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Title:
5-NITROFURFURAL DERIVATIVES
Document Type and Number:
WIPO Patent Application WO/2001/002412
Kind Code:
A1
Abstract:
The present invention relates to derivatives of 5-nitrofurfural which are useful as anticancer agents, antiviral agents, immunoprotentiators and/or as agents which may be used for combating illnesses which arise due to an elevated cell proliferation and/or for combating auto immune diseases.

Inventors:
BOERRETZEN BERNT (NO)
MOEN VIDAR (NO)
LARSEN ROLF OLAF (NO)
PETTERSEN ERIK OLAI (NO)
DUNSAED CAMILLA BRUNO (NO)
Application Number:
PCT/NO2000/000226
Publication Date:
January 11, 2001
Filing Date:
June 28, 2000
Export Citation:
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Assignee:
NORSK HYDRO AS (NO)
BOERRETZEN BERNT (NO)
MOEN VIDAR (NO)
LARSEN ROLF OLAF (NO)
PETTERSEN ERIK OLAI (NO)
DUNSAED CAMILLA BRUNO (NO)
International Classes:
A61P19/02; A61P31/00; A61P35/00; C07H5/06; C07H9/04; C07H15/26; (IPC1-7): C07H9/04; C07H5/06; C07H15/26; C07H17/04; A61P19/02; A61P31/00; A61P35/00
Domestic Patent References:
WO1992009276A11992-06-11
WO1995018607A11995-07-13
Foreign References:
EP0215395A21987-03-25
US2610181A1952-09-09
US4402965A1983-09-06
US3122535A1964-02-25
Attorney, Agent or Firm:
Lillegraven, Rita (Bryns Zacco AS P.O. Box 765 Sentrum Oslo, NO)
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Claims:
Derivatives of 5-nitrofurfural of formula (I): wherein R, and R2 are H or D or are linked together to form a 6-membered acetal ring comprising the substructure (II): wherein L is H or D; X is selected from the atoms or groups comprising H, D, OH, OR4, NH2, NHR4, NR4Rs, NHC (O) R4 or NC (O) R4Rs wherein R4 and Rs are alkyl with
1. 6 carbon atoms orcycloalkyl with 3. 6 carbon atoms and can be the same or different, or X can comprise the substructure (III) wherein L is H or D; Ri, R2 and X are interrelated such that if R, and R2 are H or D, then X is (III) and if R, and R2 are linked together to form the substructure (II), then X is H, D, OH, OR4, NH2, NHR4, NR4R5, NHC (O) R4 or NC (O) R4R5 wherein R4 and R5 are defined as above; R3 is H, D, alkyl with 1. 20 carbon atoms, cycloalkyl with 3. 6 carbon atoms, fluoroalkyl with 1. 6 carbon atoms, alkenyl with 2. 6 carbon atoms, alkynyl with 2. 6 carbon atoms, with the proviso that 2. deoxy. 2. [ (5. nitrofurfurylidene) amino]. D. glucopyranose is excluded, or a pharmaceutical acceptable salt thereof.
2. Derivatives of 5. nitrofurfural according to claim 1, wherein the derivatives are 4,6. 0. (5. nitrofurfurylidene). D. glucopyranose, 2. deoxy. 2. [(5. nitrofurfurylidene) amino]. ß. 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. d,). D. glucopyranose, 2. deoxy. 2. [(5. nitrofurfurylidene. d,) amino]. D. glucopyranose and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof.
3. Derivatives of 5. nitrofurfural useful as a therapeutic agent wherein the said derivatives are defined by formula (I), with the proviso that 2. deoxy. 2. [ (5. nitrofurfurylidene) amino]. D. glucopyranose is excluded, or a pharmaceutical acceptable salt thereof.
4. Derivatives of.
5. nitrofurfural according to claim 3, wherein the derivatives are 4,6. 0. (.
6. nitrofurfurylidene). D. glucopyranose, 2. deoxy. 2. [ (.
7. nitrofurnjrylidene) amino]. P. D. galactopyranose, 4,6. 0. (.
8. nitrofurfurylidene). D. galactopyranose, 4,6. 0. (.
9. nitrofurfurylidene). D. mannopyranose, 2. deoxy. 4,6. 0. (.
10. nitrofurfurylidene). D. glucopyranose, 2. acetamido. 2. deoxy. 4,6. 0. (.
11. nitrofurfurylidene). D. glucopyranose, 4,6. 0. (.
12. nitrofurfurylidene. d,). D. glucopyranose, 2. deoxy. 2. [(.
13. nitrofurfurylidene. d,) amino]. D. glucopyranose[(.
14. nitrofurfurylidene. d,) amino]. D. glucopyranose and/or 4,6. 0. (.
15. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof.
16. 5 Use of derivatives of 5. nitrofurfural of formula (I) or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of cancer.
17. 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]. p. 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. d,). D. glucopyranose, 2. deoxy. 2. [(5. nitrofurfurylidene. d,) amino]. D. glucopyranose[(5. nitrofurfurylidene. d,) amino]. D. glucopyranose and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of cancer.
18. Use of derivatives of 5. nitrofurfural of formula (I) or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of infections caused by virus, protozoa, fungi and other microorganisms via alteration of the immune system.
19. 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]. ß. 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. d,). D. glucopyranose, 2. deoxy. 2. [ (5. nitrofurfurylidene. d) amino]. D. glucopyranose and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of infections caused by virus, protozoa, fungi and other microorganisms via alteration of the immune system.
20. Use of derivatives of 5. nitrofurfural of formula (I) or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of diseases arising from an abnormally elevated proliferation.
21. 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]. ß. 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. dl). D. glucopyranose, 2. deoxy. 2. [(5. nitrofurfurylidene. d,) amino]. D. glucopyranose[(5. nitrofurfurylidene. d,) amino]. D. glucopyranose and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of diseases arising from an abnormally elevated proliferation.
22. Use of derivatives of 5. nitrofurfural of formula (I) or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of auto immune diseases like rheumatoid arthritis, psoriasis, psosiatic arthritis, lupus erythematosis, acne, Bechterew's arthritis, progressive systemic sclerosis (PSS), seborrhea and other auto immunic disorders like Ulcerous colitt and Morbus Chron.
23. Use according to claim 11, of 4, 6. 0. (5. nitrofurfurylidene). D. glucopyranose, 2. deoxy. 2. [ (5. nitrofurfurylidene) amino]. D. glucopyranose,<BR> 2. deoxy. 2. [(5. nitrofurfurylidene) amino]. ß. 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. d,). D. glucopyranose, 2. deoxy. 2. [ (5. nitrofurfurylidene. d) amino]. D. glucopyrano se and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for the prophylaxis and/or treatment of auto immune diseases like rheumatoid arthritis, psoriasis, psosiatic arthritis, lupus erythematosis, acne, Bechterew's arthritis, progressive systemic sclerosis (PSS), seborrhea and other auto immunic disorders like Ulcerous colitt and Morbus Chron.
24. Use of derivatives of 5. nitrofurfural of formula (I) or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for prophylactic treatment of cancers induced by viruses like hepatitis B and C, oncogene papilloma viruses and other oncogene viruses.
25. 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]. ß. 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. d,). D. glucopyranose, 2. deoxy. 2. [(5. nitrofurfurylidene. d,) amino]. D. glucopyranose[(5. nitrofurfurylidene. d,) amino]. D. glucopyranose and/or 4,6. 0. (5. nitrofurfurylidene). L. glucopyranose, or the corresponding L. isomers, or a pharmaceutical acceptable salt thereof, for the manufacture of a therapeutic agent for prophylactic treatment of cancers induced by viruses like hepatitis B and C, oncogene papilloma viruses and other oncogene viruses.
26. A pharmaceutical composition comprising a derivative of 5. nitrofurfural according to any preceding claim, and a pharmaceutically acceptable carrier, diluent and/or excipient.
27. A process for manufacture of a pharmaceutical composition, which comprises the step of incorporating a derivative of 5. nitrofurfural as defined in any preceding claim, together with a pharmaceutically acceptable carrier, diluent and/or excipient.
Description:
5-NTTROFURFURAL DERIVATIVES The present invention relates to derivatives of 5-nitrofurfural which are useful as anticancer agents, antiviral agents, immunopotentiators and/or as agents which may be used for combating illnesses which arise due to an elevated cell proliferation and/or for combating auto immune diseases. Most of the compounds of this invention are novel per se.

Most of the presently used anticancer agents are cytotoxic in their action. Although these agents have shown good results in treatment of some cancers like lymphoma, leukaemia and testicular cancer, they often produce severe and unacceptable side-effects limiting the possibility for an effective treatment. Furthermore, in several types of cancer like in solid tumours (carcinoma), chemotherapy has so far proven to be of limited value since established cytostatic drug seldom improves the prognosis for the patient. The ability of cancer cells to develop resistance against cytotoxic products is also a main reason for the failure in their use in the treatment of solid tumours. There is thus a great need for new anticancer agents having fewer side effects and having a more selective action on malignant cells.

It is known among other from EP-0215395, JP-63264411, JP-8800940, JP-55069510 and EP-0283139 that benzaldehydes and derivatives thereof exhibit a selective anticancer effect.

We have also previously found that some heteroaromatic aldehyde derivatives possess even stronger cell inactivating properties. In WO 95/18607 the anticancer action of 5-nitrofurfurylidene diacetate is disclosed. This compound exerts a stronger effect on NHIK 3025 cells than the 3-and 4-nitrobenzylidene analogues.

Although most of the present knowledge on how aldehydes react at a 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 range of O, S or N nucleofilic entities like hydroxy groups, sulfhydryl groups and amino groups to form carbonyl condensation products like acetals, mercaptals, aminals, etc.

However, with primary amines, the reaction normally take the form of Schiffs base (imine) adduct formation. It is well known that in vivo Schiffs base formation is involved in key biochemical processes like transamination, decarboxylation and other amino acid modifying reactions mediated by pyridoxal phosphate, the action of aldolase on fructose di-phosphate in the glycolysis and the condensation of retinal with rhodopsin in the process of vision. It is

also known that carbonyl condensation reactions are involved in transmembrane signalling events, for example in generating an immune response.

The formation of imines proceeds through a two-stage mechanism : The addition of the amino nucleofile to the carbonyl group to form a carbinolamine (aminohydrin) intermediate followed by a dehydration step to generate the C=N double bond. Both steps are reversible, but are facilitated at different pH values. As a consequence, the reaction occurs according to a characteristic bell-shaped pH/rate profile with the highest over-all reaction rates being found at moderate acidities. aldehyde amine carbinolamine imine However, the formation of Schiffs bases are known to take place readily also in 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 Schiffs base tend to be a reactive species itself and is prone to further reaction resulting in the addition of nucleofilic agents to the double bond. For certain sulfur-containing amines, in particular the amino acids cystein and methionine, and for glutathione, the initially formed Schiffs base can undergo reversible internal cyclization in which the sulfhydryl group adds 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). aldehyde cystein thiazolidine carboxylic acid

Evidence for reactions between carbonyl compounds and free amino groups of proteins to form reversible Schiffs base linkages was reported by G. E. Means and R. E. Feeney (Chemical Modification ofPoteins, pp. 125-138, San Francisco, Holden-Day, 1971).

Aromatic aldehydes are in general more reactive than saturated aliphatic aldehydes, and Schiffs bases can be formed even without removal of the water formed during the reaction (R. W. Layer Chem. Rev. 63 (1963), 489-510). This fact is important when considering formation of Schiffs bases under physiological conditions. Using haemoglobin as a source of amino groups Zaugg et. al. (J. Biol. Chem. 252 (1977), 8542-8548) have shown that aromatic aldehydes have a two-to threefold increased reactivity over aliphatic aldehydes in Schiffs base formation. An explanation for the limited reactivity of alkanals could be the fact that in aqueous solution at neutral pH a very large excess of free aldehyde is required to shift the equilibrium in favour of Schiffs base formation (E. Schauenstein et. al., Aldehydes in biological systems. London, Pion Ltd. 1977).

Aromatic-and heteroaromatic aldehydes readily form Schiffs base imines with membrane amino groups, and high equilibrium constants have been measured for benzaldehyde reacting 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. 15X, (1968) 323-331). Substituents possessing electron withdrawing properties in the aromatic ring will make the carbonyl carbon more electrofilic and thus more prone to addition reactions with amino nucleofiles.

Biological implications of introducing a nitro group were assessed by comparing deuterated and undeuterated 5,6- (3-nitrobenzylidene)-L-ascorbate with the unsubstituted analogue zilascorb ('H). It was shown that a far greater protein synthesis inhibition was imposed by the nitro substituted derivatives in NHIK 3025 cells (EP-0493883).

We have previously shown by radio labelling images that benzaldehyde do not enter the cell, but adhere to the cell membrane (Dornish, J. M. and Pettersen, E. O.: Cancer Letters 29 (1985) 235-243). This is in agreement with an earlier study, showing that benzaldehyde interacted with the membrane proteins of E. coli (K. Sakaguchi et. al. Agric. Biol. Chem., (1979), 43,1775-1777). It was also found that pyridoxal and pyridoxal-5-phosphate both protect the cells against the cytotoxic anti-cancer agent cis-DDP. Cis-DDP exerts its action in the nucleus within the cell. While pyridoxal in principle could penetrate the lipophilic cell

membrane, this possibility is blocked for pyridoxal-5-phosphate because of the ionic phosphate group on the latter. Pyridoxal-5-phosphate thus have to exert its protective effect by acting from outside the cell membrane. A spectral shift in the absorbance of pyridoxal-5-phosphate to lower wavelengths observed simultaneously is consistent with Schiffs base adduct formation between the aldehyde and cell membrane amino groups (J. M.

Dornish and E. O. Pettersen, Cancer Lett. 29, (1985), 235-243).

These findings suggest that aldehydes bind to amines and other nucleofilic entities on the cell membrane to form Schiffs bases and other condensation products. It is known that stimulation of cell growth is mediated by a cascade of events acting from outside the cell membrane. In the same way, the derivatives in the present patent application may act by forming adducts with ligands on the cell membrane, triggering impulses inside the cell with significance on cell growth parameters like protein synthesis and mitosis, and on the expression of tumour suppressor genes and immune responses. Since the condensation reactions are reversible, cellular effects can be modulated as a result of a shift in equilibrium involving ligating species. The presence of dynamic equlibria at a chemical level is consistent with the reversible action observed with the aldehyde derivatives.

Inhibition of the protein synthesis excerted by aromatic-or heteroaromatic aldehyde derivatives is very well studied in vitro by the inventors. In solid tumours the reduced protein synthesis may result in a lack of vital proteins which lead to cell death. In normal cells there is a potential capacity for protein synthesis which is higher than in most cancer cells of solid tumours. This is demonstrated by comparison of the cell cycle duration in normal stem cells, which is often below I Oh, and thus shorter than that of most cancer cells of solid tumours, which is typically 30-150h (see Gustavo and Pileri in: The Cell Cycle and Cancer. Ed.: Baserga, Marcel Dekker Inc., N. Y. 1971, p 99). Since cells, as an average, double their protein during a cell cycle, this means that protein accumulation is higher in growth-stimulated normal cells than in most types of cancer cells.

Keeping in mind this difference between normal and cancer cells, there is another difference of similar importance: while normal cells respond to growth-regulatory stimuli, cancer cells have reduced or no such response. Thus, while normal cells, under ordinary growth conditions, may have a reserve growth potential, cancer cells have little or no such reserve.

If a protein synthesis inhibition is imposed continuously over a long period of time on normal cells as well as on cancer cells, the two different types of cells may respond differently: Normal tissue may make use of some of its reserve growth potential and thereby maintain normal cell production. Cancer tissue however, have little or no such reserve. At the same time the rate of protein accumulation in most cancer cells is rather low (i. e. protein synthesis is only slightly greater than protein degradation). Therefore the protein synthesis inhibition could render the tumour tissue imbalanced with respect to protein accumulation, giving as a result a negative balance for certain proteins. During continuous treatment for several days this will result in cell inactivation and necrosis in the tumour tissue while normal tissue is unharmed.

To date, the most tested compounds inducing reversible protein synthesis inhibition and displaying anti-cancer activity are zilascorb (2Hj (EP-0283139 and Pettersen, et. al., Anticancer Res., vol 11, pp. 1077-1082,1991) and 4,6-0-benzylidene-D-glucopyranose (M.

Kochi et. al., Cancer Treat. Rep., vol. 69, no. 5, pp. 533-537,1985 and T. Tatsumura et. al., Br. J. Cancer, vol. 62, pp. 436-439,1990). Also, the compound 5-nitrofurfurylidene diacetate have shown strong anticancer activity in vitro. For instance, in a culture of NHIK 3025 cervix carcinoma cells, at 1 h exposure with 0.1 mM drug concentration, the rate of the protein synthesis was approx. 35 % of control (WO 95/18607). However, with only about 25 M concentration of 2-deoxy-2- [ (5-nitrofurfurylidene) amino]-D-glucopyranose, compound 2 of the present invention, a relative rate of protein synthesis at 35 % of the control value was displayed with this model (see Fig. 5A). Astonishingly strong effects are also displayed by measuring the surviving fraction of NHIK 3025 cells after 20 h exposure of compound 2 (see Fig. 2). At 50 uM drug concentration, the surviving fraction is diminished by 4 decades. Thus, compound 2 exhibits stronger effects than measured with any other reversible protein synthesis inhibitor tested with this model. Figure 5A shows that compounds 2 and 3 are more effective as protein synthesis inhibitors than the formerly known compound, 5-nitrofurfurylidene diacetate by a factor of about an order of magnitude for low drug doses.

It has been known for a long time that some nitrofuryl-and nitroimidazol derivatives possess strong antiseptic properties. Of the many 5-nitrofurfuryl derivatives which have been prepared, the semicarbazone gained wide application as a topically applied therapeutic agent

(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-Nitrofurfuryliden)-l-aminohydantoin is a chemotherapeutic agent still in use to cure 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 derivatised with a methylmorpholine entity.

Moreover, bis (5-nitrofurfurylidene) acetone guanylhydrazone (Nitrovin) is widely in use as a feed additive. The compound 5-nitrofurfurylidene-D-glucosamine and its inhibitory effects upon bacteria as well as pathogenic fungi is disclosed in US 3 122 535. Although the chemical structure is ambiguous without specified anomeric stereochemistry, it probably anticipates compound 2 in the present patent application. All the other claimed compounds (1 and 3-7 in the table) are novel per se to the best of our knowledge.

None of these prior art compounds are known to possess anticancer activity, nor are they known to have immunostimulating properties. They are, however, well-known as antibacterial, antifungal or antiprotozoal agents. Their action as bacteriostatic agents rely on the inhibition of vital bacterial enzyme systems, a mechanism completely different from the indirect way of action through stimulation of the host's immune system, as revealed for the presently claimed compounds.

We have now surprisingly found that the products of this invention show astonishingly stronger effect (by about one order of magnitude) concerning inhibition of protein synthesis and cell survival than known in the prior art (see Example 1 and 2). We have also found that the reversibility of the effect of these compounds are not pronounced as for the products previously known. This could mean that the binding of these aldehydes to the cell surface is stronger than the binding of those previously known and hence, could obtain a more efficient therapeutic treatment for the invented compounds.

It is known from UK Patent application 9026080.3 that benzaldehyde compounds, previously known as anti cancer agents may be used for combating diseases resulting from an abnormally elevated cell proliferation.

Dermatologic abnormalities such as psoriasis are often characterised by rapid turnover of epidermis. While normal skin produces about 1250 new cells/day/cm of skin consisting of

about 27,000 cells, psoriatic skin produces 35,000 new cells/day/cm2 from 52,000 cells. The cells involved in these diseases are however"normal"cells reproducing rapidly and repeatedly by cell division. While the renewal of normal skin cells takes approximately 311 hours, this process is elevated to take about 10 to 36 hours for psoriatic skin.

Today psoriasis, inflammatory diseases, rheumatic diseases and other auto immune disorders are treated with corticosteorids, NSAIDs and in serious cases, with immunosupressive agents like cytostatica and cyclosporins. All these drugs can give serious side-effects. It is thus a great need for effective products giving less side-effects.

It is known that aromatic-and heteroaromatic aldehydes and certain acetal derivatives thereof have a growth-inhibitory effect on human cells which is by its nature reversible.

Growth inhibition induced by these compounds is primarily due to a reduction in the protein synthesis by cells. (Pettersen et al., Eur. J. Clin. Oncol., vol. 19, pp. 935-940,1983 and Cancer Res., vol. 45, pp. 2085-2091,1985). The inhibition of protein synthesis is only effective as long as these agents are present in the cellular microenvironment.

This leads to the effect that the normal cells are left without damage after treatment with the above compounds. The ratio effect/side effect (therapeutic index) for this class of products may be dependent of the degree of this reversibility, and to optimize the therapeutic benefit of the products, these properties must be taken into consideration.

The ability of cells to transmit signals via cell-contact (adhesion) dependent mechanisms has been studies over many years. These mechanisms are especially important for the reactivity of circulating cells like lymphocytes, macrophages etc., and also for instance for methastatic cells to be anchored in tissues to establish new tumours. The possibility to alter the adhesion characteristic of cells which are monitoring the immune or inflammatory response might be of great therapeutic value for the treatment of many diseases like rheumatoid arthritis, psoriasis, psoriatic arthritis, lupus erythematosis, acne, Bechterew's arthritis, progressive systemic sclerosis (PSS), seborrhea and other auto immunic disorders like Ulcerous colitt and Morbus Crohn, etc.

The immune system is carefully designed to identify and eliminate any material recognised as non-self, whether it originates from a bacteria-, virus-or protozoal infection, or abnormal cells like cancer. In order to provide a specific response to the huge range of biotic variation represented by the invaders, the immune system has to be highly diversified. However, over-stimulation of this finely tuned system can lead to various allergic-and inflammatoric reactions and cause auto-immune diseases. The rejection of beneficial transplants is also difficult to overcome. It is therefore a big therapeutic challenge to modulate the immune system, either by up-regulating or down-regulating a specific response.

In an immunological recognition process, a fragment of a foreign protein is confined in 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 of a T helper cell. To activate a T helper cell, at least two signals must be provided: The primary signal is mediated by the antigen itself, via the class II MHC complex and augmented by CD4 co-receptors. The second signal can be provided by a specific plasma-membrane bound signalling molecule on the surface of the APC. A matching co-receptor protein is located on the surface of the T-helper cell. Both signals are needed for the T-cells to be activated. When activated, they will stimulate their own proliferation by secreting interleukin growth factors and synthesising matching cell-surface receptors. The binding of interleukins to these receptors then directly stimulates the T-cells to proliferate.

In the 1980'ties, it was recognised that a cyclodextrin benzaldehyde inclusion complex could stimulate the immune system by augmentation of the lymphokine activated killer cells in a murine model (Y. Kuroki et. al., J. Cancer Res. Clin. Oncol. 117, (1991), 109-114).

Studies made in vitro have later revealed the nature of the chemical reactions at the APC-donor/T-cell receptor interaction site responsible for the second co-stimulatory signal, and that these take form of carbonyl-amino condensations (Schiffs base formation).

Moreover, these interactions can be mimiced by synthetic chemical entities. These findings open up for new therapeutic opportunities for artificially potentiating the immune system. In WO 94/07479 use of certain aldehydes and ketons which forms Schiffs bases and hydrazones with T-cell surface amino groups are claimed. In EP 0609606 A1 the preferred immuno stimulating substance is 4- (2-formyl-3-hydroxyphenoxymethyl) benzoic acid

(Tucaresol), a compound originally designed to cure sickle cell anaemia. This substance is administered orally and is systemically bioavailable.

It is well known that glycoproteins bound to the cell plasma membrane have the ability to associate with sugar molecules dissolved in the extracellular fluid. Affinity to such receptors will contribute to enrich and anchor sugar/aldehyde derivatives to the cell surface. Reactive aldehyde is released through hydrolysis and become available for in situ Schiffs base formation with amino groups protruding from the cell surface. In the present patent application, 5-nitrofurfural is derivatised with biologic acceptable carbohydrates like glucose, glucosamine, galactosamine and other sugars, deoxysugars and aminosugars by either an acetal-or an imine linkage. The sugar moiety will also contribute by improving stability and enhancing bioavailability of the aldehyde function to the target cells.

The immune stimulating effect of the invented compounds may also be used in the treatment of certain virus diseases in combination with other anti-viral therapy like anti-viral drugs or vaccines. Many virus types, after the first infection, incorporate with the cell nucleus and are inactive for a long period of time. Oncogenic viruses like hepatitis B and C, certain retro virus and certain papilloma virus may cause development of cancer. In these latent period it is very difficult to cure the virus infection. These viruses can often be triggered by immune responses to cause viremia, and in this stage make it possible to get rid of the virus infection.

The ability of the aldehyde derivatives to trigger the immune response may be used in combination with antivirals or vaccines to develop a treatment for these diseases.

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

A second object of the invention is to provide new compounds being able to potentiate immune responses giving a possibility to combat infectious diseases caused by virus, bacteria, fungus and other micro organisms.

A third object of the invention is to provide compounds for prophylaxis or treatment of cancer and diseases related to immune disorders not giving toxic side-effects.

A fourth object of the invention is to provide compounds for effective and favourable prophylaxis and/or treatment of cancer in tissues and cells having receptors with affinity to corresponding sugar moieties.

A fifth object of the invention is to provide compounds for treatment of diseases related to the immune system likepsoriasis, bowel inflammations, arthritis, SLE, PSS etc.

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

The compounds of the present invention have the general formula (I): wherein R, and R2 are H or D or are linked together to form a 6-membered acetal ring comprising the substructure (II): wherein L is H or D.

X is selected from the atoms or groups comprising H, D, OH, OR4, NHz, NHR4, NR4Rs, NHC (O) R4 or NC (O) R4R5 wherein R4 and Rs are alkyl with 1-6 carbon atoms or cycloalkyl with 3-6 carbon atoms and can be the same or different. X can optionally comprise the substructure (III):

wherein L is H or D; Ri, R2 and X are interrelated such that if R, and R2 are H or D, then X is (III). If R, and R2 are linked together to form the substructure (II), then X is H, D, OH, OR4, NH2, NHR4, NR4R5, NHC (O) R4 or NC (O) R4Rs wherein R4 and Rs are defined as above.

R3 is H, D, alkyl with 1-20 carbon atoms, cycloalkyl with 3-6 carbon atoms, fluoroalkyl with 1-6 carbon atoms, alkenyl with 2-6 carbon atoms, alkynyl with 2-6 carbon atoms, or a pharmaceutical acceptable salt thereof.

It is understood that any stereo isomer according to the formula is comprised in the present invention.

With exception of compound 2, all compounds according to the present invention are new per se.

Detailed description of the invention The invention is further explained below by a table, preparation-and biological examples and attached figures. CompoundNo.ChemicalStructureName H 04, 6-0- (5-Nitrofurfurytidene)- 0 D-glucopyranose 0 4H ON OH OH HO H HOoX HA NOH 2-Deoxy-2- [ (5-nitrofurfurylidene)- amino]-D-glucopyranose H -1) 0 OH OH -0 HO OH 3N2-Deoxy-2- [ (5-nitrofurfurylidene)- amino]-beta-D-galactopyranose H -11 of, 02N 0 4, 6-0- (5-Nitrofurfurylidene)- D-galactopyranose on H 0 0 HO OHOH H O OH 4, 6-0-(5-Nitrofurfurylidene)- 5°D-mannopyranose 0 HO onOH CompoundNo.ChemicalStructureName H vo 2-Deoxy-4, 6-0- 60 0 (5-nitrofurfurylidene)- HOHOD-glucopyranose 02NOH H 2-Acetamido-2-deoxy- 0HOw 4, 6-0-(5-nitrofurfurylidene)- 02ND-glucopyranose CH, D 8 So 4, 6-0-(5-Nitrofurfurylidene-d1)- D-glucopyranose 0 HO.-C \ Lr POOH OH HA 02N OH OH Ho' 9 N OH 2-Deoxy-2-[(5-nitrofurfurylidene-d1)- amino]-D-glucopyranose 0 0 NO2 Oh 100" 10N z o 0 4, 6-0- (5-Nitrofurfurylidene)- O +° OH L-glucopyranose HO H

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

As is well known, aldehydes undergo acid facilitated condensation reactions with carbohydrates to generate sugar acetals. In stead of the aldehyde itself, a lower dialkyl acetal of the aldehyde is most often used, and the reaction forced to completion by evaporating the concomitantly formed lower alcohol under reduced pressure. However, because the nitro group exerts a highly deactivating effect on the formyl group, the formation of sugar acetals with 5-nitrofurfural is very sluggish. Elevated temperatures and prolonged reaction times will easily lead to product isomerisation and degradation, resulting in very complex reaction mixtures. It is therefore more preferable to apply protection strategies. 4,6-0-benzylidene-D-glucopyranose was already available in-house and may be chosen as a convenient starting material. By acetylating the remaining hydroxy groups and removing the benzylidene group, the 1,2,3-tri-O-protected sugar was obtained in high yield.

The acetalisation of this key intermediate with 5-nitrofurfural can now be performed under more forced reaction conditions without devastating side reactions. The new 5-nitrofurfurylidene sugar acetal (Compound 1) was finally obtained following base catalysed 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 synthesised in one step by regulating pH in a methanolic suspension of glucosamine hydrochloride and adding 5-nitrofurfural.

The compounds of formula (I) wherein L is deuterium may be prepared as described above, but starting with 5-nitrofurfural which is deuterated in the formyl position. The preparation of the deuterated aldehyde may be performed according to one of the examples given in EP 0 552 880 A1, of which the umpolung strategy is the most prefered method: The aldehyde is protected as its 1,3-dithioacetal, BuLi is added to generate the lithium salt which is subsequently quenched with D20 and the protecting group removed. In the following steps

the thus formed 5-nitrofurfural-d, is used as a starting material in analogy with the undeuterated counterpart.

The acetalisation reactions may be carried out in a dipolar, aprotic solvent such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethoxy ethane or the like. However, in order to conveniently remove water, an azeotrope forming solvent such as chloroform, dichloromethane or ethylacetate is preferred.

The catalyst may be a mineral acid, e. g. sulphuric acid, an organic acid, e. g. para-toluene sulfonic acid, an acidic ion exchanger 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 were achieved by using'H and"C NMR operation at 300 or 400 MHz proton frequencies.

The following examples are illustrative of how the compounds of the present invention may be prepared.

Compound 1: 4.6-0-(5-Nitrofurfurvlidene)-D-glucopyranose 1,2,3-tri-O-Acetyl-4,6-0-benzylidene-D-glucopyranose 4,6-0-Benzylidene-D-glucopyranose (25.0 g, 0.093 mol) was dissolved in a mixture of acetic anhydride (370 ml) and pyridine (750 ml) and left at 20°C for 24 hours. The reaction mixture was evaporated, and the residue repeatedly diluted with toluene and evaporated.

The raw product was recrystallised from heptane/ethylacetate. The yield was 32.2 g, 87% of the theoretical. The identity was confirmed by'H NMR spectroscopy.

1,2,3-tri-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 ethylacetate (250 ml) under N2.10% Pd on active carbon (1.2 g) was added and H2 bubbled through at 1 bar, 20°C for 20 hours. The reaction mixture was filtered and evaporated. The raw product was repeatedly diluted/evaporated with CHCI3 and CH2C12 to remove traces of ethylacetate. NMR analysis indicated the debenzylation to be quantitative and an a to ß ratio of 1: 3. The identity was confirmed by'H NMR spectroscopy.

1,2,3-tri O-Acetyl-4,6-0- (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 CH2CI2 (250 ml) together with a catalytic amount ofpara-toluene sulphonic acid. The reaction mixture was boiled for 20 hours and the reflux drained via 4A molecular sieve (80 ml). A small portion of triethylamine and silica gel (90 g, 200-500 um) was added and the solvent removed by evaporation. The product was chromatographed on silica gel (1100 ml, 20-45 um) eluting with heptane/ethylacetate (60/40). 250 ml fractions were collected and analysed (TLC). The product was located in fractions 11-23, which were combined and evaporated. The residue formed crystals and was vacuum-dried. The yield was 6.78 g, 31% of the theoretical. The identity was confirmed by'H NMR spectroscopy.

4,6-O- (5-Nitrofurfurylidene)-D-glucopyranose To a solution of 1,2,3-tri-O-acetyl-4,6-O- (5-nitrofurfuryliden)-D-glucopyranose (6.78 g, 0.0158 mol) in methanol (400 ml) was added NaOCH3 (0.13 g, 0.0024 mol). The reaction mixture was stirred at 20°C for 2 hours (TLC indicated the reaction to be nearly completed within 20 min.), and then stored at 5°C for 20 hours. A minor precipitate was formed. 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

volume of the filtrate was reduced to 15 ml and a second precipitate isolated. The two crops were combined to give 3.96 g product, 83% of the theoretical yield.

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

'H-and 13C NMR (400 MHz; DMSO-d6), 5 rel. to TMS: 7.67 (d, 1H, 02N-CH=CH-CH=) 6.89 (dd, 1H, 02N-CH=CH-CH=), 6.85 (d, 0.5H, OH-1 II), 6.58 (d, 0.5H, OH-1 I), 5.79 (s+s, 1H, acetal-HI+II), 5.29,5.21,5.17 and 4.85 (m+d+d+d, 0.5H+0.5H+0.5H+0. SH, OH-2+OH-3), 4.95 (t, 0.5H, H-1 1), 4.43 (t, 0.5H, H-1 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-4 1+1, H-5 II and H20), 3.27-3.21 and 3.03-2.95 (m+m, 0.5H+0.5H, H-2 1+11); 153.34 and 152.11 (furfuryl =CH-O-CH=), 114.02 and 112.80 (furfuryl =CH-CH=), 98.45,94.81, 94.70 and 94.07 (acetal C 1+11 and sugar C-1 1+11), 82.47 and 81.68 (sugar C-4 1+11), 76.62 and 73.47 (sugar C-2 1+11), 73.68 and 70.27 (sugar C-3 1+11), 69.18 and 68.82 (sugar C-6 1+11) and 66.16 and 62.46 (sugar C-5 1+11).

Compound 2: 2-Deoxv-2-f (5-nitrofurfurylidene) aminol-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 was quickly formed. 5-Nitrofurfural (3.53 g, 25.0 mmol) was then added portion wise and the reaction mixture was seen to darken rapidly to a deep brown colour as the aldehyde dissolved. After stirring for about 20 min at room temperature a pale brown precipitate was seen to form and the reaction was allowed to stir overnight under nitrogen. The mixture was then filtered with the aid of water pump vacuum and washed with ethanol until the filtrate was colourless (approx. 50 ml) to give a beige coloured solid which was dried under a stream of nitrogen (1.95 g, 52%).

'HNMR 5H (300 MHz; DMSO-d6) 2.88 (t) and 3.10-3.85 (m) (a-and ß-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 0-H-1, OH-3,

OH-4, OH-6), 6.43 (d, a-OH-1), 6.71 (d, (3-OH-1), 7.28 (1H, m, ArH) and 7.78 (1H, m, ArH), 8.16 (s, ß-CH=N) and 8.27 (s, a-CH=N).

Compound 3: 2-Deoxv-2-[(5-nitrofilrfurvlidene) aminol-ß-D-galactopYranose 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 was quickly formed. 5-Nitrofurfural (3.59 g, 25.4 mmol) was then added portion wise and the reaction mixture was seen to darken rapidly to a deep brown colour as the aldehyde dissolved. After approx. 1.5 h some precipitate was seen to form and this thickened over the course of the next half an hour. The reaction mixture was left to stir overnight at room temperature under an atmosphere of nitrogen. It was then filtered and the brown residue washed with ethanol (20 ml) and then, whilst continuing suction, dried under a stream of nitrogen for 20 minutes to give a cream powder containing only the ß-isomer (2.45 g, 64%).

'H NMR AH (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-1, 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 oct'H} (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-NO2 and C-C=N).

Compound 4: 4. 6-OS-Nitrofurfurylidene)-D-galactopyranose 4,6-O-Benzylidene-D-galactopyranose (0.093 mol) is dissolved in a mixture of acetic anhydride and pyridine and left at ambient temp. over night. The reaction mixture is evaporated, and the residue repeatedly diluted with toluene and evaporated. The raw product is recrystallised and the structure of the formed 1,2,3-tri-O-acetyl-D-galactopyranose is confirmed by NMR analysis. This intermediate (0.0517 mol) is dissolved in ethylacetate under N2. A stoichiometric excess of 10% Pd on

active carbon is added and H2 bubbled through at ambient temp. over night. The reaction mixture is filtered and evaporated. The raw product is repeatedly diluted and evaporated with CHCI3 and CH2CI2 to remove traces of ethylacetate. The structure of the formed 1,2,3-tri O-acetyl-4,6-0- (5-nitrofurfurylidene)-D-galactopyranose 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 CH2CI2 together with a catalytic amount ofpara-toluene sulphonic acid. The reaction mixture is boiled over night and the reflux drained via 4A molecular sieve. A small portion of triethylamine and silica gel are added and the solvent removed by evaporation. The product is chromatographed on silica gel eluting with heptane/ethylacetate. Fractions are collected and analysed (TLC) and the product fractions combined and evaporated. The structure of 1,2,3-tri-O-acetyl-4,6-0- (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 stirred at ambient temp. to completion (monitoring by TLC). The thus formed final product is isolated by appropriate work-up and dried in vacuo. The structure of 4,6-0- (5-nitrofurfurylidene)-D-galactopyranose is confirmed by NMR analysis.

Compound 5: 4,6-0- (5-Nitrofurfurvlidene)-D-mannopvranose This product is synthesised as described above, except that galactose is replaced by mannose.

Compound 6 : 2-Deoxv-4. 6-0-(5-nitrofurfirvlidene)-D-zlucopvranose This product is synthesised as indicated above, starting from 2-deoxyglucose Compound 7: 2-Acetamido-2-deoxv-4. 6-0- (5-nitrofurfurvlidene)-D-glucopyranose This product is synthesised as indicated above, starting from glucosamine hydrochloride.

Compound 8: 4.6-0-(5-Nitrofurfurvlidene-d,)-D-glucopvranose 5-Nitrofurfural, 1,3-propanedithiol (1 eqv.), para-toluene sulfonic acid (cat. amounts) and toluene (2 ml pr. mmol aldehyde) are mixed and boiled under reflux until the reaction is completed (approx. 20 h). Hexane and a minor amount of diisopropyl ether are added and the solution cooled in order to precipitate the product. Crystals of 5-nitrofurfural-1,3-propane dithioactal are filtered off and dried. The identity is confirmed by NMR spectroscopy.

5-Nitrofurfural-1,3-propane dithioacetal is dissolved in dry tetrahydrofurane (approx 6 ml pr. 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 butyl lithium in hexane (1.5 eqv.) is added slowly from a syringe while keeping the reaction mixture below-50°C. When the reaction is completed (approx. 4 h), D20 (1 ml pr. mmol thioacetal) is added and the reaction mixture stirred and allowed to come to room temperature. A precipitate of 5-nitrofurfural-1,3-propane dithioacetal-d, is filtered off and dissolved in dichloromethane.

The solution is washed with diluted hydrochloric acid and water, and then dried. This crude product can optionally be recrystallised from an appropriate solvent. The identity and deuterium content is determined by NMR spectroscopy.

5-nitrofurfural-1,3-propane dithioacetal-d"HgCI2 (2.2 eqv.) and HgO (1.1 eqv.) are dissolved in a 9: 1 mixture of acetonitril and water and boiled at reflux for approx. 2 h. After cooling, insoluble mercury salts are filtered off and the filtrate washed with 5 % ammonium acetate solution. 5-nitrofurfural-d, is formed as a precipitate, which is filtered off and recrystallised from an appropriate solvent. The identity is confirmed by NMR spectroscopy.

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

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

Compound 9: 2-Deoxv-2-[(5-nitrofurfurvlidene-dl) aminol-D-glucopyranose 5-Nitrofurfural-d, is prepared as described above and is condensed with glucosamine hydrochloride according to the procedure given in example 2.

Compound 10: 6-0-(5-Nitrofurfurvlidene !-L-glucopvranose 4,6-O-Benzylidene-L-glucopyranose L-Glucose (5.14 g, 28.6 mmol) was warmed in DMF (20 ml) to 95 °C until a clear solution was 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 minutes to the stirred glucose solution under a regulated water pump vacuum of 80 mbar. The DMF was then evaporated under vacuum (2 mbar) at 65 °C to give a very pale yellow oil to which was added sodium bicarbonate (345 mg) followed by stirring for 5 minutes. Warm water (67 °C, 15 ml) was added with stirring (magnetic bead) to the oil at 65 °C and then the flask was shaken in the warm water bath until the oil appeared to have dissolved. The reaction flask 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 : to stand for 40 minutes. A white precipitate formed during this time which was isolated by vacuum filtration (decanted from the amorphous material), washed with cold spring 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 analysed by gas chromatography and appeared to be 99% pure desired product.

'H NMR, 8 (DMSO-d6) rel. to TMS: 7.55-7.25 (m, 5H, Ar-H), 6.85 (s, 0.48 H, OH-1-P), 6.55 (s, 0.33H, OH-1-a), 5.25 (d, 0.48 H, OH-3-P), 5.20 (d, 0.49 H, OH-2- (3), 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-ß), 4.20-4.05 (m+m, 0.53 H +0.42 H, H-6'a+ß), 3.85-3.73 (m, 0.44 H, H-5-a), 3.72-3.57 (m,

1.27 H, H-6"-a+ß and H-3-a), 3.45-3.20 (m, 7.8 H, H-3-P, H-4-a+p, H-5-ß and H-2-a) and 3.10-2.98 (m, 0.56 H, H-2-P).

Starting with 4,6-O-benzylidene-L-glucopyranose, the title compound is prepared as described for the analogous route leading to compound 1.

Description of the figures Figure 1: Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 4 or 20h with compound 1. Cells were treated in open plastic Petri dishes incubated in CO2-incubators at 37°C. The plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard errors are indicated by vertical barrs in all cases where they exceed the symbols. From the data cell survival is down to 20 % following 4h treatment with 1 mM of compound 1. For comparison, the same survival of 20 % is obtained with only 0.3mM of compound 1 when treatment time is increased to 20h. On a drug-dose basis the effect thus, is about 3 times stronger when treatment is extended to 20 as compared to 4h.

Figure 2: Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with compounds 2 or 3. The technique and statistics are as described in figure 1. The effect of these two compounds are similar. The effect is stronger than the effect of compound 1. A direct comparison between figures 1 and 2 shows that 10% survival following 20h treatment is obtained with 500 uM of compound 1 and with only 10 M of compounds 2 and 3. Thus, on a concentration basis the latter two compounds are 50 times more effective than the former one.

Figure 3: Cell survival as measured by colony-fonning ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with compound 3 and tucaresol. The technique and statistics are as described in figure 1. The data indicate that cell survival is about 30 % following treatment with 1000 iM tucaresol as compared with 7 tM of compound 3. Thus

compound 3 induces a stronger effect than tucaresol by a factor of about 140 on a drug dose basis.

Figure 4: Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, as measured by amount of incorporated [3H]-valine during a pulse period of 1 h starting either immediately following addition of test compound (in this case compound 1) or starting 3h later, with test compound present during the pulse. Cells were pre-labelled with ['4C]-valine for at least 4 days in order to have all cellular protein labelled to saturation. Incorporated amount of [3H] was related to incorporated amount of [14C] so that protein synthesis was calculated as per cent of total amount of protein. Rate of protein synthesis is given as per cent of that in an untreated control. The plotted values for protein synthesis represent mean values from 4 simultaneously and similarly treated wells. Standard errors are indicated by vertical barrs in all cases where they exceed the symbols. The data indicate that compound 1 induces a protein synthesis inhibition which increases linearly with increasing concentration of drug. The inhibition of protein synthesis appears to be similar whether the pulse period started immediately following initiation of drug treatment or 3h later.

Figure 5: Rate of protein synthesis as measured by amount of incorporated [3H]-valine during a pulse period of 1 h starting immediately following addition of test compounds (in this case compounds 2 and 3 together with similar data for 5-nitrofurfurylidene diacetate).

Cell treatment, calculation of protein synthesis and statistics were as described under the heading of figure 4. 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 differs with respect to scaling on the abscissa. The data indicate that both compounds 2 and 3 are efficient inhibitors of protein synthesis. Both compounds reduce protein synthesis to a low level of approximately 35% of the control level for a drug concentration as low as about 25 I1M. In the dose region from 0 to 25 AM the inhibition of protein synthesis increases linearly with drug concentration. A further increase in drug concentration above 25 pM does not induce any further increase in response. In comparison a concentration of 100 u. M of 5-nitrofurfurylidene diacetate reduces the rate of protein synthesis to about 50%. As estimated from the initial slopes of the curves, compounds 2 and 3 are about one order of magnitude more effective than 5-nitrofurfurylidene diacetate as a protein synthesis inhibitor in these cells. A comparison

between figures 4 and 5B indicate that 4 mM of compound 1 induces the same degree of inhibition of protein synthesis as does 25 M of compounds 2 and 3. Thus, the latter two compounds are 160 times more effective as the former one on a purely drug dose basis.

Figure 6: Rate of protein synthesis as measured by amount of incorporated [3H]-valine during a pulse period of lh starting at different times following addition of 25 u. M of test compounds (in this case compounds 2 and 3). Cell treatment, calculation of protein synthesis and statistics were as in figure 4. The control value of 100% is plotted at time 0, since this is the level of the rate of protein synthesis just before the drugs are added to the cells. The next point is plotted at time 1/2h, i. e. in the midst of the 0-lh pulse. After 4h treatment drugs were removed and fresh, drug-free medium was added to the cells.

During the first h following removal of drug the last pulse with [3H]-valine was given and comparison of the values representing the two last time points indicates the degree of reversibility of the protein synthesis inhibition of the compounds. While both compounds show increased inhibition of protein synthesis with time after start of treatment, both are also reversible to some extent. However, the increase in protein synthesis following removal of compounds is not as fast as is seen 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. Tumor volumes were calculated by the formula volume = (length x width2)/2 where tumor length and width were measured by calipers twice a week. At day 1 and each following day each animal was given one i. v.-injection per day, 7 days a week, either with isotonic saline without drugs (placebo) or with isotonic saline containing either 0.125 or 0.938 mg of compound 1 per ml saline. The body weight of each animal was approximately 25 g and the volume of each injection was 0.2 ml, giving a final 1 or 7.5 mg of compound 1 pr kg body weight each day. Each experimental point represent the mean values of tumor volumes from 6 animals, each tumour volume taken relative to the volume of the same tumour at day 1. Standard errors are given as vertical barrs, but represent, in this case, biological variation in tumor growth rate between individual animals in addition to uncertainty in measurement. Both groups given compound 1 shows a slightly reduced rate of tumor growth as compared with the placebo-treated animals. The effect is somewhat more pronounced with 1 than with 7.5 mg/kg drug, indicating that the anti-tumor

effect may be optimal at a certain dose region with reduced effect for lower as well as higher drug doses.

Biological experiments Example 1 Biological materials and methods used to demonstrate the effect.

Cell Culturing Techniques Human cells, NHIK 3025, originating form 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 cultivated in Eagel's Minimal Essential Medium (MEM) supplemented with 15% foetal calf serum (Gibco BRL Ltd). The cells are routinely grown as monolayers at 37°C in tissue culture flasks. In order to maintain cells in continuous exponential growth, the cells were trypsinised and recultured three times a week.

Cell Survival Cell survival was measured as the colony forming ability. Before seeding, the exponentially growing cells were trypsinised, suspended as single cells and seeded directly into 5 cm plastic dishes. The number of seeded cells was adjusted such that the number of surviving cells would be approximately 150 per dish. After about 2 h incubation at 37°C, the cells had attached to the bottom of the dishes. Drug treatment was then started by replacing the medium with medium having the desired drug concentration. Following drug treatment the cells were rinsed 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 CO2-incubator, the cells were fixed in ethanol and stained with methylene blue before the colonies were counted.

The survival measurements shown in figures 1 and 2 indicate that these drugs inactivate cells with increasing effect for increasing drug doses. The effect also increases with increasing treatment time (figure 1). Compounds 2 and 3 are, however, far more effective than compound 1, by a factor of 50 on a concentration basis (figure 2).

From figure 3 it is seen that compound 3 induces a stronger cell inactivating effect than tucaresol by about a factor of 140 on a drug dose basis.

Example 2 Protein Svnthesis The rate of protein synthesis was calculated as described previously (Ronning, O. W. et al., J. Cell Physiol., 107: 47-57,1981). Briefly, cellular protein was labelled to saturation during a minimum 4 day preincubation with ['4C] valine of constant specific radioactivity (0.5 Ci/mol). In order 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 ['4C] valine by intracellular valine and proteolytically generated valine will be negligible (Ronning, O. W., et al., Exp. Cell Res. 123: 63-72,1979). The rate of protein synthesis was calculated from the incorporation of [3H] valine related to the total ['4C] radioactivity in protein at the beginning of the respective measurement periods and expressed as percentage per hour (Ronning, O. W. et al., J. Cell Physiol., 107: 47-57,1981).

Compound 1 induces an inhibition of protein synthesis which increases linearly with increasing dose up to 4 mM, the highest dose tested (figure 4). Whether pulsing with [3H]-valin was done the first or the third hour after start of treatment the effect was the same. Thus, compound 1 induces an inhibition of protein synthesis which is constant as long as the compound is present in the cell culture.

Compounds 2 and 3 induces protein synthesis inhibition at extremely low concentrations. A significant effect is seen at concentrations as low as on the order of

10 ßM (figure 5B) and full effect, with a low level of protein synthesis of 35% of that in the control, is found at concentrations above 25 tM (figure 5A). Figure 5A also shows that, at low drug doses as estimated from the initial slopes of the curves, compounds 2 and 3 are about one order of magnitude more effective than 5-nitrofurfurylidene diacetate as a protein synthesis inhibitor in NHIK 3025 cells.

From figure 6 both of the compounds 2 and 3 are seen to induce increasing protein synthesis inhibition with increasing treatment time. Both drugs are seen to be reversible to some extent since the rate of protein synthesis increases immediately after removal of drugs. However, protein synthesis is not completely restored during the first hour after treatment, the increase is only up to a level of 50% of that of control cells. It is possible that a fraction of the cells are irreveribly inactivated with respect to protein synthesis, but without beeing lysed during the first 3 hours of treatment. These cells would then contribute to the amount of ["C], but not to that of ['H] in the final sample, and thereby mask the possible reversibility of inhibition of protein synthesis of cells surviving the 3h treatment.

Example 3 Experiments on human xenografts in nude mice Drugs were tested in the treatment of three human cancer xenografts implanted into female, athymic 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 cultivated shortly in vitro before being implanted into nude mice. The tumour lines were passaged as s. c. implants in nude mice. Mice which should be used in experiments were 8-9 weeks of age at the time of tumour implantation. Small tumour pieces were implanted s. c. on the left flank of the animals. Animals with growing tumours (tumour volumes 25-110 mm3) were randomly assigned to drug-treated or control groups, with the average tumour size among the groups being approximately equal. The antitumour activity was measured by tumour volume growth curves and histological evaluation of some

tumours. During treatment tumours were measured 2 times a week by measuring two perpendicular diameters using calipers. Tumour volume was estimated by the formula: volume = (length x width2)/2. The tumour volume growth curves were generated by standardising the tumour sizes in the different groups by obtaining relative tumour volume (RV) calculated by the formula RV = Vx/Vl, where Vx is the tumour volume at day x and VI is the initial volume at the start of the treatment (day 1), and plotting the mean volume with standard errors for each treatment group as a function of time. An exponential curve was fitted to the relative tumour volume growth data and the interval during which the tumour volume in each group increased to twice its volume, tumour volume doubling time (TD), was determined from the fitted curve (loge2/k, where k is the estimated rate constant for the process.) Histological evaluation was based on macroscopic examination of the tumour and light microscopic examination of small tumour sections (6-8 mm thick) embedded in paraffin and stained 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 inhibition of tumour volume growth is stronger with 1 mg/kg than with 7.5 mg/kg daily dose in these animals.

Conclusions: The products of this invention react with active groups on the cell surface, e. g. amino-, hydroxy-or sulfhydryl groups in a substantial more efficient way than products previously known, to form carbonyl condensation products like Schiffs bases, acetals, mercaptals, thiazolidenes, aminals etc. on the cell surface. This can influence on the signal machinery of cells and can be used in the treatment of diseases like cancer, immunological disorders, microbial infections etc.

The aldehyde derivatives of this invention react with certain groups on the cell surface, e. g. with free amino groups to form Schiffs bases. As many cell processes, like protein synthesis, cell-cycle progression, immune response etc. are controlled by signals from the cell-surface, these bindings will alter the behaviour of the cell. We have also shown that the aldehyde complex of the cell surface change the adhesion characteristics of the cell. We have shown

that the compounds of this invention can be useful in new therapies to combat cancer, auto immune diseases, viral infections and also infections of other microorganisms.

Administration The pharmaceutical compositions according to the present invention may be administered in anti-cancer treatment, anti-viral treatment or in treatment of diseases which arise due to abnormally elevated cell proliferation and/or for combating auto immune diseases. These pharmaceutical compositions may also be administered as immunopotentiators.

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

It is especially preferred to prepare the formulations for systemic therapy either as oral preparations or parenteral formulations.

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

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

When administered topically the compounds of formula (I) may be formulated as a lotion, salve, cream, gel, tincture, spray or the like containing the compounds of formula (I) in admixture with non-toxic, inert, solid or liquid carriers which are usual in topical preparations. It is especially suitable to use a formulation which protects the active ingredient against air, water and the like.

The preparations can contain inert or pharmacodynamically active additives. Tablets or granulates e. g. can contain a series of binding agents, filler materials, carrier substances and/or diluents. Liquid preparations may be present, for example, in the form of a sterile solution. Capsules can contain a filler material or thickening agent in addition to the active ingredient. Furthermore, flavour-improving additives as well as the substances usually used as preserving, stabilising, moisture-retaining and emulsifying agents, salts for varying the osmotic pressure, buffers and other additives may also be present.

The dosages in which the preparations are administered can vary according to the indication, the mode of use and the route of administration, as well as to the requirements of the patient. In general a daily dosage for a systemic therapy for an adult average patient will be about 0.01-500mg/kg body weight once or twice a day, preferably 0.1-100 mg/kg body weight once or twice a day, and most preferred 1-20 mg/kg weight once or twice a day.

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