NO312226B1 - Use of 4,6- <Omicron> - (benzylidene-d1) -D-glucopyranose and the corresponding 1-isomer in the prophylaxis and treatment of cancer - Google Patents

Description

This invention relates to the use of benzaldehyde derivatives for the production of anticancer agents.

Most cancer drugs used today are cytotoxic. Although these 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) has so far been shown to have 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 recognized, among other things 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 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, however, 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 involves key biochemical processes such as transamination, decarboxylation and other amino acid modifying reactions catalysed 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-arm 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 readily forms 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., 154 (1968), 323-331).

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 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 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 for normal stem cells, which is often less than 10 hours, with the cell cycle time for most cancer cells in solids; tumors, which are typically 30-150 hours (see Gustavo and Pileri in: The Cell C<y>cle 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 accumulates

protein 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 unharmed.

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) produces tumor necrosis in vivo in implants of human tumor tissue on; 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 4,6-0-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 now surprisingly found that benzaldehyde derivatives of hexose-type sugars (including 4,6-0-(benzylidene-di)-D-glucopyranose, compound 2) have an unexpectedly strong effect against cancer in certain organs or tissues. We cannot explain the mechanism of this selectivity yet, but we believe that it is connected with the sugar group in the derivatives having an affinity for certain cells or tissues. For example, compound 2 (the glucose derivative of deuterated benzaldehyde) has an astonishingly much better anti-liver cancer activity than zilascorb(H) (derivative of deuterated benzaldehyde and ascorbic acid) (see Example 6). Also in experiments with Panc-1 cells derived from human pancreatic cancer tumors, we have surprisingly found that compound 2 gives stronger protein inhibition than zilascorb(<2>H) (see example 2, fig. 3). Common to these tissue types is a high concentration of certain glucose transporters and receptors. There are no indications in the known technique that indicate a better effect in the treatment of liver and pancreatic cancer with compound 2 than with zilascorb(<2>H). On the contrary, based on known experiments presented in EP-0283139, we would expect that compound 2 would show a similar or slightly weaker effect than zilascorb(<2>H).

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 3 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. I. 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 3). 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.

In an in vzvo model where we compared compounds 1 and 2, cancer cells from the liver-invading human colorectal cancer cell line C170HM2 were injected intraperitoneally into nude mice after treatment with the two drugs. The animals treated with compound 2 had surprisingly much less tumor formation in the liver compared to those treated with compound 1 (see Example 7).

In the above experiments, we have shown that the D isotope effect may be more relevant for cancer treatment with aldehyde derivatives than was previously known. The two experiments together (examples 3 and 7) show that compound 2 and similar drugs can be particularly beneficial for the treatment of primary and secondary liver cancer.

Use of compound 1 (4,6-O-benzylidene-D-glucopyranose) i.a. against cancer of the liver is presented in US-4,882,314.1 experiment 2 in US-4,882,314 a patient who had colon cancer with metastases in the liver was cured. However, in Experiment 5 of the aforementioned US patent, a patient who had a primary liver tumor died after four months of treatment.

Later, G. Tanum et al., Am. J. Clin. Oncol. (CCT), 13(2), 1990, pp. 161-163 concluded that compound 1 is not active in patients with colorectal cancer. In this study, the treatment was given daily for two months and: the effect was evaluated by measuring the tumor size.

In an experiment conducted by the inventors on chemically induced liver cancer in Wistar rats treated intravenously for 10 days, it was surprisingly observed that massive necrotic areas had developed in 2 out of 5 animals treated with compound 2, but none of the 5 animals treated with compound 1 (see Example 6).

The inventors of the present invention also conducted a study where nude mice injected with C170HM2 cells were treated with compound 1 and compound 2. The tumor line C170HM2 is a human colorectal cell line obtained from the primary tumor of the patient. At termination, the liver was exposed while counting the visible liver tumors and measuring their total cross-sectional area. The effect of compound 2 on the liver invasion of the human colorectal tumor C170HM2 was far superior to that of compound 1 (Example 7).

Furthermore, the inventors surprisingly observed that compound 2 produced a significantly better effect on the development of liver cancer in rats than zilascorb(<2>H). After initial nitrosamine treatment and partial hepatectomy in young rats, the animals developed liver cancer within 3 to 6 months. After an additional 11 months of treatment with either zilascorb(<2>H) or compound 2, both the number of animals that developed liver cancer as well as the amount of cancerous tissue in the cancerous tumors were reduced several times more for animals treated with compound 2 than for animals that was treated with zilascorb(<2>H) (see Example 6).

Thus, it has now been found that some of the compounds of formula (I), such as compound 2 and compound 5, surprisingly provide a much better therapeutic effect on liver cancer (primary as well as metastatic) than the previously known effects.

In an inbred strain of Wistar rats (see Eker R., Acta Path. Microbiol. Scand., 34, (1954) 554-562), the animals develop kidney cancer as a result of an autosomal dominant gene. At 11 months of age, the animals were operated on to inspect which had developed cancer (50% of them had kidney cancer). Animals with tumors of 2-4 mm diameter were included in the experiment since experience indicates that such small tumors do not have necrotic areas: These rats received injections daily for 10 days, with isotonic saline solution (placebo), with saline containing zilascorb(<2> H), with the non-deuterated analogue of zilascorb(<2>H) (5,6-O-benzylidene ascorbic acid sodium salt) or with compound 2. While the tumors of animals receiving the non-deuterated analogue of zilascorb(<2> H) and those receiving placebo showed little or no necrosis after 10 days of treatment, the tumors of those animals treated with zilascorb(<2>H) or compound 2 were semi-necrotic (see Example 8).

i The chemically induced carcinogenesis (as in example 6) 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 who are infected with hepatitis B and C. It can therefore be assumed that 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 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 vz/ro 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. IWO 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, the preferred immunostimulating substance is 4-(2-formyl-3-hydroxy-phenoxymethyl)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 weakens. 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-hydroxy-phenoxymethyl)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 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. 1 and fig. 2, it was demonstrated that compound 2 was more effective than Tucaresol with respect to both measured parameters.

The immunostimulating effect of the new compounds 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 aim of the invention to provide compounds for effective and beneficial prevention and/or treatment of liver cancer (primary liver cancer as well as liver metastases from other forms of cancer, e.g. colorectal cancer). Preventive treatment can also be of great importance in preventing the development of liver cancer in people with hepatitis B or C infection.

Another aim of the invention is to provide compounds for effective and beneficial prevention and/or treatment of kidney cancer.

A third aim of the invention is to provide compounds for effective and beneficial prevention and/or treatment of pancreatic cancer.

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

The compounds according to the present invention are 4,6-O-(benzylidene-di)-D-glucopyranose (compound 2), the corresponding L-isomer, or pharmaceutically acceptable salts thereof.

Detailed description of the invention

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

Description of the figures

Fig. 1: The data represent an experiment where NHIK 3025 cells were treated with compound 2 (A) or Tucaresol (•) 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 value of colony counts from 5 parallel dishes. The standard deviations are shown if they are larger than the symbols. Fig. 2: The rate of protein synthesis compared 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. 3: The rate of protein synthesis compared to untreated control group of Panc-1 cells treated with compound 2 (■) or zilascorb(<2>H) (a). 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 standard deviations are smaller than the symbols. Fig. 4: Median adhesion force for cells exposed to different benzaldehyde derivatives. The cells were exposed to a 1 mM concentration of compounds 1 and 2. Fig. 5: 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. 6: NMRI mice were infected intraperitoneally with spleen-invading Friend erythroleukemia virus. Infected and uninfected mice were treated intraperitoneally daily with 5 mg/kg of compound 2. After 19 days of treatment, the spleen was dissected out and weighed.

Fig. 7: What proportion of the animals had liver tumor tissue at necropsy.

Fig. 8: Average amount of tumor material per live in the animals that had developed tumors. Fig. 9: Growth curves representing average body weight per animals for each group. The time scale represents the age of the animals. To make the curves clearer, the standard deviations are shown only on a few of the measurements. Fig. 10: Plotting both the frequency and the size of the liver tumors on the same curve, with a logarithmic scale on the size axis. Fig. 11: Shows the effect of compounds 1, 2 and 5 on the liver invasion of human colorectal tumor, C170HM2. Compound 5 is 4,6-O-(benzylidene-di)-2-deoxy-D-glucopyranose.

Description of the attached tables.

Table 2: Histological observations in normal tissue and cancer tissue. Group 1: untreated control.

Table 3: Histological observations in normal tissue and cancer tissue. Group 2: placebo.

Table 4: Histological observations in normal tissue and cancer tissue. Group 3: 85 mg/kg/day of compound 2.

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, D(+) or L(-) glucose is condensed with benzaldehydes or benzaldehyde equivalents to form benzylidene glucose acetals. Particularly preferred is a reacetalization strategy where the benzaldehyde is introduced protected as the dimethyl acetal instead of the benzaldehyde 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

used will in each individual case depend 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. para-toluenesulfonic 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 widely used reaction medium.

The deuterated compounds can be prepared as described above, but starting from the dimethyl acetal of benzaldehyde-di. Preparation 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 Bl.

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

Compound 1: 4, 6- O- benzylidene- D- glucopvranose

This known compound was prepared as described for compound 2, starting from undeuterated benzaldehyde dimethyl acetal. The identity was confirmed by

'H NMR spectroscopy in DMSO-d6:

8 rel. to TMS: 7.58-7.29 (5H, m, Ar-H), 6.83 (0.4H, d, OH-l-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-a), 5.00 (0.6H, H-1-a), 4.82 (0.6H, d, OH-2-a), 4.49 ( 0.4H, t, H-l-P),

4.18-4.02 (1H, m, H-6'-a+P), 3.89-3.77 (0.6H, m, H-5-a), 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-(benzylidene-dj)-D-glucopyranose

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 clumps to fall apart and become suspended. 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 of 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 a ct:p anomer ratio of approximately 7:3.

<!>H and <13>C NMR (DMSO-d6), 5 rel. to TMS: 7.55-7.28 (5.00H, m, Ar-H), 6.85 (0.27H, d, OH-l-p), 6.58 (0.71H, d, OH-l-a) , 5.24 (0.27H, d, OH-3-P), 5.19 (0.28H, d, OH-2-p), 5.61 (0.71H, d, OH-3-a ), 4.99 (0.72H, H-l-a), 4.82 (0.71H, d, OH-2-a), 4.48 (0.29H, t, H-l-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 (2.61H, m, H-3-P, H-4-a+p, H-5-p and H-2-a) 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-l-P ), 93.211 (C-l-a), 81.729 (C-4-a), 80.897 (C-4-p), 75.796 (C-2-p), 72.906 (C-2-a 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-a).

Biological experiments

Example 1

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

Cell culture calculations

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 cultured 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.

Based on fig. 1 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. 2 that compound 2 leads to stronger inhibition of protein synthesis than Tucaresol.

From fig. 3 it can be seen that compound 2 leads to stronger inhibition of protein synthesis than zilascorb (H). The cells used in the experiment are Panc-1 cells from a human pancreatico-salivary tumor (Lieber et al., Int. J. Cancer, 15: 741-747, 1975) grown in medium E2a.

Example 3

Cell attachment 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. 4 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 pyroglutamyl-glutamylglycylserylsparagine 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. Med. 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 extra-cellular 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 4

Experiments with compound 2 on NMRI mice infected with FRIEND erythroleukemia virus (FLV)

Virus: Eveline cells were provided by Prof. Gerhard Hunsman, Miinchen. We have shown that this virus, which was originally used as a source of Free end 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 was dissolved in sterile isotonic glycerol solution in a concentration corresponding to 5 mg per kg by administration of 50 microliters intrapeirtonally.

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

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. 6.

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 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 treated with compound 2 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.

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 5

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 5) 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 a very small 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.

Example 6

Effects on tumors in liver cancer in rats induced with nitrosamine

In this experiment, we tested whether 11 months of treatment with compound 2 or zilascorb(H) could prevent the development of liver cancer in rats after partial hepatectomy and treatment with diethylnitrosamine (DENA) and phenobarbital.

Materials and methods:

We used Wistar Kyoto rats from the Versuchtierzucht Institut, Hannover. A partial hepatectomy (70% removed) was performed on 4-week-old rats at Radiumhospitalet before intraperitoneal treatment with diethylnitrosamine (DENA) and 4-week feeding with phenobarbital. Most of the animals developed liver cancer within 10 months of such carcinogenic initiation. Six and a half months after this, i.e. before any cancer had developed, treatment was started at the Norwegian Institute of Occupational Health and Safety. 56 animals were randomly divided into 4 groups of 14 in each and given the following daily intravenous drug dose:

Group 1: control, no injections

Group 2: placebo, saline injections only

Group 3: 85 mg/kg compound 2

Group 4: 100 mg/kg zilascorb(<2>H)

The treatment was given in a 5-week periodic cycle: first 3 weeks daily intravenous injection, then 2 weeks break. In total, 9 complete 5-week treatment periods were performed. Total elapsed time from the first treatment to the end of the treatment and autopsy was 11 months.

At the autopsy, the volume of the liver tumors was approximately estimated, but without cutting the liver material. After fixation in formalin, Professor Jahn M. Nesland, head of the pathology department at the Norwegian Radium Hospital, first performed an accurate estimate of the tumor mass by cutting open and inspecting the tissue macroscopically and then performed a histological examination both on the tumor tissue (which appeared mainly in the liver ) as well as the normal tissue from all relevant organs and tissues.

A histological examination of the occurrence of multiple foci as well as premalignant nodules was carried out for each of the animals.

Results

Drug effects on the tumors, analyzed as the number of animals that had liver tumors at necropsy, show that there is a significantly lower frequency of animals with liver tumors in the group treated with compound 2 than in the one treated with placebo. Analysis using a one-tailed Fischer's exact test gives a p-value of 0.05. If both control groups are combined, there are 28 control animals. 25 of these developed liver cancer. Of the 14 animals treated with compound 2, 7 developed liver cancer. In this case, the number of animals is high enough for an x<2->test, which shows that the difference is significant with p = 0.015.1 the group treated with zilascorb(<2>H) developed liver cancer in 11 out of 14 animals. This is only one fewer than in the control group, and not significantly.

Fig. 9 shows average body weight measurements for each group of animals throughout the period from the partial hepatectomy and nitrosamine treatment (time 0). Both the body weight measurements and the histological evaluation of normal tissues seem to clearly indicate a total absence of side effects. The jagged shape of the body weight curves (Fig.

9) has a clear connection with the treatment, but not with the drugs. Rather, it is the injections themselves that work on the animals, since animals treated only with saline had exactly the same characteristic body weight variations as those treated with saline and drug.

That animals treated with 210 intravenous injections over an 11-month period show signs of body weight variation is not surprising. For each injection, the animals were warmed slightly under an electric heat light bulb, and were then immobilized in a specially constructed holder for the injection to take place. Although this procedure took place in a quiet room and was performed by a skilled person trained to sedate them, it is not surprising that it can cause a biological reaction in the animals.

One aspect of the adverse event investigation is the histological evaluation of normal tissue from various organs of the body. This rather comprehensive survey can be easily summarized from tables 2 to 4 (attached). In no case were any abnormalities attributable to the drug treatment found. It is worth noting that even the rat tails, where the intravenous injections were given daily for such a long time, were not damaged by the treatment.

However, an interesting conclusion can be drawn from the frequency of preneoplastic lesions in the liver. From Tables 2 to 4, all the animals developed multiple foci. This lesion is believed to be an early stage in the process of developing malignant liver cancer and does not appear to be affected by drug therapy. Moreover, preneoplastic nodules, as if restricted to a few animals, occur in all groups. The present data therefore do not indicate any drug effect on this lesion, which further strengthens the impression that compound 2 has a real cancer-specific effect on the liver.

In fig. 10, both the frequency and the size of the liver tumors are plotted on the same curve with a logarithmic scale on the size axis.

The incidence of tumor development in the liver: The number of animals in the control group and in the placebo group that developed liver cancer is 13 and 12 respectively of the 14 animals in each group (tables 2 and 3). In group 3 (85 mg/kg of compound 2), only 7 of the 14 animals developed liver cancer (Table 4). For statistical testing of the difference between group 2 (placebo) and 3, the number of cases is too low for an x<2->test. But a one-sided Fischer's exact test can be done, which shows that the two groups are significantly different (p = 0.05) with regard to the occurrence of liver cancer. This difference becomes statistically even stronger if we also include the untreated control group (group 1) in the control group. In that case, there are 28 animals in control, of which 25 developed liver cancer, and thus an x<2->test can be accepted. In this case, the difference between group 3 and the controls is significant with p = 0.015.

In group 4 (100 mg/kg zilascorb(<2>H)), 11 of the 14 animals developed liver cancer. This is only one fewer than in the placebo group, and in no way significant. Thus, we must conclude that the analysis of the development of liver cancer showed that the treatment with compound 2 significantly reduced this development, while zilascorb(<2>H) had no such effect.

The size of the developed liver tumors: The size of the liver tumors is most easily analyzed based on fig. 10, but also shown in Tables 3 to 4. Here it is seen that compound 2 has an over-proving effect: while 50% of the animals in the control and placebo groups had more than 10 cm3 of cancerous tissue, none of the animals in the group treated with compound 2 so much cancer tissue (x<2->test: p = 0.0038). Moreover, only 3 animals (i.e., 21%) in the group treated with compound 2 had more than 1 cm<3> of cancerous tissue, while 10 and 13 animals (i.e., 71 and 93%) in the placebo and control groups, respectively, had cancerous tissue above this limit (Fischer's exact test: p = 0.0002 and 0.011, respectively).

Thus, the effect of compound 2 against cancer is even clearer when tumor size is taken into account than when only the frequency of cancer development is analysed. In fact, this effect is so striking that it was evident even at autopsy, when only a quick macroscopic view of the liver was taken.

For animals treated with zilascorb(<2>H), the effect is much weaker than for those treated with compound 2, even when tumor volume is taken into account. The number of animals treated with zilascorb(<2>H) that had more than 1 cm<3> liver cancer tissue was 7. A significance test against the control and placebo groups, similarly to compound 2, gives p values of 0.036 and 0, respectively, 44. When compared with the untreated control group, the difference is therefore significant, while when compared with the placebo group, the difference is clearly not significant. It must be mentioned here that 3 of the animals in the placebo group had a tumor volume just under 1 cm<3>, and that if the basis of comparison had been 0.5 cm3 there would have been a significant difference also compared to the placebo group (see fig. 10). Nevertheless, the present analysis suggests that the effect of zilascorb(<2>H) is weak, and probably on the verge of being significant. Furthermore, there is no difference between the placebo animals and those treated with zilascorb(<2>H) in terms of the number of animals that had tumor volumes above approx. 5 cm<3>.

In a separate experiment where the rats received zilascorb(<2>H) and compound 2 for only 10 days, histological examinations of the liver tumors showed no changes in the animals treated with zilascorb(<2>H), while 2 of the 5 animals that were treated with compound 2 showed increased tumor necrosis.

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 RPMI1640 culture medium (Gibco, Paisley, UK) containing 10 vol% heat-inactivated fetal calf serum (Sigma, Poole, UK) at 37 °C in 5% CO2 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 lxl0<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 cancer research- department at the 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, 2 and 5 on liver invasion of human colorectal tumor C170HM2 is shown in Fig. 11.

Example 8

Necrotizing effect on primary rat kidney adenoma cells

In an experiment on hereditary rat kidney adenomas (Eker and Mossige, Nature 189, (1961) 858-859), severe tumor necrosis was found after 10 days of intravenous injections of 85/kg body weight of compound 2 in two of the animals. In animals that received saline solution without drug, little or no necrosis was observed.

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 benz-aldehyde 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 in certain organs such as the liver and kidneys. We believe that this phenomenon is associated with the receptor affinity of these organs for the sugar group in the derivatives.

Administration

The pharmaceutical compositions according to the present invention can be given in cancer treatment.

For this purpose, the compounds according to the present invention can be formulated in any suitable way to be administered 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, grains 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. can

tablets or granules 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 compounds may contain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

Claims (2)

1. Use of 4,6-0-(benzylidene-di)-D-glucopyranose, or the corresponding L-isomer or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prevention and/or treatment of liver cancer, kidney cancer and cancer in the pancreas. 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.