NO309815B1 - Derivatives of 5-nitrofurfural - Google Patents

Description

The present invention relates to derivatives of 5-nitrofurfural which are useful in fighting cancer, fighting viruses, as immunopotentiators and/or agents that can be used to fight diseases that arise as a result of increased cell division and/or to fight autoimmune diseases. Most of the compositions according to the invention are innovative in themselves.

Most agents used in the treatment of cancer today have a cytotoxic mode of action. Although these agents have shown good results in the treatment of a number of cancers such as lymphoma, leukemia and testicular cancer, they often cause severe and unacceptable side effects that limit the possibility of effective treatment. Furthermore, in various types of cancer such as solid tumors (carcinoma), chemotherapy has so far been shown to be of limited value, as the existing cytostatic drugs rarely improve the prognosis for the patient. The ability of cancer cells to develop resistance to cytotoxic products is also a main reason why these products are so ineffective in the treatment of solid tumours. Consequently, there is a great need for new agents for the treatment of cancer which have fewer side effects and a more selective mode of action on the malignant cells.

It is known, among other things, from EP-0215395, JP-63264411, JP-8800940, JP-55069510 and EP-0283139 that benzaldehydes and derivatives thereof have a selective anticancer effect. We have also previously found that some heteroaromatic aldehyde derivatives have even stronger properties for inactivating cells. WO 95/18607 describes the anticancer activity of 5-nitrofurfurylidene diacetate. This compound has a stronger effect on NHIK 3025 cells than the 3- and 4-nitrobenzylidene analogues.

Although most of the current knowledge of how aldehydes react at the cellular level is based on experiments with benzaldehyde, we have found that many results are valid for other aromatic and heteroaromatic aldehydes, including 5-nitrofurfural. Aldehydes react with a variety of O-, S- or N-nucleophilic units such as hydroxy groups, sulfhydryl groups and amino groups to form carbonyl condensation products such as acetals, mercaptals, aminals, etc. With primary amines, however, the reaction normally takes the form of addition of Schiff bases (in my). It is well known that formation of Schiff bases in vivo is involved in key biochemical processes such as transamination, decarboxylation and other amino acid modifying reactions influenced 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 carbonyl condensation reactions are involved in signal communication across membranes, for example in the generation of an immune response.

The formation of imines occurs in a two-step mechanism. First, the amino nucleophile is added to the carbonyl group to form a carbinolamine (aminohydrin) intermediate, followed by a dehydration step to form the C=N double bond. Both steps are reversible, but occur most easily at different pH values. As a result, the reaction takes place according to a characteristic bell-shaped curve in relation to the pH value, where the highest reaction values can be found at moderate acidity.

However, it is known that the formation of Schiff bases also occurs easily 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 tends to be reactive on its own and is subject to further reaction leading to the addition of nucleophiles to the double bond. In the case of certain sulfur-containing amines, particularly the amino acids cysteine and methionine, as well as in the case of glutathione, the Schiff base that was originally formed can undergo reversible internal cyclization where the sulfhydryl group joins 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).

Evidence for reactions between carbonyl compounds and free amino groups of proteins to form reversible Schiff base compounds 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 aliphatic aldehydes, and Schiff bases can be formed even without removing the water produced during the reaction (R.W. Layer Chem. Rev. 63 (1963), 489-510). This fact is important when considering the formation of Schiff bases under physiological conditions. By using hemoglobin as a source of amino groups, Zaugg et al. (J. Biol. Chem. 252 (1977), 8542-8548) demonstrated that aromatic aldehydes have two to three times higher reactivity than aliphatic aldehydes in the formation of Schiff bases. An explanation for the limited reactivity of alkane channels may be the fact that in aqueous solutions at neutral pH a very large excess of free aldehyde is needed to shift the balance towards the formation of Schiff bases (E. Schauenstein et al., Aldehydes in biological systems. London, Pion Ltd. 1977).

Aromatic and heteroaromatic aldehydes readily form Schiff base imines with membrane amino groups, and high equilibrium constants have been measured for benzaldehyde in reaction with amines (J.J. Pesek and J.H. Frost, Org. Magnet. Res. 8 (1976), 173-176; J. N. Williams Jr. and R. M. Jacobs, Biochem Biophys Acta. 154, (1968) 323-331). Substituents in the aromatic ring that have the property of withdrawing electrons will cause the carbonyl carbon to become more electrophilic, and therefore more available for addition reactions with amino nucleophiles. The biological side effects of introducing a nitro group were assessed by comparing deuterated and non-deuterated 5,6-(3-nitrobenzylidene)-L-ascorbate with the unsubstituted analogue zilascorb(<2>H). It was demonstrated that the nitro-substituted derivatives produced a much higher inhibition of protein synthesis in NHIK 3025 cells (EP-0493883).

Through radiolabelling, we have previously shown that benzaldehyde does not penetrate the cell, but attaches to the cell membrane (Dornish, J.M. and Pettersen, E.O.: Cancer Letters 29

(1985) 235-243). This is consistent with a previous study which showed that benzaldehyde interacted with the membrane proteins of E. coli (K. Sakaguchi et al., Agric. Biol. Chem. (1979), 43, 1775-1777). It was also demonstrated that both pyridoxal and pyridoxal-5-phosphate protect the cells against the cytotoxic cancer-fighting agent cis-DDP. Cis-DDP acts in the nucleus inside the cell. While pyridoxal can in principle penetrate the lipophilic cell membrane, this is not possible with pyridoxal-5-phosphate because the latter has an ionic phosphate group. Pyridoxal-5-phosphate must therefore exert its positive effect from outside the cell membrane. A spectral transition in the absorption of pyridoxal-5-phosphate to lower wavelengths observed at the same time is consistent with the addition of Schiff bases between the aldehyde and amino groups in the cell membrane (J.M. Dornish and E.O. Pettersen, Cancer Lett. 29 (1985), 235- 243). These results suggest that aldehydes bind to amines and other nucleophilic units on the cell membrane to form Schiff bases and other condensation products. It is known that the stimulation of cell formation is influenced by a number of events outside the cell membrane. In the same way, the derivatives in this patent application can work by adding to ligands on the cell membrane, which trigger impulses inside the cell with an effect on cell growth parameters such as protein synthesis and mitosis, and on how tumor suppressor genes and immune responses are expressed. As the condensation reactions are reversible, effects on the cell can be modulated as a result of a shift in equilibrium involving ligating units. The presence of dynamic equilibrium states at a chemical level is consistent with the reversible mode of action recorded with the aldehyde derivates.

The inventors have carried out extensive studies in vitro of inhibition of protein synthesis through aromatic or heteroaromatic aldehyde derivatives. In solid tumours, the reduced protein synthesis can result in a lack of vital proteins, which leads to cell death. In normal cells, there is a potential capacity for protein synthesis that is higher than in most cancer cells in solid tumors. This is shown by comparing the duration of the cell cycle in normal stem cells, which is often less than 10 hours, with most cancer cells in solid tumors where the duration is typically 30-150 hours (cf. Gustavo and Pileri in The Cell Cycle and Cancer, Ed.: Baserga , Marcel Dekker Inc., New York 1971, page 99). As cells on average double their protein content during a cell cycle, this means that the accumulation of protein is higher in growth-stimulated normal cells than in most types of cancer cells.

In addition to this difference between normal cells and cancer cells, there is another difference of similar importance: while normal cells respond to growth-regulating stimuli, cancer cells have reduced or no such response. This means that while normal cells under normal growth conditions may have a reserve potential for growth, cancer cells have little or no such reserve. If an inhibition of protein synthesis occurs continuously over a long period of time on both normal cells and cancer cells, the two cell types can react differently: normal tissue can utilize part of its reserve potential for growth and thus maintain normal cell production, while cancer tissue has little or no such reserve. At the same time, the rate at which protein accumulates is rather low in most cancer cells (ie protein synthesis is only slightly higher than protein degradation). An inhibition of protein synthesis can therefore create an imbalance in the tumor when it comes to the accumulation of proteins, which leads to a negative balance for certain proteins. With continuous treatment over several days, this will cause the cell to be inactivated and necrosis to occur in the tumor tissue, while normal tissue is not damaged.

Until now, the most tested compounds that induce reversible inhibition of protein synthesis and show anticancer activity include zilascorb(<2>H) (EP-0283139 and Pettersen et al., Anticancer Res., vol. 11, pages 1077-1082, 1991) and 4,6-O-benzylidene-D-glucopyranose (M. Kochi et al., Cancer Treat. Rep., vol. 69, no. 5, pages 533-537, 1985 and T. Tatsumura et al., Br. J .Cancer, vol. 62, pages 436-439, 1990). The compound 5-nitrofurfurylidene diacetate has also shown strong anticancer activity in vitro. For example, the rate of protein synthesis in a culture of NHIK 3025 cervical carcinoma cells at 1 hour exposure with a concentration of the agent of 0.1 mM was approx. 35% compared to the control substance (WO 95/18607). However, with a concentration of only about 25 µM of 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, compound 2 according to the invention, a relative rate of protein synthesis of 35% has been achieved in relation to the control value with this model (see Fig. 5A). Astonishing potency has also been demonstrated by measuring the surviving amount of NHIK 3025 cells after 20 hours of exposure to compound 2 (see Fig. 2). At a concentration of the drug of 50 (xM, the surviving amount is reduced by 4 decades. Compound 2 thus shows a stronger effect than has been measured with any other reversible inhibitor of protein synthesis with this model. Figure 5A shows that compounds 2 and 3 are more effective as easier for protein synthesis than the previously known compound, 5-nitrofurfurylidene diacetate, by a factor of about one order of magnitude for low drug doses.

It has long been known that certain derivatives of nitrofuryl and nitroimidazole have strong antiseptic properties. Among the many 5-nitrofurfuryl derivatives that have been prepared, semicarbazone has been widely used as a therapeutic agent for topical use (US 2,416,234 and US 2,927,110). A related compound with similar therapeutic use is the oxazolidinone derivative (US 2,759,931 and US 2,927,110). N-(5-nitrofurfurylidene)-1-aminohydantoin is a chemotherapeutic agent still in use to treat urinary tract infections (US 2,610,181, US 2,779,786, US 2,898,335 and US 2,927,110). In DE 1 792 459 the hydantoin ring is further derivatized with a methylmorpholine component. Furthermore, bis(5-nitrofurfurylidene)acetone-guanylhydrazone (Nitrovin) is in extensive use as an additive for The compound 5-nitrofurfurylidene-D-glucosamine and its inhibitory action on bacteria as well as on pathogenic fungi is described in US 3,122,535. Although the chemical structure is ambiguous without specific anomeric stereochemistry, this is likely a precursor to compound 2 in this patent application . All the other compounds described (1 and 3-7 in the table) are, as far as we know, innovative in themselves.

None of these compositions described previously are known to have anticancer properties, nor are they known to have immunostimulating properties. However, they are well known as antibacterial, antifungal or antiprotozoal agents. The mode of action as bacteriostatic agents is dependent on the inhibition of vital enzyme systems in the bacteria, a mechanism which is completely different from the indirect mode of action through stimulation of the host's immune system which is described by the compounds according to the invention.

We have now surprisingly found that the products according to the invention have a surprisingly greater effect (by about one order of magnitude) in terms of inhibition of protein synthesis and cell survival than was previously known (see examples 1 and 2). We have also found that the reversibility of the action of these compounds is not apparent, as with the products that have been described previously. This may mean that the binding of these aldehydes to the cell surface is stronger than the binding to the previously known substances, and consequently can provide a more effective therapeutic treatment with the compounds according to the invention.

It is known from British patent application 9026080.3 that benzaldehyde compounds, formerly known as anticancer agents, can be used to combat diseases caused by abnormally high cell division.

Dermatological abnormalities such as psoriasis are often characterized by frequent replacement of the epidermis. While normal skin produces approx. 1250 new cells/day/cm of skin consisting of around 27,000 cells, psoriatic skin produces 35,000 new cells/day/cm from 52,000 cells. However, the cells involved in these disorders are "normal" cells that reproduce rapidly and repeatedly through cell division. While the renewal of a normal skin cell takes around 311 hours, this process goes much faster and takes around 10-36 hours in psoriatic skin.

Today, psoriasis, inflammatory conditions, rheumatic disorders and other autoimmune diseases are treated with corticosteroids, NS AID agents and in severe cases with immunosuppressive agents such as cytostatics and cyclosporins. All of these drugs can cause serious side effects. There is therefore a great need for effective products that produce fewer side effects.

It is known that aromatic and heteroaromatic aldehydes and certain acetal derivatives thereof have an inhibitory effect on the growth of cells in humans, and that this inhibitory effect is reversible in nature. The inhibition of growth caused by these compounds is primarily due to a reduction of the cells' protein synthesis (Pettersen et al., Eur. J. Clin. Oncol., vol. 19, pages 935-940, 1983 and Cancer Res., vol. 45, page 2085-2091, 1985). The inhibition of protein synthesis is only effective as long as these agents are present in the microenvironment of the cells.

This leads to the effect that the normal cells remain undamaged after treatment with the above compounds. The relationship between effects and side effects (the therapeutic index) for this class of products may depend on the degree of such reversibility, and in order to optimize the therapeutic benefits of the products, one must take these characteristics into account.

The cells' ability to transmit signals via mechanisms that depend on cell contact (adhesion) has been studied for many years. These mechanisms are particularly important for the reactivity of circulating cells such as lymphocytes, macrophages, etc. and also, for example, for metastatic cells to become anchored in tissue and create new tumours. The ability to change the adhesion characteristics of cells that register an immune response or an inflammatory response can be of great therapeutic value in the treatment of many disorders such as rheumatoid arthritis, psoriasis, psoriatic arthritis, lupus erythematosus, acne, Ankylosing spondylitis, progressive systemic sclerosis (PSS), seborrhea and other autoimmune diseases such as ulcerative colitis and Crohn's disease etc.

The immune system is carefully designed to identify and eliminate any material it recognizes as foreign, whether it originates from a bacterial, viral or protozoan infection or abnormal cells such as cancer cells. In order to be able to provide a specific response to the enormous range of biotic variation represented by the invaders, the immune system must be highly diversified. However, overstimulation of this fine-tuned system can lead to various allergic and inflammatory reactions and lead to autoimmune diseases. Rejection of favorable transplants is also a problem that is difficult to solve. It is therefore a major therapeutic challenge to modulate the immune system by regulating a specific response either upwards or downwards.

In an immunological recognition process, a fragment of a foreign protein is enclosed within the groove of the class II MHC protein on the surface of an antigen-presenting cell

(APC). Attached to this MHC-antibody complex is also the receptor for a T helper cell. To activate a T-helper cell, at least two signals must be given. The first signal comes from the antigen itself via the class II MHC complex and is amplified by CD4 coreceptors. The second signal can be given by a special signal molecule bound in the plasma membrane on the surface of the APC. A corresponding coreceptor protein is located on the surface of T-

the helper cell. Both signals are necessary for the T cells to be activated. When they are activated, they will stimulate their own proliferation by secreting interleukin growth factors and synthesizing corresponding receptors on the cell surfaces. The binding of interleukins to these receptors then directly stimulates the T cells to proliferate. 1 In the 1980s, it was discovered that a cyclodextrin-benzaldehyde 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). Studies carried out in vitro have later shown which chemical reactions at the site of interaction between the APC donor and T cells are responsible for the second, costimulatory signal, and that these take the form of carbonyl-amino condensations (formation of Schiff bases). Furthermore, these interactions can be replicated through synthetic chemical entities. These findings open up new therapeutic possibilities for artificial potentiation of the immune system. WO 94/07479 describes the use of certain aldehydes and ketones which form Schiff bases and hydrazones with amino groups on the surface of T cells. EP 0609606 A1 describes as preferred immunostimulating substance 4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid (Tucaresol), a compound originally developed to cure sickle cell anemia. This substance is administered orally and is systemically bioavailable.

It is well known that glycoproteins bound to the cell's plasma membrane have the ability to associate with sugar molecules dissolved in the extracellular fluid. Susceptibility to such receptors will help to attract and anchor sugar/aldehyde derivatives to the cell surface. Reactive aldehyde is released through hydrolysis and becomes available for formation of Schiff bases in situ with amino groups protruding from the cell surface. In the present patent application, 5-nitrofurfural is derivatized with biologically acceptable carbohydrates such as glucose, glucosamine, galactosamine and other sugars, deoxysugars and aminosugars, through either an acetal or an imine bond. The sugar component will also help to improve stability and increase the bioavailability of the aldehyde function to the target cells.

The immunostimulating effect of the compounds according to the invention can also be utilized in the treatment of certain viral diseases combined with other antiviral therapy such as antiviral drugs or vaccines. Many virus types are incorporated with the cell nucleus after the first infection 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 the latency period for these, it is very difficult to cure the viral infection. Such viruses can often be triggered by immune responses and cause viraemia, enabling clearance

the viral infection at this stage. The ability of the aldehyde derivatives to trigger the immune response

can be used in conjunction with antiviral drugs or vaccines to develop a treatment for these diseases.

It is a main purpose of the invention to produce new compounds for the prophylaxis and/or treatment of cancer and disorders related to the immune system.

Another purpose of the invention is to produce new compounds that are capable of potentiating immune responses that provide the opportunity to fight infectious diseases caused by viruses, bacteria, fungi and other microorganisms.

A third purpose of the invention is to produce compounds for the prophylaxis and/or treatment of cancer and disorders related to the immune system which do not produce toxic side effects.

A fourth purpose of the invention is to produce compounds for effective and beneficial prophylaxis and/or treatment of cancer in tissues and cells that have receptors with attraction towards corresponding sugar components.

A fifth purpose of the invention is to produce compounds for the treatment of disorders linked to the immune system, such as psoriasis, stomach inflammations, arthritis, SLE, PSS, etc.

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

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

there R! and R 2 is H or D, or is linked to form a 6-membered acetal ring comprising the substructure (II):

where L is H or D.

X is selected from the atoms or groups of atoms comprising H, D, OH, OR4, NH2, NHR4, NR4R5, NHC(0)R4 or NC(0)R4R5 where R4 and R5 are alkyl with 1-6 carbon atoms or cycloalkyl with 3- 6 carbon atoms, and can be identical or different. X may alternatively comprise the substructure (III):

where L is H or D;

R 1 , R 2 and X are interrelated in such a way that if R 1 and R 2 are H or D, then X is (III). If R 1 and R 2 are linked to form the substructure (II), X is either H, D, OH, OR 4 , NH 2 , NHR 4 , NR 4 R 5 , NHC(0)R 4 or NC(0)R 4 R 5 where R 4 and R 5 are defined as above.

R 3 is H, D, alkyl of 1-20 carbon atoms, cycloalkyl of 3-6 carbon atoms, fluoroalkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, or a pharmaceutically acceptable salt thereof.

It appears that any stereoisomer according to the formula is covered by the present invention.

With the exception of compound 2, all the compounds according to the present invention are innovative in themselves.

Detailed description of the invention

The invention is described in more detail below through a table and examples showing the preparation and biological examples, as well as the attached figures.

Manufacturing

According to the present patent application, various sugars, deoxysugars and aminosugars are condensed with 5-nitrofurfural to form sugar acetal or sugar imine derivatives.

It is well known that aldehydes undergo acid-facilitated condensation reactions with carbohydrates to form sugar acetals. Instead of the aldehyde itself, a lower dialkyl acetal of the aldehyde is most often used, and the reaction is completed by evaporating the lower alcohols that are formed at the same time under reduced pressure. As the nitro group has a very deactivating effect on the formyl group, however, the formation of sugar acetals with 5-nitrofurfural occurs very slowly. Higher temperatures and longer reaction times easily lead to isomerization and degradation of the product, resulting in very complex reaction mixtures. It is therefore more advantageous to use protection strategies. 4,6-O-benzylidene-D-glucopyranose was already available with us and can be chosen as a suitable starting material. By acetylating the remaining hydroxy groups and removing the benzylidene group, a high yield of 1,2,3-tri-O-protected sugar was obtained. The acetalization of this important intermediate stage with 5-nitrofurfural can now be carried out under more stringent reaction conditions without destructive side reactions. The new 5-nitrofurfurylidene sugar acetal (compound 1) was finally produced after base-catalyzed removal of the acetyl protecting groups.

The amino group is sufficiently reactive to allow imine condensation reactions to take place directly, even with deactivated aldehydes. Compound 2 was synthesized in one step by adjusting the pH of a methanolic suspension of glucosamine hydrochloride and adding 5-nitrofurfural.

The compounds according to formula (I) where L is deuterium can be prepared as described above, however, starting with 5-nitrofurfural which is deuterated in the formyl position. The preparation of the deuterated aldehyde can be carried out according to one of the examples given in EP 0 552 880 A1, where the C/mpø/img strategy is the preferred method. The aldehyde is protected as its 1,3-dithioacetal, BuLi, is added to generate the lithium salt which is then quenched with D 2 O and the protecting group is removed. In the subsequent steps, 5-nitrofurfural-di formed in this way is used as starting material analogously to the corresponding non-deuterated substance. The acetalization reactions can be carried out in a dipolar, aprotic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, dimethoxyethane or the like. In order to be able to remove water in a suitable manner, however, an azeotrope-forming solvent such as chloroform, dichloromethane or ethyl acetate is preferred. The catalyst can be a mineral acid, e.g. sulfuric acid, an organic acid, e.g. para-toluenesulfonic acid, an acidic ion exchange resin, e.g. Amberlyst 15, a Lewis acid mineral clay, e.g. Montmorillonite K-10 or a resin supported super acid, e.g. Nafion NR 50.

The reaction conditions are adjusted to reflect the characteristics of each individual compound as described in the examples below.

Identification of the products was carried out using 1 H and <13>C NMR methods at proton frequencies of 300 or 400 MHz.

The following examples serve to illustrate how the compounds according to the present invention can be prepared.

Compound 1: 4, 6- 0-( 5- nitrofurfurvlidene)- D- glucopvranose

1,2,3-tri-O-acetyl-4,6-O-benzylidene-D-glucopyranose

4,6-O-benzylidene-D-glucopyranose (25.0 g, 0.093 mol) was dissolved in a mixture of acetic anhydride (370 mL) and pyridine (750 mL) and allowed to stand at 20 °C for 24 h. The reaction mixture was evaporated, and the residue repeatedly diluted with toluene and evaporated. The crude product was recrystallized from heptane/ethyl acetate. The yield was 32.2 g, 87% of the theoretical yield. The identity was confirmed by <!>H NMR spectroscopy.

1,2,3-tir-O-acetyl-D-glucopyranose

1,2,3-tri-O-acetyl-4,6-O-benzylidene-D-glucopyranose (20.4 g, 0.0517 mol) was dissolved in ethyl acetate (250 mL) under N 2 . 10% Pd on activated carbon (1.2 g) was added and H 2 bubbled through at 1 bar, 20 °C for 20 h. The reaction mixture was filtered and evaporated. The crude product was repeatedly diluted/evaporated with CHCl 3 and CH 2 Cl 2 to remove traces of ethyl acetate. NMR analysis indicated that the debenzylation was quantitative and that the ratio of a to P was 1:3. The identity was confirmed by 1 H NMR spectroscopy.

1,2,3-tri-O-acetyl-4,6-O-(5-nitrofurfurylidene)-D-glucopyranose

1,2,3-tri-O-acetyl-D-glucopyranose (max. 0.0517 mol) and 5-nitrofurfural (7.3 g, 0.052 mol) were dissolved in CH 2 Cl 2 (250 mL) along with a catalytic amount of para -toluenesulfonic acid. The reaction mixture was boiled for 20 hours and the reflux (80 mL) was decanted via a 4 Å molecular sieve. A small amount of triethylamine and silica gel (90 g, 200-500 µm) was added and the solvent removed by evaporation. The product was chromatographed on silica gel (1100 ml, 20-45 µm) and eluted with heptane/ethyl acetate (60/40). Fractions of 250 ml were collected and analyzed (TLC). The product was found in fractions 11-23, which were combined and evaporated. The residual material formed crystals and was vacuum dried. The yield was 6.78 g, 31% of the theoretical yield. The identity was confirmed by <1>H NMR spectroscopy.

4,6-O-(5-nitrofurfurylidene)-D-glucopyranose

To a solution of 1,2,3-tri-O-acetyl-4,6-O-(5-nitrofurfurylidene)-D-glucopyranose (6.78 g, 0.0158 mol) in methanol (400 mL) was added added NaOCH 3 (0.13 g, 0.0024 mol). The reaction mixture was stirred at 20 °C for 2 h (TLC indicated that the reaction was almost complete within 20 min) and then stored at 5 °C for 20 h. A minor precipitation occurred. The volume was reduced to 50 ml by evaporation and the reaction mixture cooled to 5 °C. The precipitate thus formed was filtered, washed with cold methanol and dried in vacuo. The filtrate volume was reduced to 15 ml and another precipitate isolated. The results of the two precipitation steps were combined to give 3.96 g of the product, 83% of the theoretical yield.

GC of the TMS derivatives showed an isomer ratio of 2:1, while the NMR data was closer to a 1:1 ratio indicating that an isomerization had taken place in the DMSO-d6 solution. In a separate batch, the corresponding figures were 4:1 (GC) and 1.7:1 (NMR).

'h and <13>C NMR (400 MHz; DMSO-d6), δ relative to TMS: 7.67 (d, 1H, O2N-CH=Ctf-CH=) 6.89 (dd, 1H, O2N- CH=CH-C#=), 6.85 (d, 0.5H, OH-\ II), 6.58 (d, 0.5H, OH-\ I), 5.79 (s+s, 1H, acetal-tf I+II), 5.29, 5.21, 5.17 and 4.85 (m+d+d+d, 0.5H+0.5H+0.5H+0.5H, 0# -2+Otf-3), 4.95 (t, 0.5H, H- l I), 4.43 (t, 0.5H, H- l II), 4.19-4.06 (m, 1H, H- 6' I+II), 3.83-3.74 (m, 0.5H, H- 5 I), 3.71-3.56 (m, 1.5H, H- 6" I +II and H- 3 I), 3.42-3.29 (m, 5H, H- A I+II, H- 5 II and H 2 O), 3.27-3.21 and 3.03-2, 95 (m+m, 0.5H+0.5H, H- 2 I+II); 153.34 and 152.11 (furfuryl =CH-0-CH=), 114.02 and 112.80 (furfuryl = CH-CH=), 98.45, 94.81, 94.70 and 94.07 (acetal C I+II and sugar C-1 I+II),

82.47 and 81.68 (sugar C-4 I+II), 76.62 and 73.47 (sugar C-2 I+II), 73.68 and 70.27 (sugar C-3 I+II) , 69.18 and 68.82 (sugar C-6 I+II) and 66.16 and 62.46 (sugar C-5 I+II).

Compound 2: 2- deoxy- 2-[( 5- nitrofurfurvlidene) arninol- D- glucopvranose

To a stirred suspension of glucosamine hydrochloride (2.70 g, 12.5 mmol) in methanol

(24 mL) at room temperature was added 1,8-diazabicyclo[5.4.0]undec-7-ene (2.24 mL, 15.0 mmol) and a solution quickly formed. 5-nitrofurfural (3.53 g, 25.0 mmol) was then added portionwise and the reaction mixture quickly darkened to a deep brown color as the aldehyde dissolved. After stirring for approx. After 20 minutes at room temperature, a light brown precipitate could be seen to form, and the reaction mixture was allowed to stir overnight under nitrogen. The mixture was then filtered using water pump vacuum and washed with ethanol until the filtrate was colorless (ca. 50 mL) to give a beige solid which was dried under a stream of nitrogen (1.95 g, 52%).

'H NMR 8H (300 MHz; DMSO-d6) 2.88 (t) and 3.10-3.85 (m) (a- and (3- H-2, H-3, H-4, H- 5, H-6), 4.46 (t), 4.58 (t), 4.78 (t), 4.86 (d) and 4.95-5.05 (m) (a- and P - H-1, OH-3, OH-4, OH-6), 6.43 (d, a-OH-1), 6.71 (d, p-OH-1), 7.28 (1H, m ArH ) and 7.78 (1H, m ArH), 8.16 (s, p-CH=N) and 8.27 (s, α-CH=N).

Compound 3: 2- deoxv- 2-[( 5- nitrofurfurvlidene) aminol- B- D- galactopuranose

To a stirred suspension of galactosamine hydrochloride (2.74 g, 12.7 mmol) in methanol

(25 mL) at room temperature was added 1,8-diazabicyclo[5.4.0]undec-7-ene (2.28 mL, 15.3 mmol) and a solution quickly formed. 5-nitrofurfural (3.59 g, 25.4 mmol) was then added portionwise and the reaction mixture quickly darkened to a deep brown color as the aldehyde dissolved. After approx. After 1.5 hours you could see that a certain result had formed, and this thickened during the next half hour. The reaction mixture was allowed to stir overnight under an atmosphere of nitrogen. It was then filtered and the brown residue washed with ethanol (20 mL) and then, with suction continuing, dried under a stream of nitrogen for 20 min to give a cream-colored powder containing only the P isomer (2.45 g, 64 %).

<!>H NMR 5H (300 MHz; DMSO-d6) 3.18 (1H, t, H-3), 3.44-3.72 (5H, m, H-2, H-4, H-5 and H-6), 4.57 (1H, d, either OH-3 or OH-4) and 4.60-4.75 (3H, m, H-l, OH-6 and either OH-3 or OH-4 ), 6.63 (1H, d, OH-1), 7.28 (1H, d, ArH), 7.78 (1H, d, ArH) and 8.15 (1H, s, CH=N); 13C NMR 6C (75 MHz; DMSO-d6) 60.7 (C-6), 67.0, 71.2, 75.2 and

75.3 (C-2, C-3, C-4, C-5), 95.7 (C-1), 114.0 and 116.4 (arom. CH), 150.4 (CH=N), 151.9 and 152.3 (C-NO 2 and C-C=N).

Compound 4: 4, 6- Q -(" 5- nitrofurfurylidene)- D- galactopuranose

4,6-O-benzylidene-D-galactopyranose (0.093 mmol) is dissolved in a mixture of acetic anhydride and pyridine, and allowed to stand at ambient temperature overnight. The reaction mixture is evaporated, and the residual material is repeatedly diluted with toluene and evaporated. The crude product is recrystallized and the structure of the 1,2,3-tri-O-acetyl-D-galactopyranose thus formed is confirmed by NMR analysis. This intermediate (0.0517 mol) is dissolved in ethyl acetate under N2. A stoichiometric excess of 10% Pd on activated carbon is added, and H2 is bubbled through at ambient temperature overnight. The reaction mixture is filtered and evaporated. The crude product is diluted and evaporated repeatedly with CHCl 3 and CH 2 Cl 2 to remove traces of ethyl acetate. The structure of 1,2,3-tri-O-acetyl-4,6-O-(5-nitrofurfurylidene)-D-galactopyranose thus formed is confirmed by NMR analysis. The 4,6-O-deprotected tri-O-acetyl sugar (max. 0.0517 mol) and 5-nitrofurfural (0.052 mol) are dissolved in CH 2 Cl 2 together with a catalytic amount of para-toluenesulfonic acid. The reaction mixture is boiled overnight and the reflux is drained off via a molecular sieve of 4 Å. A small amount of triethylamine and silica gel are added and

the solvent is removed by evaporation. The product is chromatographed on silica gel and eluted with heptane/ethyl acetate. Fractions are collected and analyzed (TLC), and the product fractions combined and evaporated. The structure of 1,2,3-tri-O-acetyl-4,6-O-(5-nitrofurfurylidene)-D-galactopyranose is confirmed by NMR analysis.

To a solution of this in methanol, NaOCH3 (0.0024 mol) is added and the reaction mixture is stirred at ambient temperature until complete (monitored by TLC). The end product thus formed is isolated using suitable methods and dried in vacuo. The structure of 4,6-O-(5-nitrofurfurylidene)-D-galactopyranose is confirmed by NMR analysis.

Compound 5: 4, 6- 0-( 5- nitrofurfurvlidene)- D- mannopvranose

This product is synthesized as described above, except that galactose is replaced by mannose.

Compound 6: 2- deoxy- 4, 6- O-( 5- nitrofurfurylidene)- D- glucopvranose

This product is synthesized as described above, starting from 2-deoxyglucose.

Compound 7: 2- acetamido- 2- deoxy- 4, 6- O-( 5- nitrofurfurylidene)- D- glucopvranose

This product is synthesized as described above, starting from glucosamine hydrochloride.

Compound 8: 4, 6- 0-( 5- nitrofurfurvlidene-d1)- D- glucopvranose

5-nitrofurfural, 1,3-propanethidiol (1 eq.), para-toluenesulfonic acid (cat. amounts) and toluene (2 ml per mmol aldehyde) are mixed and boiled under reflux until the reaction is complete (approx. 201). Hexane and a small amount of diisopropyl ether are added and the solution is cooled to precipitate the product. Crystals of 5-nitrofurfural-1,3-propanedithioacetal are filtered off and dried. The identity is confirmed with NMR spectroscopy.

5-nitrofurfural-1,3-propanedithioacetal is dissolved in dry tetrahydrofuran (ca. 6 mL per mmol) under argon in a dry three-necked round-bottomed flask equipped with a septum. The solution is cooled with acetone/dry ice and 1.6 M butyllithium in hexane (1.5 eq.) is added slowly from a syringe while the reaction mixture is kept below -50 °C. When the reaction is finished (approx. 41), D 2 O (1 ml per mmol thioacetal) is added and the reaction mixture is stirred and allowed to reach room temperature. A yield of 5-nitrofurfural-l,3-propanedithioacetal-d! filtered off and dissolved in dichloromethane. The solution is washed with dilute hydrochloric acid and water, and then dried. This crude product can optionally be recrystallized from a suitable solvent. The identity and deuterium content are determined with NMR spectroscopy.

5-nitrofurfural-1,3-propanedithioacetal-d!, HgCl2 (2.2 eq.) and HgO (1.1 eq.) are dissolved in a 9:1 mixture of acetonitrile and water and boiled at reflux for approx. 2 hours. After cooling, insoluble mercury salts are filtered off and the filtrate is washed with a 5% solution of ammonium acetate. 5-nitrofurfural-di is formed by precipitation, filtered off and recrystallized from a suitable solvent. The identity is confirmed with NMR spectroscopy.

1,2,3-tri-O-acetyl-D-glucopyranose is prepared as described in the preparation of compound 1.

etc

1,2,3-tri-O-acetyl-4,6-0-(5-nitrofurfurylidene-d1)-D-glucopyranose is prepared by condensing 1,2,3-tri-O-acetyl-D-glucopyranose with 5 -nitrofurfural-di and the title compound finally prepared by removing the acetyl protecting groups according to the procedure described in Example 1.

Compound 9: 2- deoxv- 2- r( 5- nitrofurfurvlidene- dj) aminol- D- glucopvranose

5-nitrofurfural-d! is prepared as described above and condensed with glucosamine hydrochloride according to the method described in example 2.

Compound 10: 4, 6- O-( 5- nitrofurfurylidene)- L- glucopvranose

4,6-O-benzylidene-L-glucopyranose

L-glucose (5.14 g, 28.6 mmol) was heated in DMF (20 mL) at 95 °C until a clear solution formed. The reaction flask was then transferred to a water bath at 65 °C and p-toluenesulfonic acid (33 mg, 0.17 mmol) was added. Benzylidene dimethyl acetal (4.7 mL, 31 mmol) was then added dropwise by syringe over 20 min to the stirred glucose solution under a regulated water pump vacuum of 80 mbar. DMF was then evaporated under vacuum (2 mbar) at 65 °C to give a very pale yellow oil to which sodium bicarbonate (345 mg) was added, followed by stirring for 5 min. Hot water (67°C, 15 mL) was added with stirring (magnetic stirrer) to the oil at 65°C and the bottle shaken in a hot water bath until the oil appeared to have dissolved. The reaction bottle was then placed under a stream of cold water for approx. 5 minutes. After only one or two minutes, an amorphous mass formed. The aqueous mixture was placed in an ice water bath and left for 40 minutes. A white precipitate formed during this time and was isolated by vacuum filtration (decanted from the amorphous material), washed with cold tap water (25 mL) followed by cold iso-propanol (5 °C, 2 x 5 mL) and dried under a stream of nitrogen to give 1.85 g of a dry white powder. This product was silylated and analyzed by gas chromatography and proved to be the desired product with a purity of 99%.

1H NMR, δ (DMSO-d6) vs. TMS: 7.55-7.25 (m, 5H, Ar-H), 6.85 (s, 0.48 H, OH-1-6), 6.55 (s, 0.33H, OH-1-a), 5.25 (d, 0.48 H, OH-3-6), 5.20 (d, 0.49 H, OH-2-6) , 5.10 (d, 0.35 H, OH-3-a), 4.98 (d, 0.35 H, H-1-a), 4.82 (d, 0.34 H, OH-2-a) , 4.48 (d, 0.51 H, H-1-8), 4.20-4.05 (m+m, 0.53 H +0.42 H, H-6' a+G), 3, 85-3.73 (m, 0.44 H, H-5-a), 3.72-3.57 (m, 1.27 H, H-6"-a+B and H-3-a) , 3.45-3.20 (m, 7.8 H, H-3-13, H-4-a+B, H-5-6 and H-2-a) and 3.10-2.98 (m, 0.56 H, H-2-B)

Starting from 4,6-O-benzylidene-L-glucopyranose, the title compound was prepared as described by the analogous procedure which led to compound 1.

Description of the figures

Figure 1: Cell survival as measured by colony-forming ability of human cervical carcinoma cells, NHIK 3025, after treatment for 4 or 20 hours with compound 1. The cells were treated in open plastic Petri dishes incubated in CO 2 incubators at 37 °C. The survival values plotted represent means from five dishes that were treated in the same way at the same time. Standard deviations are shown with vertical bars in all cases where they lie outside the symbols. Based on the data, cell survival is down to 20% after 4 hours of treatment with 1 mM of compound 1. For comparison, the same survival of 20% is obtained with only 0.3 mM of compound 1 when the treatment time is extended to 20 hours. Based on the drug doses, the effect is thus around 3 times greater when the treatment is extended from 4 hours to 20 hours. Figure 2: Cell survival as measured by colony-forming ability for cervical carcinoma cells from humans, NHIK 3025, after treatment for 20 hours with compound 2 or 3. The technique and statistics are described in figure 1. The effect of these two compounds is similar. The effect is stronger than for compound 1. A direct comparison between figures 1 and 2 shows that 10% survival is obtained after 20 hours of treatment with 500 iM of compound 1 and only 10 iM of compounds 2 and 3. Measured by concentration, the latter two are the compounds thus 50 times more effective than the former. Figure 3: Cell survival as measured by colony-forming ability of human cervical carcinoma cells, NHIK 3025, after treatment for 20 hours with compound 3 and tucaresol. The technique and statistics are described in figure 1. The data indicate that the cell survival is about 30% after treatment with 1000 iM tucaresol compared to 7 iM of compound 3. Thus, compound 3 produces a stronger effect than tucaresol by a factor of about 140 measured by drug dose . Figure 4: The rate of protein synthesis by human cervical carcinoma cells, NHIK 3025, as measured by the amount of incorporated [ H]-valine during a 1 hour pulse period starting either immediately after the addition of the test compound (in this case compound 1) or starting 3 hours later with the test compound present during the pulse. The cells were pre-labeled with [<14>C]-valine for at least 4 days to allow all cellular protein to be labeled to saturation point. The incorporated amount of [ H] was proportional to the incorporated amount of [<14>C] so that protein synthesis was calculated as a percentage of the total amount of protein. The rate of protein synthesis is given as a percentage compared to the untreated control. The values plotted for protein synthesis represent average values from 4 wells treated simultaneously and in the same way. Standard deviations are shown with vertical bars in all cases where they lie outside the symbols. The data show that compound 1 induces an inhibition of protein synthesis that increases linearly as the concentration of the drug increases. The inhibition of protein synthesis appears to be similar regardless of whether the pulse period starts immediately after the drug treatment begins or 3 hours later. Figure 5: The rate of protein synthesis as measured by the amount of incorporated [ H]-valine during a pulse period of 1 hour starting immediately after the addition of the test compounds (in this case compounds 2 and 3 together with corresponding data for 5-nitrofurfurylidene diacetate). Cell processing, calculation of protein synthesis and statistics were as indicated in the description of Figure 4. The data for compounds 2 and 3 represent two different experiments, one in panel A and one in panel B. Note that panels A and B differ in terms of the scale of X axis. The data indicate that both compounds 2 and 3 are effective facilitators of protein synthesis. Both compounds reduce protein synthesis to a low level of approx. 35% of the control level for a drug concentration as low as about 25 µM. In the dosage range from 0 to 25 iM, the inhibition of protein synthesis increases linearly with the drug concentration. A further increase of the drug concentration beyond 25 iM does not lead to a further increase of the response. In comparison, a concentration of 5-nitrofurfurylidene diacetate of 100 µM reduces the rate of protein synthesis to about 50%. As calculated from the original slope of the curves, compounds 2 and 3 have an efficiency that inhibits protein synthesis in these cells which is about one order of magnitude higher than that of 5-nitrofurfurylidene diacetate. A comparison between Figures 4 and 5B indicates that 4 mM of compound 1 promotes the same degree of inhibition of protein synthesis as 25 µM of compounds 2 and 3. The latter two compounds are therefore 160 times more effective than the former based on drug dose alone. Figure 6: The rate of protein synthesis as measured by the amount of incorporated [ H]-valine during a pulse period of 1 hour starting immediately after the addition of 25 µM of the test compounds (in this case compounds 2 and 3). Cell treatment, calculation of protein synthesis and statistics were as indicated in the description of Figure 4. The control value of 100% is plotted as time 0, as this is the level of the rate of protein synthesis just before the drugs are added to the cells. The next point is plotted as 1/2 hour, i.e. halfway through the cycle from 0-1 hours. After 4 hours, the treatment drugs were removed and new, fresh, drug-free medium was added to the cells. In the first hour after removal of the drug, the last pulse of [<3>H]-valine was given, and comparison of the values representing the last two time points indicates the degree of reversibility of the compounds' inhibition of protein synthesis. While both compounds show increased inhibition of protein synthesis with time after the start of treatment, both are also to some extent reversible. However, the increase in protein synthesis after removal of the compounds is not as rapid as that observed after removal of benzaldehyde (see Pettersen et al., Eur J Cancer Clin. Oncol. 19 (1983), 935-940).

Figure 7: Relative tumor volume as a function of the number of days after the first i.v. injection. The tumor volume was calculated based on the formula volume = (length x width<2>)/2 where tumor length and width were measured with calipers twice a week. On day 1 and each subsequent day, each animal was given one i.v. injection per day, 7 days per week, of either drug-free isotonic saline (placebo) or isotonic saline containing either 0.125 or 0.938 mg of compound 1 per ml of saline. The body weight of each animal was approx. 25 g, and the volume of each injection was 0.2 ml, giving a total delivery of 1 mg or 7.5 mg of compound 1 per kg body weight each day. Each point in the experiment represents mean values of tumor volumes from 6 animals where each tumor volume was measured relative to the volume of the same tumor on day 1. Standard deviations are given as vertical lines, but in this case represent the biological variation in tumor growth rate between individual animals in addition to the uncertainty in the measurement. Both groups that were given compound 1 show a slightly reduced tumor growth compared to the group of animals that were treated with placebo. The effect is somewhat more prominent with 1 mg drug per kg than with 7.5 mg/kg, which may indicate that the anti-tumour effect may be optimal within a certain dosage range, with reduced effect both at lower and higher drug doses.

Biological experiments

Example 1

Biological materials and methods used to demonstrate efficacy Cell culture techniques

Human cells, NHIK 3025, originating in a cervical carcinoma in situ (Nordbye, K. and Oftebro, R., Exp. Cell Res., 58: 458,1969, Oftebro R. and Nordbye K., Exp. Cell

Res., 58: 459-460, 1969) were grown in Eagel's Minimal Essential Medium (MEM) supplemented with 15% fetal calf serum (Gibco BRL Ltd). The cells are routinely grown as monolayers at 37 °C in tissue culture flasks. To maintain continuous exponential growth of the cells, the cells were trypsinized and recultured three times a week.

Cell survival

Cell survival was measured as colony-forming ability. Prior to seeding, the exponentially growing cells were trypsinized, pelleted as single cells and seeded directly on 5 cm plastic dishes. The number of germ cells was adjusted so that the number of surviving cells should be around 150 per dish. After approximately two hours of incubation at 37 °C, the cells had attached to the bottom of the dishes. Drug treatment was then initiated by replacing the medium with medium having the desired concentration of the drug. After drug treatment, cells were washed once 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 labeled with methylene blue before colonies were counted.

The measures of survival shown in Figures 1 and 2 indicate that these drugs inactivate cells with increasing efficacy at increasing drug doses. The effect also increases when the treatment time increases (figure 1). However, compounds 2 and 3 are far more effective than compound 1, by a factor of 50 relative to the concentration (Figure 2).

From Figure 3, it can be seen that compound 3 creates a stronger inactivating effect on the cells than tucaresol by a factor of around 140 in relation to the drug dosage.

Example 2

Protein synthesis

The rate of protein synthesis was calculated as described previously (Ronning, O.W. et al., J. Cell Physiol., 107: 47-57, 1981). Briefly, cell protein was labeled to saturation point during a pre-incubation of at least 4 days with [<14>C]-valine of constant specific radioactivity (0.5 Ci/mol). To keep the specific radioactivity at a constant level, a high concentration of valine (1.0 mM) was used in the medium. At this concentration of valine, the dilution of [<14>C]-valine due to intracellular valine and proteolytically generated valine becomes negligible (Ronning, O.W. et al., Exp. Cell Res. 123: 63-72, 1979) . The rate of protein synthesis was calculated from the content of [H<3>]valine in relation to the total [<l4>C] radioactivity in the protein at the beginning of the relevant measurement periods, and expressed as a percentage per hour (Ronning, O.W. et al ., J. Cell Physiol., 107: 47-57, 1981).

Compound 1 promotes an inhibition of protein synthesis that increases linearly with increasing dose up to 4 mM, which was the highest dose tested (Figure 4). The effect was the same regardless of whether pulsing with [<3>H]-valine was performed in the first or third hour after the start of treatment. Compound 1 thus leads to an inhibition of protein synthesis which is constant as long as the compound is present in the cell culture.

Compounds 2 and 3 promote inhibition of protein synthesis at very low concentrations. A significant effect can be registered at concentrations as low as 10 uM (figure 5B), and full effect with a low level of protein synthesis of 35% compared to the control substance, is registered at concentrations above 25 uM (figure 5A). Figure 5A also shows that at low drug doses as estimated from the initial flanks of the curve, compounds 2 and 3 have an efficiency that is about one order of magnitude higher than 5-nitrofurfurylidene diacetate which inhibits protein synthesis in NHIK 3025 cells.

From Figure 6, it can be seen that both compounds 2 and 3 increase the inhibition of protein synthesis when the treatment time increases. Both drugs are shown to be somewhat reversible, as the rate of protein synthesis increases immediately after the drugs are removed. However, protein synthesis is not fully restored during the first hour after treatment, the increase is only to a level of 50% of the control cells. It is possible that a part of the cells are deactivated irreversibly in terms of protein synthesis, but without being lysed during the first 3 hours of treatment. These cells will then contribute to the amount of [<14>C] but not to the amount of [<3>H] in the final sample, thus masking the possible reversibility of the inhibition of protein synthesis in cells that survive the three-hour treatment.

Example 3

Experiments on xeno<g>rafts from humans on nude mice

The drugs were tested in the treatment of three xenografts of human cancer cells implanted in athymic female mice. The cell lines used are SK-OV-3 ovarian carcinoma, A-549 lung carcinoma and Caco-2 colorectal carcinoma. They were purchased from the American Type Culture Collection and cultured briefly in vitro before being implanted into nude mice. The tumor lines were transferred as subcutaneous implants in nude mice. The mice to be used in the experiments were 8-9 weeks old when the tumors were implanted. Small pieces of tumor were implanted subcutaneously on the animals' left side. Animals with growing tumors (tumor volume 25-110 mm ) were randomly placed in a drug-treated group or a control group, where the average tumor size was approximately equal between the groups. The antitumor activity was measured from growth curves for tumor volume as well as histological evaluation of some tumors. During treatment, the tumors were measured 2 times a week by measuring two perpendicular diameters using calipers. The tumor volume was calculated based on the formula volume = (length x width)/2. The growth curves for tumor volume were generated by standardizing the tumor sizes in the different groups by finding relative tumor volume (RV) calculated from the formula RV = Vx/Vl, where Vx is the tumor volume on day x and VI is 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 applied to the data for relative growth in tumor volume, and the interval where the tumor volume in each group increased to twice the volume, the tumor volume doubling time (TD) was calculated from this curve (loge2/fc, where k is the calculated rate constant for the process). Histological assessment was based on macroscopic examinations of the tumor and light microscope examinations of small sections of the tumor (6-8 mm thick) embedded in paraffin and marked with hematoxylin and eosin.

Figure 7 shows that compound 1 induces a growth inhibitory effect on SKOV-3 ovarian carcinoma grown as xenografts in nude mice. These data indicate that the inhibition of tumor volume growth in these animals is stronger at a daily dose of 1 mg/kg than at a daily dose of 7.5 mg/kg.

Conclusions:

The products according to this invention react with active groups on the cell surface, e.g. amino, hydroxy or sulfhydryl groups in a significantly more efficient manner than previously known products, to form carbonyl condensation products such as Schiff bases, acetals, mercaptals, thiazolidenes, aminals, etc. on the cell surface. This can affect the signaling apparatus in the cells and can be used in the treatment of disorders such as cancer, immunological disorders, microbial infections, etc.

The aldehyde derivatives according to the invention react with certain groups on the cell surface, e.g. with free amino groups to form Schiff bases. As many cellular processes, such as protein synthesis, cell cycle progression, immune response, etc. are controlled by signals from the cell surface, these bonds alter the behavior of the cell. We have also demonstrated that the aldehyde complex in the cell surface changes the cell's adhesion characteristics. We have demonstrated that the compounds according to the invention can be useful in new therapies to fight cancer, autoimmune diseases, viral infections as well as infections by other microorganisms.

Administration

The pharmaceutical compositions according to the invention can be administered in cancer treatment, antiviral treatment or in the treatment of disorders that arise as a result of abnormally high cell division and/or to combat autoimmune diseases. These pharmaceutical compositions can also be administered as immunopotentiators.

To this end, the compounds of formula (I) may be formulated in any suitable manner for administration to a patient, either alone or mixed with suitable pharmaceutical carriers or adjuvants.

It is particularly preferred to prepare the substances for systemic therapy either for peroral or parenteral administration.

Suitable enteral forms of administration will be in the form of tablets, capsules, e.g. soft or hard gelatin capsules, granules, grains or powders, syrups, suspensions, solutions or suppositories. The preparation takes place in a well-known manner by mixing one or more of the compounds of formula (I) with non-toxic, inert, solid or liquid carriers.

Suitable parenteral administration forms for the compounds of formula (I) are solutions for injection or infusion.

For topical administration, the compounds of formula (I) may be in the form of a lotion, ointment, cream, gel, tincture, spray or the like containing the compounds of formula (I) mixed with non-toxic, inert, solid or liquid carriers which are common in topical preparations. It is particularly advantageous to use a form that protects the active ingredient from air, water and the like.

The preparations may include inert or pharmacodynamically active ingredients. Tablets or granules can e.g. include a series of binders, fillers, carriers and/or diluents. Liquid preparations can, for example, be present in the form of a sterile solution. Capsules may contain a filler or thickener in addition to the active ingredient. In addition, flavor-enhancing additives may also be present, as well as substances that are usually used as preservatives, stabilisers, humectants and emulsifiers, salts to vary the osmotic pressure, buffers and other additives.

The dosages in which the preparations are administered may vary according to the indication, the method of use and the method of administration, as well as according to the patient's needs. In general, the daily dose for systemic therapy for an average adult patient will be around 0.01-500 mg/kg body weight once or twice daily, preferably 0.1-100 mg/kg body weight once or twice daily, and most advantageously 1-20 mg /kg body weight once or twice daily.

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

Claims (16)

1. Derivatives of 5-nitrofurfural characterized by the formula (I): there R.! and R 2 is H or D, or is linked to form a 6-membered acetal ring comprising the substructure (II): where L is H or D; X is selected from atoms or groups comprising H, D, OH, OR4, NH2, NHR4, NR4R5, NHC(0)R4 or NC(0)R4R5 where R4 and R5 are alkyl of 1-6 carbon atoms or cycloalkyl of 3-6 carbon atoms and may be identical or different, or X may comprise the substructure (III): where L is H or D; Rb R2 and X are interrelated in such a way that if R! and R 2 is H or D, X is (III) and if R! and R 2 are linked together to form the substructure (II), in which case X is H, D, OH, OR 4 , NH 2 , NHR 4 , NPmR 5 , NHC(0)R 4 or NC(0)R 4 R 5 where R 4 and R 5 are defined as described above; R3 is H, D, alkyl of 1-20 carbon atoms, cycloalkyl of 3-6 carbon atoms, fluoroalkyl of 1-6 carbon atoms, alkenyl of 2-6 carbon atoms, alkynyl of 2-6 carbon atoms, provided that 2-deoxy-2 -[(5-nitrofurfurylidene)amino]-D-glucopyranose is excluded, or a pharmaceutically acceptable salt thereof. 2. Derivatives of 5-nitrofurfural according to claim 1, characterized in that the derivatives are 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-6-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D -galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6 -0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene-d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-di)amino]-D- glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof. 3. Derivatives of 5-nitrofurfural which are useful as therapeutic agents, characterized in that said derivatives are defined through formula (I), provided that 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose is excluded, or a pharmaceutically acceptable salt thereof. 4. Derivatives of 5-nitrofurfural according to claim 3, characterized in that the derivatives are 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-6-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D -galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6 -0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene-d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-d0amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof. 5. Use of derivatives of 5-nitrofurfural of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of cancer. 6. Use according to claim 5 of 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-6-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy -4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5- nitrofurfurylidene-d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-d1)amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of cancer. 7. Use of derivatives of 5-nitrofurfural of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of infections caused by viruses, protozoa, fungi or other microorganisms via changes of the immune system. 8. Use according to claim 7 of 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-B-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4 ,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene- d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-di)amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of infections caused by viruses, protozoa, fungi or other microorganisms via alterations of the immune system. 9. Use of derivatives of 5-nitrofurfural of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of disorders resulting from abnormally high cell division. 10. Use according to claim 9 of 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-B-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4 ,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene- d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-di)amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of disorders resulting from abnormally high cell division. 11. Use of derivatives of 5-nitrofurfural with formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of autoimmune diseases such as rheumatoid arthritis, psoriasis, psoriatic arthritis, lupus erythematosus, acne, Bekhterev's arthritis, progressive systemic sclerosis (PSS), seborrhea and other autoimmune diseases such as ulcerative colitis and Crohn's disease. 12. Use according to claim 11 of 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-B-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4 ,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene- d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-d i )amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylaxis and/or treatment of autoimmune diseases such as rheumatoid arthritis, psoriasis, psoriatic arthritis, lupus erythematosus, acne, Ankylosing spondylitis, progressive systemic sclerosis (PSS), seborrhea and others autoimmune diseases such as ulcerative colitis and Crohn's disease. 13. Use of derivatives of 5-nitrofurfural of formula (I), or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylactic treatment of cancers caused by viruses such as hepatitis B and C, oncogenic papillomaviruses and other oncogenic virus. 14. Use according to claim 13 of 4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene)amino]-D-glucopyranose, 2-deoxy-2- [(5-nitrofurfurylidene)amino]-B-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-galactopyranose, 4,6-0-(5-nitrofurfurylidene)-D-mannopyranose, 2-deoxy-4 ,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 2-acetamido-2-deoxy-4,6-0-(5-nitrofurfurylidene)-D-glucopyranose, 4,6-0-(5-nitrofurfurylidene- d1)-D-glucopyranose, 2-deoxy-2-[(5-nitrofurfurylidene-di)amino]-D-glucopyranose and/or 4,6-0-(5-nitrofurfurylidene)-L-glucopyranose, or the corresponding L-isomers, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic agent for the prophylactic treatment of cancers caused by viruses such as hepatitis B and C, oncogenic papillomaviruses and other oncogenic viruses. 15. A pharmaceutical composition characterized in that it comprises a derivative of 5-nitrofurfural according to one of the above patent claims, as well as a pharmaceutically acceptable carrier, diluent and/or excipient. 16. A method for producing a pharmaceutical composition characterized in that it comprises a step of incorporating a derivative of 5-nitrofurfural according to one of the above patent claims, in a pharmaceutically acceptable carrier, diluent and/or excipient.