NO309305B1 - Use of benzaldehyde derivatives in the manufacture of pharmaceutical preparations for the prevention and / or treatment of cancer, as well as certain new benzaldehyde derivatives - Google Patents

Description

This invention relates to a new use of benzaldehyde derivatives which

anticancer agents. Some of the compounds used in accordance with this invention are novel perse.

Most cancer drugs used today are cytotoxic. Although these

the agents have shown good results in the treatment of some forms of cancer such as lymphoma, leukemia and testicular cancer, they often produce serious and unacceptable side effects that limit the possibility of effective treatment. In addition, chemotherapy for a number of forms of cancer, such as in solid tumors (carcinoma)

so far proved to be of limited value, as established cytostatics rarely improve the prognosis for the patient. The ability of cancer cells to develop resistance to cytotoxic products is also a main reason why they cannot be used for the treatment of solid tumors. There is therefore a need for new cancer drugs with fewer side effects and a more selective effect on cancer cells.

It is known i.a. from EP-0215395, JP-63264411, JP-8800940, JP-55069510 and EP-0283139 that benzaldehydes and derivatives thereof have a selective effect against

cancer.

Aldehydes react with a number of 0-, 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, however, the reaction will normally lead to a

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 visual 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 follows a dehydration step 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 i

proteins during the formation of reversible Schiff bases was 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 ones

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. 5(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. 35(1990), 309-318).

We have previously shown by photographing reactions between radioactively labeled reagents that the benzaldehyde does not enter the cells, but is bound to the cell membrane (Dornish, J.M. and Pettersen, E.O., Cancer Letters 25(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 spectral grayscale 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 benzaldehyde derivatives' inhibition of protein synthesis has been very well studied in vitro in our research group. In solid tumours, the reduced protein synthesis can result in a lack of vital proteins leading to cell death. Normal cells have a potential capacity for protein synthesis that is greater than in most cancer cells in solid tumors. This can be shown by comparing the cell cycle time of normal stem cells, which is often less than 10 hours, with the cell cycle time of most cancer cells in solid tumors, which is typically 30-150 hours (see Gustavo and Pileri in: The Cell Cycle of Cancer, ed .: Baserga, Marcel Dekker Inc., N.Y. 1971, p. 99). Since cells on average double their protein content during a cell cycle, this means that more protein accumulates in growth-stimulated normal cells than in most types of cancer cells.

With this difference between cancer cells and normal cells in mind, there is another difference of similar importance to consider: while normal cells respond to growth-regulatory stimuli, cancer cells have no or a reduced such response. While normal cells under ordinary growth conditions can have a reserve growth potential, cancer cells will therefore have little or no such reserve. If protein synthesis is continuously inhibited over a long period of time in both cancer cells and normal cells, the two cell types will be able to react differently. The normal tissue will be able to use some of the reserve growth potential and thus maintain normal cell production. But cancerous tissue has little or no such reserve. At the same time, the rate of accumulation of protein in cancer cells is quite low (i.e. protein synthesis occurs only slightly faster than protein breakdown). Therefore, the inhibition of protein synthesis may be enough to make the cancer tissue unbalanced with respect to protein accumulation, resulting in a negative balance for certain proteins. During continuous treatment for several days, this will result in inactivation of the cells and necrosis in the tumor tissue while the normal tissue remains undamaged.

To date, 5,6-benzylidene-di-ascorbic acid [zilascorb(<2>H)] is the most tested of the compounds that provide reversible inhibition of protein synthesis and show activity against cancer. The protein synthesis inhibitory activity of this known compound is described in detail by Pettersen et.al. (Anticancer Res., Vol. 11, pp. 1077-1082, 1991) and in EP-0283139. Zilascorb(<2>H) causes tumor necrosis in vivo \ implants of human tumor tissue in nude mice (Pettersen et al., Br. J. Cancer, vol. 67, pp. 650-656, 1993). In addition to zilascorb(<2>H), the closest known compound for cancer treatment is 4,6-O-benzylidene-D-glucopyranose (compound 1). These two compounds have a known general activity against cancer and have been tested clinically against a number of cancers. However, no particular cancerous organs or tissues proved more amenable to treatment than others with these compounds, so developing the drug commercially was not warranted.

We have found that certain of our products, such as compound 8, (2-acetamido-4,6-0-(benzylidene-di)-2-deoxy-D-glucopyranose) produce an unexpectedly good effect in a nude mouse model (see example 3, table 1). 3 out of 8 mice were free of tumors, which is an extraordinary result for similar experiments on immunosuppressed species. The reason for this effect may be that the acetamide group has a high affinity for hyaluronic acid receptors. It is known that malignant tumors are rich in hyaluronic acid and therefore also rich in the associated receptors.

We have also found in our experiments that the deuterated analogue of these compounds is significantly more effective than the corresponding proton analogue. The difference in effect is very striking in our cell binding experiment (see Example 5 and also Example 7). When a hydrogen atom is substituted with the twice as heavy deuterium isotope, the kinetic properties of the molecule change, since the rate of breaking the C-D bond is lower than that of breaking the C-H bond. It is known i.a. from M.l.Blake et.al., J.Pharm. Pollock. 64 (1975), 367-391 that the pharmacological function of drugs can be changed by deuteration.

It is also known (EP 0 283 139 and Anticancer Res. 15:1921-1928 (1955)) that when the acetal proton in 4,6-O-benzylidene-D-glucopyranose is replaced by deuterium (compound 1/compound 2) this can act into both protein synthesis and the cell survival fraction measured in vitro. We believe that a possible explanation for this deuterium isotope effect at the chemical level comes from the fact that the deuterated benzaldehyde is oxidized more slowly to the inactive benzoic acid, resulting in a longer half-life of the deuterated active ingredient at the cellular level. However, one must use drug concentrations greater than 6 mM to see a significant difference in the fraction of NHIK 3025 cells that survive when exposed to compound 1 compared to the survival fraction for compound 2. The difference in protein synthesis inhibition was very small when these cells were exposed to concentrations of 1-10 mM.

Now the inventors did a completely different kind of experiment. The adhesion force between NHIK 3025 cells and substratum was measured after pre-incubation of the cells in solutions of compounds 1 and 2 (see example 5). Even at a concentration as low as 1 mM, an astonishing D isotope effect was seen. Surprisingly, compound 2 significantly reduced the adhesion force to 1/3 compared to the control, while compound 1 did not produce any significant reduction. The inventors believe that compound 2 can interfere with the biosynthesis of integrins so that the cell's ability to bind to the substrate is reduced. Integrins are structural transmembrane proteins that are important for binding cells to the extracellular matrix and for interactions between cells. Inhibiting the function of the integrins can thus directly affect the cancer cells' ability to form metastases. The experiment suggests that the integrins may be particularly sensitive to inhibition of protein synthesis. Compound 2 can therefore be used to prevent metastatic processes during the development of cancer.

The chemically induced carcinogenesis has a mechanism similar to the carcinogenesis of certain virus types such as hepatitis B and C, certain papilloma viruses, certain herpes viruses etc. This is especially the case in the development of liver cancer in patients infected with hepatitis B and C. One can therefore assume that a preventive treatment of these patients with products according to the invention can prevent or delay the development of liver cancer. Also, the fact that these products have a low toxicity profile (eg compound 2) would make them suitable for such treatment.

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 v/fro 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. IN

WO 94/07479 patent claimed 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 is the preferred immunostimulatory substance 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 investigations of the effects in vivo

resulted in a bell-shaped dose/response profile (H.Chen and J.Rhodes, J. Mol.

With. (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 efficient binding of APC to T-

the cell is 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 it

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 disclosure, 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. Surprisingly enough, this 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. 4 and fig. 5, it was demonstrated that compound 2 was more effective than Tucaresol with respect to both measured parameters.

It is a main aim of the invention to provide a new use of benzaldehyde derivatives of formula I for the production of a therapeutic agent for the prevention and/or treatment of cancer.

Another aim of the invention is to provide, by the new application, therapeutic agents for the prevention or treatment of cancer without the compounds having toxic side effects.

A third aim of the invention is to provide, through the new application, therapeutic agents for effective and beneficial prevention and/or treatment of cancer in tissues and cells that have receptors with affinity for corresponding sugar groups.

A fourth objective is to provide therapeutic agents for effective and beneficial prevention of liver cancer in persons with hepatitis B or C infection.

A fifth aim of the invention is to provide new compounds which can be utilized in the application according to the invention.

These and other aims 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(s) is selected from the group consisting of alkyl with 1-6 carbon atoms, CF3) fluorine, chlorine, nitro, cyano, OR<1>, SR<1>, N(R1)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)R2]2 where A is H or D and R2 is CF3 or alkyl with 1-6 carbon atoms, or C(0) R 3 where R 3 is H, D, CF 3 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(O)R<2>]2; and R<1> and R2 are as defined above;

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

or a pharmaceutically acceptable salt thereof, with the proviso that 4,6-0-benzylidene-D-glucopyranose, 4,6-0-(benzylidene-di)-D-glucopyranose, 4,6-0-benzylidene-D- allose and derivatives of 4,6-O-benzylidene-D-allose are excluded.

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

Fig. 1: The data represent an experiment where NHIK 3025 cells were treated with compound 8 (O) or compound 9 (•) for 20 hours at 37°C while attached to plastic petri dishes. Surviving fraction means the proportion of cells that were able to form a macroscopic colony after the treatment. Each point represents the mean of colony counts from 5 parallel dishes. The standard deviations are smaller than the size of the symbols. Fig. 2: The data represent an experiment where NHIK 3025 cells were treated with compound 5 (O) or compound 7 (#) for 20 hours at 37°C while attached to plastic petri dishes. Surviving fraction means the proportion of cells that were able to form a macroscopic colony after the treatment. Each point represents the mean of colony counts from 5 parallel dishes. The vertical bars indicate standard deviations and are shown if they are larger than the symbols. Fig. 3: The data represent an experiment where NHIK 3025 cells were treated with compound 12 (■) for 20 hours at 37°C while attached to plastic petri dishes. Surviving fraction means the proportion of cells that were able to form a macroscopic colony after the treatment. Each point represents the mean of colony counts from 5 parallel dishes. The vertical bars indicate standard deviations and are shown if they are larger than the symbols. Fig. 6: Shows average tumor growth curves for the tumor cell line SK-OV-3 ovarian carcinoma implanted in nude mice. The mice were treated daily intravenously with 1 mg/kg of compound 8 (T) and 7.5 mg/kg of compound 8 (A). ■, the control group, received 0.9% NaCl. Each data point represents the mean tumor volume for 4 to 5 mice relative to the day 1 tumor volume. The vertical bars represent standard deviation. Figures 7-12 show morphological appearance of SK-OV-3 tumors from each of the following 3 groups: the animals treated with placebo (Figures 7 and 8), the group treated with 1 mg/kg/day of compound 8 (figures 9 and 10) and the group treated with 7.5 mg/kg/day (figures 11 and 12). The tumors were fixed in formalin, embedded in paraffin, cut into 6 mm slices and stained with hematoxylin and eosin. The magnification is 40 times. Fig. 13: Shows average growth curves of spheres of the T-47D breast carcinoma cell line. The spheres were treated with 0.1 mM of compound 8 (A) and 1.0 mM of compound 8 (T) dissolved in the medium. ■, control. Each data point represents the mean sphere volume of 6 to 11 spheres. The vertical bars represent standard deviation. Fig. 14 shows microscopic photographs of sections of three NHIK 3025 cell spheres that received different treatments, an untreated control (A), one treated with 0.1 mM of compound 8 for 4 days (B) and one treated with 1.0 mM of compound 8 for 4 days (C). Fig, 15-18: The data show how large a fraction of nuclei within each of the interphase steps, G1, S and G2, have the RB protein bound in the nucleus after treatment with compound 8.

Fio. 19-20: The protein synthesis rate relative to control cells for NHIK 3025 cells (Figure 19) and T-47D cells (Figure 20). Each point represents the average of measurements from 4 parallel samples. The standard deviations are shown with vertical bars if they are larger than the symbols.

Fig. 21: Median adhesion force for cells exposed to different benzaldehyde derivatives. The cells were exposed to a 1 mM concentration of compounds 1 and 2.

Fio. 23: Shows the effect of compounds 1 and 5 on the liver invasion of human colorectal tumor, C170HM2.

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- benzylidene- D- qulcopvranose

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-cc ), 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-cc), 5.00 (0.6H, H-1-a), 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 3: 4. 6- O- benzylidene- D- chalactopyranose

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-oc), 4.56 (0.5H, d, H-1-P), 4.23+4.18 (0.5H+0.5H, d+d, H-4-a+P), 4.14-3 .98 (2H, m, H-6-ot+p), 3.94-3.79 (1.5H, m, H-2-cc, 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-oc + C-3-P), 66.579 (C-5-P) and 62.886 (C-5-a).

Compound 4: metvl- 4. 6- Q- benzvlidene- a- D- mannopvranoside

Methyl α-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), 5 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, H-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 (CH3).

Compound 5: 4, 6-( Benzvlidene-di)- 2- deoxy- D-alukoDvranose

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),

in benzaldehyde dimethylacetal-d-i (11.7 g, 76.4 mmol) and paratoluenesulfonic acid (70 mg,

0.37 mmol) was mixed under N2 to a white slurry. When heated 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.

Then the distillation was stopped, the apparatus rebuilt without the column and the DMF remained

removed by short path distillation at 50-55 °C, max. vacuum. The rest was a pale yellow syrupy mass.

in 1/4 of the syrup mass was dissolved in weakly alkaline (NaHC03) methanol/water 60/40 and

purified on a Merck LiChroprep RP-8 reversed phase column with methanol/water 60/40 as eluent. Product fractions were concentrated to remove methanol and freeze-dried to a white, woolly solid. Products from four separate syntheses were combined in a yield of 3.5 g, 23% of theory.

in

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), δ (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' l+ll and H-3 II), 3.42-3.18 (2.5 H, H-4 l+ll and H-5 II and H2O), 2.10-1.86 ( m, 1H,

H-2 1+ll) and 1.62-1.34 (m, 1H, H-2' 1+ll); 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-O-(4-carbomethoxybenzvlidene)-D-alucopyranose

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 neck bottle. 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 rotavapor bottle. 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), δ (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,

i 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, 80,965.9. 95.9.9.S

Compound 7: 4. 6- 0- benzvlidene- 2- deoxv- D- alucopvranose

12-deoxy-D-glucose (10.0 g, 60.9 mmol), dry DMF (34 mL),

in benzaldehyde dimethyl acetal (11.6 g, 76.2 mmol) and paratoluenesulfonic acid (70 mg, 0.37 mmol) were mixed under N 2 to a white slurry. The reaction mixture was stirred at room temperature for 30 minutes and by heating to 45-50 °C the solid substance was gradually dissolved. It was connected to a vacuum pump to remove methanol through a cooled column (to prevent loss of benzaldehyde dimethyl acetal) and

in 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), δ (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 HsO), 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- qlukopvranose

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, 70,549, 68,15, 67,15, 67,1,1, 67,1,1,1. (sugar-C) and 23.123 and 22.674 (CH3).

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 µJ) were mixed in a 500 mL three-necked bottle to a yellow mush which turned into a solution 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 f3-anomers. <1>H and <13>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-alactopvranose

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 <13>C NMR (DMSO-d6), 8 rel. to TMS: 7.52-7.30 (m, 5H, Ar-H), 6.62 (0.5H, d, OH-1-0), 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+1 H+0.5H+1 H+2H, m+m+m+m+m, H-2-H-6+H 2 O); 138.753. 68.862, 68.486 and 67.730 (C-2, C-3 and C-6) and 65.829 and 62.068 (C-5).

Compound 11: 4, 6- Q-( benzvlidene-di)- D- mannopyranose

Benzaldehyde-di was prepared and converted to benzaldehyde-dimethyl acetal-d! 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 <1>H, <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, 5 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, in 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- benzvlidene- 2- deoxy- a- D- aalactopvranose

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 beaker and placed under vacuum (0.06 mbar) for 72 hours to give the pure desired product as only the oc isomer (1.74 g, 83%).

<1>H NMR 5H (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 6C{<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- glucopvranose

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), 8 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-3-1, 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

Salicylicaldehyde (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 µl) are mixed in a 500 mL three-necked flask. The reaction mixture is stirred at room temperature for 15 minutes and refluxed for a further 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 volatile substances are removed (water jet pump) and the formed salicylaldehyde dimethyl acetal is distilled in a vacuum (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 N 2 at 50 °C. The apparatus is connected to a vacuum pump and the evaporation of methanol starts. The temperature is maintained at 55-60 °C and the vacuum is gradually regulated down to make the reaction complete. 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 15: 2- deoxy- 4. 6- 0-( 2- hvdroxvbenzvlideneVD- glucopvranose

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 (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 16: 2- acetamido- 2- deoxy- 4. 6- O-( 2- hydroxybenzvlidene)-D- glucopyranose

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(+)-galactose (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-alalactopvranose 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 (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 19: 2-acetamido-2-deoxy-4.6-(2-hydroxybenzvlidene)-D-alactopvranose

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-mannopuranose 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

Biological materials and methods used to show the action of the compounds.

Cell culture techniques

Human cells, NHIK 3025, from a cervical tumor in situ (Nordbye, K and Oftebro, R. Exp. Cell Res., 58:458, 1969, Oftebro, R. and Nordbye K., Exp. Cell Res., 58:459 -60, 1969) were cultured in Eagel's Minimal Essential Medium (MEM) supplemented with 15% fetal calf serum (Gibco BRL Ltd). Human breast cancer cells, T-47D, (Keydar, I. et al., Er. J. Cancer, vol. 15, pp. 659-670, 1979) were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, 0.2 u /ml insulin, 292 mg/ml L-glutamine, 50 u/ml penicillin, 50 mg/ml streptomycin. The cells are routinely grown as monolayers at 37 °C in tissue culture flasks. To keep the cells in continuous exponential growth, they were trypsinized and recultured three times per week.

Cell survival factor

The survival factor was measured as the ability to form colonies. Before seeding, the exponentially growing cells were trypsinized, suspended as single cells and seeded directly onto 5 cm plastic dishes. The number of seeded cells was adjusted so that the number of surviving cells would be approximately 150 per slice. After approximately 2 hours of incubation at 37°C, the cells had adhered to the bottom of the slides. The drug treatment was then started by replacing the medium with a medium having the desired drug concentration. After the drug treatment, the cells were washed again with warm (37 °C) Hank's balanced salt solution before fresh medium was added. After 10 to 12 days at 37°C in a CO 2 incubator, cells were fixed in ethanol and stained with methylene blue before colonies were counted.

Figures 1-3 show surviving cell fraction for NHIK 3025 cells treated for 20 hours with either compounds 8 and 9 (Figure 1), compounds 5 and 7 (Figure 2) or compound 12 (Figure 3). The data suggest that all compounds lead to inactivation of the cells in approximately the same dose ranges as zilascorb(<2>H) (Pettersen et al., Anticancer Res. Vol. 11, (1991), pp. 1077-1082).

Based on fig. 4 it can be seen that compound 2 gives a higher degree of cell inactivation than Tucaresol.

Example 2

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. 5 that compound 2 leads to stronger inhibition of protein synthesis than Tucaresol.

Example 3

Experiments with human implant on naked mice

The drugs were tested by treating three human cancer implants on

female bristleless mice. The cell lines used are SK-OV-3 ovarian carcinoma, A-549 lung cancer and Caco-2 colorectal cancer. They were purchased from the American Type Culture Collection and cultured for a short time in vitro before being implanted in the mice. The tumor lines were sent as s.c. implants on nude mice. The mice used

in the experiments were 8-9 weeks old at the time of implantation. Small pieces of tumor were implanted s.c. on the left flank of the animals. Animals with growing tumors (tumor volume 25-110 mm<3>) were randomly assigned to drug and control groups, but it was taken into account that the average tumor size in each of the groups should be approximately the same. The antitumor activity of the drug was measured by growth curves for the tumor volume and histological evaluation of some of the tumors. During the treatment, the tumors were measured 2 times per week by measuring two diameters perpendicular to each other with calipers. The tumor volume was estimated with the formula: volume = (length x width)/2. The growth curves were created by standardizing the tumor volume in the different groups by calculating relative tumor volume (RV) according to the formula RV = Vx/V1, where Vx is the tumor volume on day x and V1

the initial volume at the start of treatment (day 1), and plot the mean volume with standard deviation for each treatment group as a function of time. An exponential curve was fitted to the relative volume growth data and the time interval for tumor volume doubling in each group,

the tumor volume doubling time (TD), was calculated from the adaptation course/en (loge2//c,

where k is the estimated rate constant for the process.) The histological evaluation was based on macroscopic examination of the tumor and light microscopic examination of small tumor sections (6-8 mm thick) embedded in paraffin and stained with hematoxylin and eosin.

Table 1 shows the tumor volume doubling time (TD) in humans

tumor implants on nude mice treated daily intravenously with the indicated drugs and doses.

Fig. 6 shows average tumor growth curves for the tumor line SK-OV-3 ovarian carcinoma implanted in nude mice, where the mice were treated daily with 1 mg/kg and 7.5 mg/kg of compound 8. The curves show a significant growth inhibitory effect at both doses. Fig. 7-12 shows microscope photographs of tumors from each group. These images show a general finding for this compound, namely that there is a difference in tumor cell necrosis between the control tumors and those treated with compound 8. Since the treated tumors necrotize with the treatment, the drug effect is actually even stronger than that shown by the growth curves.

Example 4

Multicellular spheres and activation of retinoblastoma protein (pRB)

The spheres were initiated by transferring suspended single cells to a 25 cm<2>

tissue culture flask containing 12 ml medium. The flask was then placed on a tilt table (MIXER 440, Swelab Instrument) inside a walk-in incubator room at 37 °C. The flip speed was adjusted to 10 flips in 18 seconds. During the tilting, the suspended cells were prevented from sticking to the bottom of the flask. Instead, many cells could adhere to each other and form small cell clumps of typically 50-100 cells each after about 24 hours of rocking. The small clumps were then transferred to another 25 cm<2> tissue culture flask. In this case, the bottom of the flask was previously covered (i.e. coated) with a thin layer of 1.3% sterilized agar (Bacto-Agar, Difco Laboratories,

USA). The clumps of cells settled on top of the agar layer and failed to

stick to this. However, the cells in the clumps stuck to each other and began to divide. After 1 week, the volume of the lumps had doubled several times and they were round, like balls. During this period, the medium was changed 3 times a week and the spheres were transferred to new agar-coated flasks once a week.

When the balls had become about 400 mm in diameter (after 2 to 3 weeks of cultivation) they were

transferred to small microwells with one sphere per well together with 1 ml medium.

Care was taken to ensure that all the selected balls were approximately the same size. The wells were also coated with agar to prevent the beads from sticking to the bottom. The different wells were supplied with new medium containing the test substance in the chosen concentration and then the diameter of each of the spheres was measured once a day. This was done by microscopy, with fixed contrast optics and a grating with known line separation (the distance between two neighboring lines) on one of the eyepieces. In each of the groups there were 8-12 parallel balls. Each day, relative sphere volume (the volume on day n divided by the volume on day 1) was calculated for each individual sphere and growth curves were plotted with the average relative volume for all spheres in a group as a function of time after the start of treatment.

Table 2 shows the volume doubling time (TD) for T-47D bullets treated in 259

hours with compound 8 at the indicated doses. Table 2 shows the volume doubling time

for beads of T-47D cells either untreated (dose = 0 mM) or continuously treated with 0.1 or 1.0 mM of compound 8 in the medium. Compound 8 demonstrably increases the doubling time of spheres (i.e. inhibits sphere growth) in a dose-dependent manner, as the effect is clearly stronger with 1.0 mM than with 0.1 mM of the drug.

Fig. 13 shows average growth curves for the sphere volume of the cell line T-47D breast cancer where the spheres were treated with 0.1 mM and 1.0 mM compound 8 dissolved in the medium.

With NHIK 3025 cell spheres, it was not found that the increase in sphere volume with time was reduced in the same way as with T-47D cell spheres. Instead, the spheres treated with compound 8 disintegrated after a relatively short period of treatment (7 to 10 days). To elucidate the reason for this strong effect, we performed an experiment where we treated spheres of NHIK 3025 cells for only 4 days and then made histological sections of the spheres. The results of this experiment are shown in fig. 14.

Fig. 14 shows microscope photographs of cross-sections of 3 NHIK 3025 cell spheres that had received different treatments, an untreated control (A), one treated with 0.1 mM of compound 8 for 4 days (B) and one treated with 1.0 mM of compound 8 for 4 days (C). The spheres were fixed in 4% formaldehyde and embedded in paraffin before 6 mm thick sections were made and stained with hematoxylin and eosin.

Both spheres treated with compound 8 show significant central areas where the cells appear to be in apoptosis. Apoptotic figures were not found in any of the control spheres, but were widespread in those treated with compound 8.

In both cell types that were treated with compound 8, there is a clear effect of the drug, but it differs from one cell type to the other. For spheres of T-47D cells, there is a reduced volume growth in medicinal spheres which is dose-dependent. In spheres of NHIK 3025 there is no reduction in the growth of the sphere volume due to the medication, the volume of the treated spheres rather increases more quickly compared to control spheres over a period of 9 days. In the treated spheres, however, a significant fraction undergoes apoptosis, so that waste from dead cells in this case makes up much of the sphere volume. The rapid increase in volume in these spheres probably comes from changes in osmotic pressure after dissolution of the cell fragments. Due to the swelling, these spheres become unstable and disintegrate after about 9 days.

The reason why the two cell types react differently cannot be determined with certainty. But it is possible that part of the reason may be an important genetic difference between the two cell types when it comes to the regulation of cell growth and cell proliferation. T-47D cells functionally express pRB, the retinoblastoma protein, a normal tumor suppressor gene important for regulating cell cycle progression in normal cells. This gene is often defective in cancer cells, and NHIK 3025 is one of the cell types that has a defective pRB function. We have observed that pRB can be activated to slow down cells under stress conditions even after the cells have entered the S phase of the cell cycle, suggesting that this gene can protect cells from the inactivating effect of stress in combination with DNA synthesis ( see Åmellem, Sandvik, Stokke and Pettersen, British Journal of Cancer 77

(1998) 862-872). In the present investigation, the benzaldehyde derivative acts as a growth-inhibiting stress effect. Thus, it is possible that the T-47D cells are protected by the functional pRB gene, and therefore do not undergo apoptosis, while NHIK 3025 cells with a defective pRB function do not avoid apoptotic death.

To test the activation of pRB, we used two different cell types that both express pRB normally, T-47D and MCF-7. The nuclear bound pRB protein was measured by flow cytometry. We simultaneously measured DNA and the data were presented in histograms with the two parameters DNA versus pRB. Fixation and staining were performed as described by Åmellem, Sandvik, Stokke and Pettersen, British Journal of Cancer 77(1998) 862-872. Cells that had been extracted with detergent were quickly prepared by resuspending the cells in 1.5 ml low-salt detergent buffer. The extracted nuclei were fixed in 4% paraformaldehyde for 1 h before pRB was bound with PMG3-245 monoclonal antibody (Pharmingen) which recognizes both the hypophosphorylated and hyperphosphorylated forms of the protein. DNA was stained with Hoechst 33258 and the pRB antibody with streptavidin-FITC. The nuclei were measured in a FACStartplus flow cytometer (Becton-Dickinson) with two argon lasers (Spectra Physics) set to r.h.v. 488 nm and UV.

The data in fig. 15-18 show which fraction of the nuclei in each of the three interphase stages, G1, S and G2, has the RB protein bound in the nucleus after treatment with compound 8. When it is bound in this way, it is assumed that pRB regulates the cell out of the cell cycle, i.e. that it takes over cell cycle control. (See Åmellem, 0., Stokke, T., Sandvik, J.A. & Pettersen, E.O.: The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phase during hypoxic stress. Exp. Cell Res. 227 (1996) 106-115.) The figures show data for two types of human breast cancer cells, MCF-7 (Figs. 15 and 16) and T-47D (Figs. 17 and 18) and for drug treatment times of 24 hours (Figs. 15 and 17) and 48 hours (fig. 16 and 18). For both cell types, compound 8 at concentrations above 0.5 mM increases the fraction of nuclei that have bound pRB in all interphase stages. The effect increases with increasing treatment time and is therefore far more marked after 48 than after 24 hours of treatment. This is believed to indicate that the substance activates the cell cycle regulatory function of pRB, resulting in reduced cell cycle progression. However, the dependence on the drug dose is complicated, showing a maximal effect for a dose of compound 8 of around 1 mM and a decrease for higher doses. We therefore see a bell-shaped dose-response curve in much the same way as for compound 10 in the animal experiment on SK-OV-3 implants in nude mice (see table 1). Although it is not yet known why pRB activation is reduced for compound 8 at doses above 1 to 1.5 mM, it is interesting to note that we have found a relatively strong protein synthesis inhibition for this drug at these doses. After 24 hours of treatment of NHIK 3025 cells with 1.5 mM of compound 8, the rate of protein synthesis is 70% of the rate in the control cells (see Fig. 19).

Fig. 20 shows that the protein synthesis after 24 hours of treatment with either 1.5 or 2.5 mM of compound 8 is respectively 75 and 50%, but increases to normal again in approximately 6 hours after the drug is removed. Perhaps the regulatory action of pRB at concentrations in the range of 1.5 to 2.5 mM is itself overridden by the inhibition of the cell cycle that inevitably follows as a result of the reduced inhibition of protein synthesis (see Rønning, Ø.W., Lindmo, T ., Pettersen, E.O. & Seglen, P.O.: Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells. J. Cell Physiol. 107 (1981) 47-57).

Example 5

Cellebindina's measurements

The cell attachment forces were measured with the manipulative force microscope. (G. Sagvolden. Manipulation force microscope. Doctoral thesis, University of Oslo, 1998, and G. Sagvolden, I. Giaever and J. Feder. Characteristic protein adhesion forces on glass and polystyrene substrates by atomic force microscopy. Langmuir 14 (21), 5984-5987, 1998) NHIK 3025 cancer cells were cultured briefly in a CO 2 -independent medium containing 15% fetal calf serum. The cells were exposed for 20 hours to a 1 mM concentration of compound 1 or compound 2 before being released from the cell culture flasks with trypsin. The cells were kept in suspension and seeded in compound 1 or compound 2 medium on polystyrene tissue culture substrates 90 minutes after the trypsin reaction was stopped. The adhesion force between cell and substrate was measured by detaching the cells using an inclined atomic force microscope boom that acted as a force transducer. The cells were detached one at a time and each of the cells was detached only once.

The maximum force applied to each of the cells was recorded as a function of the time elapsed since the cells were seeded on the substrate. Fig. 21 shows the median of a group of 19 force measurements as a function of the average time for cells exposed to compound 1 or compound

2 together with the adhesion force of cells not exposed to these <->compounds. Compound 2 strongly reduces the adhesion force at this concentration, while compound 1 shows no significant effect. The effect of the compounds is mainly to reduce the adhesion force of the cells, but not the time course of the adhesion.

The reduced ability to adhere to the substrate may be related to blocking the integrin-based cell anchoring. It has been shown that such blockage can lead to programmed cell death in both hepatoma and melanoma tumors. (Paulsen JE, Hall KS, Rugstad HE, Reichelt KL and Elgjo K, The synthetic hepatic peptides pyroglutamylglutamylglycylserylsparagine and pyroglutamylglutamylgylcylserylaspartic acid inhibit growth of MH1C1 rat hepatoma cells transplanted into buffalo rats and athymic mice. Cancer Res. 52

(1992) 1218-1221 and Mason MD, Allman R and Quibell M, "Adhesion molecules in melanoma - more than just superglue?" J. Royal Soc. With. 89 (1992) 393-395).

The adhesion force between NHIK 3025 cells and the substrate was measured after preincubation of the cells in solutions of compounds 1 and 2. Even at 1 mM concentration, a surprising D-isotope effect could be seen. Surprisingly, compound 2 significantly reduced the adhesion force to 1/3 compared to the control, while compound 1 did not produce any significant reduction. The inventors believe that compound 2 may have affected the biosynthesis of integrins and thus reduced the cell's ability to bind to the substrate. Integrins are structural transmembrane proteins that are essential for binding cells to an extracellular matrix and for interactions between cells. Inhibiting the function of the integrins can therefore directly affect the metastatic ability of cancer cells. The experiment suggests that integrins may be particularly sensitive to inhibition of protein synthesis. Compound 2 can therefore be advantageously used to prevent metastasis processes in the development of cancer.

Example 7

The effect on liver-invasive colorectal cancer in nude mice

Material and methods

The evaluated cell line, C170HM2, is an established human colorectal cell line (S.A.Watson et al., Eur.J.Cancer 29A (1993), 1740-1745) and was originally obtained from a primary tumor in a patient. C170HM2 cells were maintained in vitro in RPMI 1640 culture medium (Gibco, Paisley, UK) containing 10 vol% heat-inactivated fetal calf serum (Sigma, Poole, UK) at 37°C in 5% CO 2 and humidified conditions. Cells from semiconfluent monolayers were harvested with 0.025% EDTA and washed twice in the above culture medium.

C170HM2 cells harvested from semiconfluent monolayers of cells were resuspended in 1x10<6>/ml sterile phosphate buffered saline, pH 7.4 [PBS] and injected in a 1 ml volume into the peritoneal body cavity of 20 MFI male nude mice (raised in the Cancer Research Department of University of Nottingham). The mice were identified with an electronic tagging system (RS Biotech DL2000 Datalogger). On day 10 after the injection, the mice were randomly assigned to either a placebo control group or experimental groups.

The drugs were dosed intravenously from day 10 and continued until treatment was terminated. The experiment was terminated on day 40 after implantation. The mice were weighed at regular intervals during the experiment.

At the end, the liver was uncovered, visible liver tumors were counted and the total cross-sectional area was measured. The tumors were also photographed. No liquefaction of the tumors had occurred, so they were dissected from the normal liver tissue, weighed and fixed in formal saline. Peritoneal nodules were dissected out and the weight and cross-sectional area measured.

A detailed pathological assessment of the tumors was performed.

The effect of compounds 1 and 5 on liver invasion of human colorectal tumor C170HM2 is shown in Fig. 23.

Conclusions

The benzaldehyde derivatives which according to this invention react to form 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 can be useful in new treatments to fight cancer.

We have found that the hexose derivatives of benzaldehydes are surprisingly more effective than the derivatives of other carbohydrates in treating cancer. We believe that this phenomenon is associated with the receptor affinity of these organs for the sugar group in the derivatives.

Administration

The therapeutic agents produced by the application according to the present invention can be given in cancer treatment.

For this use, 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 administered externally, the compounds of formula (I) may be formulated as a solution, ointment, cream, syrup, tincture, spray or the like containing the compounds of formula (I) with the addition of non-toxic, inert solid or liquid carriers which are customary in local 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 (6)

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, 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(0)R<3> where R<3> is H, D, CF3 or alkyl with 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(O)R<2>]2; and R<1> and R<2> are as defined above; R is H, CF3 or alkyl of 1-6 carbon atoms; or any steroisomer thereof, or a pharmaceutically acceptable salt thereof, with the proviso that 4,6-O-benzylidene-D-glucopyranose, 4,6-O-(benzylidene-d-i)-D-glucopyranose, 4, 6-benzylidene-allose and derivatives of 4,6-benzylidene-D-allose are excluded, for the preparation of a therapeutic agent for the prevention and/or treatment of cancer. 2. Use according to claim 1, for the production of a therapeutic agent for the preventive treatment of cancer caused by viruses such as hepatitis B and C, oncogenic papillomaviruses and other oncogenic viruses. 3. Use according to claim 1 or 2 where the benzaldehyde derivative is, 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-d-i)-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-galactopyranose, 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 of these. 4. A benzaldehyde derivative characterized in that it is 4,6-O-benzylidene-D-galactopyranose, methyl-4,6-0-benzylidene-oc-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-(benzyliclene-cli)-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-galactopyranose, 4,6-0-(2-hydroxybenzylidene)-D- mannopyranose, 4,6-O-(2-acetoxybenzylidene)-D-glucopyranose and/or 4,6-O-(2,3-dihydroxybenzylidene)-D-glucopyranose or the corresponding L-isomers or a pharmaceutically acceptable salt thereof. 5. A pharmaceutical composition characterized in that it includes a benzaldehyde derivative according to claim 4, and a pharmaceutically acceptable carrier, diluent or auxiliary substance. 6. A benzaldehyde derivative characterized in that it is 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, 4,6-0-(3- nitrobenzylidene)-D-glucopyranose, in 4,6-O-(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-galactopyranose, 4,6-0-(2-hydroxybenzylidene)-D-mannopyranose, 4,6-0-(2-acetoxybenzylidene )-D-glucopyranose, 4,6-0-(2,3-dihydroxybenzylidene)-D-glucopyranose or the corresponding L-isomers or a pharmaceutically acceptable salt thereof.