by Prof. Gerhard Hunsman, Munich. We have

showed that this virus, which was originally used as a source for Friend helper virus, contains a defective virus of the same size as the Spleen Focus Forming Virus (SFFV) which causes erythroleukemia in NMRI mice after 4-8 weeks.

Mice: The NMRI mice came from the old Bomholt Gård in Denmark, and were purchased via SIFF. The mice arrived on 6 May and participated in the experiment from 11 May. They were infected intraperitoneally with 50 microliters of Evelin culture supernatant. The treatment was started after 24 hours. Compound 2 and compound 5 were dissolved in sterile isotonic glycerol solution in a concentration corresponding to 5 mg per kg by administration of 50 microliters intraperitoneally.

The experiment was set up as follows:

10 mice uninfected control

10 mice infected control

5 mice uninfected, treated with compound 2

10 mice infected, treated with compound 2

5 mice uninfected, treated with compound 5

10 mice infected, treated with compound 5

The mice received intraperitoneal injections once a day for 19 days. From June 1 to June 16, when they were euthanized, they received no treatments. The mice were euthanized on 16 June. Blood samples were taken (for later analysis). The spleen was removed and weighed (see Table 1 below). A piece of the spleen was frozen in nitrogen to make thin sections and a piece was fixed in formalin.

The results are also shown in fig. 3.

As can be seen, there is a significant difference in the spleen weight for the infected animals compared to the uninfected ones. The spleen weight of the uninfected animals treated with compound 2 or compound 5 is above the weight of the uninfected control animals, although this is not significant. It is seen that infected animals that were treated with compound 2 actually have a lower average spleen weight than the uninfected animals that were treated in the same way (here it is assumed that the result is due to an animal in the control group that had a relatively large spleen).

A histological examination revealed that the uninfected control animals had normal spleen anatomy. All the animals in the infected untreated group had invasion of pathological leukemic cells in the red pulp. The spleens from both uninfected control groups that were treated with h.v. compounds 2 and 5, have enlarged germinal centers, which is interpreted as an expression of immune stimulation. No leukemic changes are found in the spleen of the infected group treated with compound 2.1 the group treated with compound 5, the animal with the largest spleen (308 g) had leukemic changes, while all of the infected control group had leukemic changes.

The results are encouraging considering the aggressive nature of FLV in mice and also when comparing the effects of the drugs with the effects of azidothymine and other viral treatments.

Example 3

Proliferation of peripheral blood mononuclear cells

The inventors performed an experiment in which peripheral blood mononuclear cells were exposed to superantigen together with benzaldehyde, deuterated benzaldehyde, compound 2 or zilascorb(<2>H). Superantigen is used as a highly active standard for the propagation of T cells and is presented to T cells by means of antigen-presenting cells.

The experiment showed (see Figure 2) that adding benzaldehyde, deuterated benzaldehyde or compound 2 significantly increased the proliferation of peripheral blood mononuclear cells in a bell-shaped dose-dependent manner, while very little effect was found with zilascorb(<2>H). The fact that we were able to increase the propagation signal from the superantigen suggests that the compounds work by helping to stimulate the T cells.

Conclusions

The benzaldehyde derivatives according to this invention react to Schiff bases with certain groups on the cell surface, e.g. free amino groups. Since many cell processes, such as protein synthesis, cell cycle, immune response, etc., are controlled by signals from the cell surface, these bonds will change the behavior of the cell. We have also shown that the benzaldehyde complexes on the cell surface change the adhesion characteristics of the cell. We have shown that the compounds of this invention may be useful in new treatments to combat viral infections and possibly also infections by other microorganisms.

Administration

The therapeutic agents produced by the present invention can be given by antiviral treatment. These therapeutic agents can also be given as immunomodulators.

For this purpose, the compounds of formula (I) may be formulated in any suitable manner for administration to a patient, either alone or with the addition of suitable pharmaceutical carriers or excipients.

It is particularly preferred that the formulations for systemic therapy are prepared either as oral preparations or parenteral formulations.

Suitable enteral preparations will be tablets, capsules, e.g. soft or hard gelatin capsules, granules or powders, gels, suspensions, solutions or suppositories. Such preparations will be prepared in a known manner by mixing one or more of the compounds of formula (I) with non-toxic, inert solid or liquid carriers.

Suitable parenteral preparations of the compounds of formula (I) are injection or infusion solutions.

When given externally, the compounds of formula (I) can be formulated as a solution, ointment, cream, syrup, tincture, spray or the like containing the compounds of

in formula (I) with the addition of non-toxic, inert solid or liquid carriers common in topical preparations. It is particularly well suited to use a formulation that protects the active ingredient from air, water or the like.

The preparations may contain inert or pharmacodynamically active additives. E.g. tablets or granules may contain a series of binders, fillers, carrier substances and/or diluents. Liquid preparations can, for example, be available in the form of a sterile solution. Capsules may contain a filler or thickener in addition to the active ingredient. Furthermore, the preparation may also contain flavor-enhancing additives as well as such substances that are usually used as preservatives, stabilizers, emulsifiers or humectants, salts to vary the osmotic pressure, buffers and other additives.

The dosage may vary according to the disease, the method of use and the route of administration, as well as the needs of the patient. In general, a daily dose in a systemic therapy for an average adult patient will be about 0.01-500 mg/kg body weight once or twice a day, preferably 0.5-100 mg/kg body weight once or twice a day and preferably 1-20 mg/kg weight once or twice a day.

If desired, the pharmaceutical preparation of the compound of formula (I) may contain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

Claims (2)

1. Use of a benzaldehyde derivative of formula I: where L is H or D; Ar is phenyl or mono- or di-substituted phenyl, where the substituent (e) is selected from the group consisting of alkyl with 1-6 carbon atoms, CF3, fluorine, chlorine, nitro, cyano, OR1, SR<1>, N(R <1>)2 or COOR<1> where R<1> is H, CF3 or alkyl with 1-6 carbon atoms, or OC(0)R2, CA(OR2)2, CA[OC(0)R<2> ]2, NHC(0)R<2> or N[C(0)R<2>]2 where A is H or D and R2 is CF3 or alkyl with 1-6 carbon atoms, or C(0)R3 where R3 is H, D, CF3 or alkyl of 1-6 carbon atoms; Y is selected from the atoms or group consisting of H, D, fluorine, chlorine, nitro, OR<1>, OC(0)R<2>, N(R<1>)2, NHC(0)R<2> or N[C(0)R<2>]2 where R1 and R<2> are as defined above; R is H, CF3 or alkyl of 1-6 carbon atoms; or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prevention and/or treatment of infections by viruses, protozoa, fungi and other microorganisms by means of modulation of the immune system. 2. Use according to claim 1, where the benzaldehyde derivative is 4,6-O-benzylidene-D-glucopyranose, 4,6-O-(benzylidene-di)-D-glucopyranose, 4,6-O-benzylidene-D- galactopyranose, methyl-4,6-0-benzylidene-α-D-mannopyranoside, 4,6-0-(benzylidene-di)-2-deoxy-D-glucopyranose, 4,6-0-(4-carbomethoxybenzylidene)-D- glucopyranose, 4,6-0-benzylidene-2-deoxy-D-glucopyranose, 2-acetamido-4,6-0-(benzylidene-di)-2-deoxy-D-glucopyranose, 2-acetamido-2-deoxy- 4,6-0-(3-nitrobenzylidene)-D-glucopyranose, 4,6-0-(benzylidene-di)-D-galactopyranose, 4,6-0-(benzylidene-di)-D-mannopyranose, 2- acetamido-4,6-0-benzylidene-2-deoxy-α-D-galactopyranose, 4,6-0-(3-nitrobenzylidene)-D-glucopyranose, 4,6-0-(2-hydroxybenzylidene)-D-glucopyranose, 2-deoxy-4,6-0-(2-hydroxybenzylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(2-hydroxybenzylidene)-D-glucopyranose, 4,6-0- (2-hydroxybenzylidene)-D-galactopyranose, 2-deoxy-4,6-0-(2-hydroxybenzylidene)-D-galactopyranose, 2-acetamido-2-deoxy-4,6-0-(2-hydroxybenzylidene)- D-galactopyran ose, 4,6-0-(2-hydroxybenzylidene)-D-mannopyranose, 4,6-0-(2-acetoxybenzylidene)-D-glucopyranose and/or 4,6-0-(2,3-dihydroxybenzylidene)- D-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof.