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  • C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms
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  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/18—Acyclic radicals, substituted by carbocyclic rings

Definitions

• the present invention relates to benzaldehyde derivatives 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.
• Some of the compounds of this invention are novel per se.
• 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.
• 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.
• the reaction normally take the form of Schiff s base (imine) adduct formation.
• Schiff s base in vivo Schiff s 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.
• carbonyl condensation reactions are involved in transmembrane signalling events, for example in generating an immune response.
• R— N— R' + H 2 0 aldehyde amine carbinolamine imine
• the Schiff s 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.
• the initially formed Schiff s 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).
• aldehydes bind to amines and other nucleofilic entities on the cell membrane to form Schiff s 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.
• 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 and non- toxic way of action observed with the benzaldehyde derivatives.
• normal cells 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).
• the protein synthesis inhibition may be enough to 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.
• Compound 1 4,6-O-Benzylidene-D-glucopyranose. These two compounds are known to possess a general anti-cancer activity and have been tested in clinical trials against a number of cancer diseases. However, no particular cancer afflicted organs or tissues projected as more suitable for treatment with these compounds, and commercial development was not justified.
• benzaldehyde derivatives of sugars of the hexose type (including 4,6-O-(Benzylidene-d ⁇ )-D-glucopyranose, Compound 2) give an unexpected strong effect on cancer in certain organs or tissues. We cannot yet explain the mechanism for this selectivity, but we believe that this is connected to the affinity of the sugar moiety of the derivatives to certain cells or tissues.
• the chemical induced carcinogenesis has a similar mechanism as the cancerogenesis induced by certain virus types like hepatitis B and C, certain papilloma virus, certain herpes virus etc. Especially this will be the case in the development of liver cancer in hepatitis B and C infected patients. It is therefore presumable that a prophylactic treatment of these patients with products of this invention could prevent or delay the development of liver cancer. Also the fact that these products show a low toxic profile would make them suitable for such a treatment.
• benzaldehyde compounds previously known as anti cancer agents may be used for combating diseases resulting from an abnormally elevated cell proliferation. Such compounds also exert an effect on cells having an abnormally elevated cellular proliferation rate, and accordingly the compounds may be used for treatment of diseases such as psoriasis, inflammatory diseases, rheumatic diseases and other auto immune disorders like Ulcerous colitt and Morbus Crohn, and allergic dermatologic reactions.
• Dermatologic abnormalities such as psoriasis are often characterised by rapid turnover of epidermis. While normal skin produces about 1250 new cells/day/cm 2 of skin consisting of about 27,000 cells, psoriatic skin produces 35,000 new cells/day/cm 2 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.
• aromatic 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. The synthesis of cellular protein is, for instance, rapidly restored to its normal level following removal of the agent from the cells (i.e. within 1 h in most cases).
• 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.
• the immune system 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.
• 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.
• 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.
• Tucaresol 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.
• the potential of Tucaresol in curing a number of diseases including bacteria-, virus- and protozoal infections, auto-immune related illness and cancer is presently being investigated (H.Chen and J. Rhodes, J.Mol.Med (1996) 74:497-504) and combinational strategies where Tucaresol is administered together with a vaccine to cure chronic hepatitis B, HIV and malignant melanoma are currently under development.
• aldehydes are intrinsically unstable due to oxidation.
• 4-(2-Formyl-3-hydroxyphenoxymethyl) benzoic acid (Tucaresol) which is disclosed in EP-0609606, is considerably more potent in vivo than in vitro. This may be because of the drug's susceptibility to oxidation in aqueous solutions in vitro (H.Chen and J. Rhodes, J.Mol.Med (1996) 74:497-504).
• Many aldehydes are too reactive to be administered as such, and benzaldehyde, even proven to be an active anti cancer drug in vitro, is highly irritating and unsuitable for direct in vivo application.
• the aldehyde carbonyl group will react rapidly with nucleofilic entities predominantly present in all body fluids. These unwanted by-reactions could lead to fast drug metabolisation and difficulty in controlling serum level of the active drug. Controlling the drug at a cellular level within a narrow concentration window is crucial for achieving an effective immune potentiation.
• Tucaresol is orally administered as an unprotected aldehyde, and one might suspect drug deterioration and difficulties in controlling pharmacokinetics.
• the benzaldehyde derivatives 4,6-benzylidene-D-glucose and the deuterated analogue have proven to possess high bioavailability either administered i.v. or per oz. Bioavailability measured as serum level after oral administration of Compound 2 to BALB mice was 93-99% (CB. Dunsaed, J.M. Dornish and E.O. Pettersen, Cancer Chemother. Pharmacol. (1995) 35: 464-470). Moreover, the glucose moiety can possess affinity to receptors present at the cell surface, thereby improving drug availability at a cellular level.
• the free aldehyde can easily be released by hydrolysis of the acetal, making the carbonyl group available for Schiff s base formation at the target ligands.
• the aldehydes are derivatised with biologically acceptable carbohydrates like glucose, galactose and others to form acetals.
• the sugar moiety will thus contribute by improving stability and enhancing bioavailability of the aldehyde function to the target cells. This surprisingly leads to more effective carbonyl condensation reactions and easier controllable pharmacokinetics by using our compounds as compared with previously known compounds.
• 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 benzaldehyde derivatives to trigger the immune response may be used in combination with antivirals or vaccines to develop a treatment for these diseases.
• Another 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 prophylactic treatment to prevent the development of liver cancer in persons with Hepatitis B or C infection.
• a fifth 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 sixth object of the invention is to provide compounds for treatment of diseases related to the immune system like psoriasis, bowel inflammations, arthritis, SLE, PSS etc.
• the compounds of the present invention have the general formula (I):
• L is H or D
• Ar is phenyl or substituted phenyl with 1-3 substituents, the substituents which are the same or different, are selected from the group comprising 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, phenyl, halogen, nitro, cyano, NH 2 , NHR 1 , N(R') 2 , NHC(O)R' or N[C(O)R'] 2 wherein R 1 which is the same or different, is alkyl with 1-20 carbon atoms, or fluoroalkyl with 1-6 carbon atoms, OR 2 or OC(O)R 2 wherein R 2 is H, D, alkyl with 1-20 carbon atoms, or fluoroalkyl with 1-6 carbon atoms, SR 2 , CA(OR 1 ) 2 or CA[OC(
• Y is selected from the atoms or groups comprising 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, fluoro, chloro, nitro, OR 2 , OC(O)R 2 , SR 2 , NH 2 , NHR 1 , N(R ] ) 2 wherein R 1 is the same or different, NHC(O)R' or N[C(O)R'] 2 wherein R 1 is the same or different;
• R 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.
• Fig. 1 The data represent an experiment where NHIK 3025-cells were treated with Compound 8 (O) or Compound 9 (•) for 20 hours at 37°C while attached to plastic Petri dishes.
• Surviving fraction means fraction of cells able to form a macroscopic colony following treatment. Each point represents the mean value of colony counts from 5 parallel dishes. The standard errors are smaller than the size of the symbols.
• Fig. 2 The data represent an experiment where NHIK 3025-cells were treated with Compound 5 (O) or Compound 7 (*) for 20 hours at 37 °C while attached to plastic Petri dishes.
• Surviving fraction means fraction of cells able to form a macroscopic colony following treatment. Each point represents the mean value of colony counts from 5 parallel dishes. Vertical bars indicate standard errors and are shown when exceeding the symbols.
• Fig. 3 The data represent an experiment where NHIK 3025-cells were treated with Compound 12 ( ⁇ ) for 20 hours at 37°C while attached to plastic Petri dishes.
• Surviving fraction means fraction of cells able to form a macroscopic colony following treatment. Each point represents the mean value of colony counts from 5 parallel dishes. Vertical bars indicate standard errors and are shown when exceeding the symbols.
• Fig. 4 The data represents an experiment where NHIK 3025-cells were treated with Compound 2 ( ⁇ ) or Tucaresol (•) for 20 hours at 37°C while attached to plastic Petri dishes.
• Surviving fraction means fraction of cells able to form a macroscopic colony following treatment. Each point represents the mean value of colony counts from 5 parallell dishes. The standard errors are shown when exceeding the size of the symbols.
• Fig. 5 The rate of protein synthesis relative to untreated control of NHIK 3025-cells treated with Compound 2 ( ⁇ ) or Tucaresol (A) for 1 hour at 37°C. The rate of protein synthesis was measuered by amount of [ 3 H]-valine incorporated during the first hour after start of drug treatment. Protein synthesis rate was measured relative to the total amount of protein in the cells. Data are representative for one experiment performed in quadruplicate. Standard errors are indicated when exceeding the size of the symbols.
• Fig. 6 Mean tumour growth curves of tumour line SK-OV-3 ovarian carcinoma xenograft implanted in nude mice are shown. Mice were treated daily i.v. with 1 mg/kg Compound 8 (T) and 7.5 mg/kg Compound 8 (A). ⁇ , Control group received 0.9% NaCl. Each data point represents the mean tumour volume of 4 to 5 mice related to the tumour volume at day 1. Vertical bars represent standard error.
• Fig. 7-12 show morphologic appearance of SK-OV-3 tomours from each of the following 3 groups: The placebo-treated group of animals (figures 7 and 8), the group treated with 1 mg/kg/day of Compound 8 (figures 9 and 10) and the group treated with 7.5 mg/kg/day (figures 11 and 12).
• Tumours were fixed in formalin, embedded in paraffin, sliced in 6 mm slices and stained with haematoxylin and eosin. Magnification is 40 times.
• Fig. 13 Mean spheroide volume growth curves of cell line T-47D breast carcinoma are shown. The spheroids were treated with 0.1 mM Compound 8 (A) and 1.0 mM Compound 8 (T) dissolved in medium. ⁇ ,Control. Each data point represents the mean spheroid volume of 6 to 11 spheroids. Vertical bars represent standard error.
• Fig. 14 shows microscopic photographs of sections of 3 differently treated NHIK 3025 cell spheroids, one untreated control (A), one treated with 0.1 mM Compound 8 for 4 days (B) and one treated with 1.0 mM Compound 8 for 4 days (C).
• Fig. 15-18 The data show the fraction of nuclei within each of the interphase stages, Gl, S and G2, having the RB-protein bound in the nucleus following treatment with Compound 8.
• Fig. 19-20 Rate of protein synthesis relative to that of control cells for NHIK 3025 cells (figure 19) and T-47D-cells (figure 20). Each point represent the mean of measurements from 4 parallell samples. Standard errors are indicated by vertical bars when exceeding the symbols.
• Fig. 21 Median adhesion forces for cells exposed to different benzaldehyde derivatives. The cells were exposed to a 1 mM concentration of Compounds 1 and 2.
• Fig. 22 Peripheral blood mononuclear cells and Superantigen in Ex Vivo 10 medium were exposed to either benzaldehyde, deuterated benzaldehyde, Compound 2 or zilascorb( 2 H). The proliferation of peripheral blood mononuclear cells was measured as incorporation of tritiated thymidine at different drug concentrations.
• Fig. 23 NMRI mice were infected i.p. with spleen invading Friend erythroleukemia virus. Infected- and uninfected mice were treated i.p. daily with 5 mg/kg of either Compound 2 or Compound 5. After treatment for 19 days, spleens were dissected out and weighted.
• Fig. 24 The effect of Compound 1 , 2 and 5 on the liver invasion of human colorectal tumour, C170HM2 is shown.
• Fig. 25 Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or Compound 13 (•) is shown.
• Fig. 26 Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or Compound 14 (•) is shown.
• Fig. 27 Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, treated with Compound 1 or Compound 21 as measured by amount of incorporated [ 3 H]-valine during a pulse period of lh starting either immediately following addition of test compound (closed symbols) or starting 2h later (open symbols).
• Fig. 28 Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, treated with Compound 2 or Compound 22 as measured by amount of inco ⁇ orated [ 3 H]-valine during a pulse period of lh starting either immediately following addition of test compound (closed symbols) or starting 2h later (open symbols).
• Fig. 29 Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (•) or Compound 21 (O) is shown.
• Fig. 30 Cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 2 (O) or Compound 22 (A) is shown.
• Fig. 31 Cell survival as measured by colony-forming ability for human breast carcinoma cells, T47-D, after treatment for 20h with either L-glucose (•) or Compound 21 (O) is shown.
• aldehydes undergo acid facilitated condensation reactions with alcohols to generate acetals. Water is concomitantly formed as a co-product.
• the reaction is reversible, and in solution, an equilibrium mixture of aldehyde/alcohol and acetal/water is formed. The position of the equilibrium will mainly be determined by the reactivity and concentration of each species.
• one of the products acetal or water, is normally removed from the reaction mixture.
• various sugars, deoxysugars and aminosugars are condensed with aldehydes or aldehyde equivalents to form neutral-acetal derivatives.
• Particularly preferred is a re-acetalisation strategy, where the aldehyde protected as its dimethyl acetal is used instead of the aldehyde itself.
• Methanol is then formed as co-product.
• the reaction mixture is moderately heated at reduced pressure to remove the methanol once it is formed. In most cases, these reaction conditions will drive the equilibrium smoothly in favour of the acetal.
• Acetalisation of sugars will normally lead to mixtures of regio- and stereo isomers. Ring contraction transformations may also occur, leading to mixtures of pyranoses and furanoses, and, in some cases, di-acetalisation adducts are formed. As a consequence, unless protection strategies are applied, very complex reaction mixtures are often encountered. However, surprisingly pure product fractions were prepared following appropriate work-up, especially by using liquid chromatography. Identification of the products were achieved by using GC-MS-spectroscopy and various NMR techniques.
• the specific reaction conditions, solvent and catalyst used will in each individual case depend on the solubility and reactivity of the reactants and of the properties of the product.
• the catalyst may be a mineral acid, e.g. sulphuric acid, an organic acid, e.g. p ⁇ r ⁇ -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 may conveniently be carried out in a dipolar, aprotic solvent such as dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethoxyetane or the like.
• a dipolar, aprotic solvent such as dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethoxyetane or the like.
• R ⁇ r ⁇ -toluene sulfonic acid in dimethyl formamide constituted the preferred and most applied reaction condition.
• the compounds of formula (I) wherein L is deuterium may be prepared as described above, but starting with the dimethyl acetal of an aldehyde which is deuterated in the formyl position.
• the preparation of deutero-benzaldehyde may be performed by a modified Rosenmund reduction using D 2 gas in a deuterated solved, as described in EP 0 283 139 Bl.
• Deuterated benzaldehyde derivatives with substituents in the phenyl ring may be prepared according to examples given in EP 0 493 883 Al and EP 0 552 880 Al.
• Benzaldehyde-di was prepared and converted to benzaldehyde dimethylacetal-di as described in EP 0 283 139 Bl.
• the residue was cooled to approx. 40°C and ice/water (2.9 L) added within 5 min. The temperature dropped below 0°C and a precipitate was formed, partly as big lumps.
• the mixture was transferred to a beaker and additional 8-9 L ice/water added in order to make the lumps fell apart and form a suspension.
• the suspension was filtered on two notches and the two filter cakes left over night on the filters with water jet vacuum connected, each filter cake being flushed with N 2 via an inverted funnel.
• the filter cakes were spread on two boards and dried at 32°C for 20 hours in a vacuum oven. The vacuum was first set at 13 mbar, then regulated down to 1 mbar.
• the crude product was recrystallised (in order to remove di-benzylidene acetals) and water-washed (to remove DMF and glucose) until these contaminants were eliminated. Accordingly, the crude product (500 g) was dissolved in hot dioxane (800 ml) and the solution added via a folded filter to boiling chloroform (9 L). The solution was allowed to cool, first to ambient temperature, then in an ice bath overnight. The precipitate was filtered off, dried for 2 hours on the filter (flushing with N 2 as described previously) and dried further overnight at 31°C in vacuo on a rotavapor.
• the product (142 g) was suspended in ice/water (1 L), filtered on a nutch (washing with 200 ml ice/water) and dried on the filter overnight as described previously. It was then grounded, sieved (0.5 mm grid size) and dried in vacuo for 5 hours at 31 °C on a rotavapor.
• the product (96 g) once again was suspended in ice/water (500 ml), filtered (washing with 150 ml ice/water) and dried (7 hours under N 2 flush). It was finally grounded on a mortar, sieved (0.5 mm) and dried in a vacuum oven.
• Methyl- ⁇ -D-mannopyranoside (18.1 g, 0.093 mol), benzaldehyde dimethylacetal (21.0 g, 0.138 mol) and dry DMF (90 ml) were mixed with stirring at 50-55°C in a distillation apparatus.
• R ⁇ r ⁇ -toluene sulfonic acid (ca. 0.1 g) was added and 10 min. thereafter a water jet was connected to distil off methanol. After 4 hours, the reaction mixture was evaporated to form a white solid. The residue was washed with dibutyl ether, filtered and the filter cake dissolved in acetonitrile. A precipitation started and the mixture left in a refrigerator for 5 days. Thereafter, the precipitation was filtered off and the filtrate evaporated. The residue was purified on a Lobar C RP-8 column, eluting with 30 % acetonitrile in water. Product fractions from 4 separate runs were freeze dried and combined.
• Benzaldehyde-di was prepared and converted to benzaldehyde dimethylacetal-di as described in EP 0 283 139 B 1.
• Methyl 4-formylbenzoate 100 g, 0.609 mol
• methanol 91.5 g, 2.86 mol
• trimethyl ortoformate 71 g, 0.67 mol
• cone, hydrochloric acid 165 ⁇ l
• the slurry transformed into a slightly yellow solution within few minutes and the temp, spontaneously increased from 15°C to 30°C.
• the reaction mixture was refluxed at 58°C for another 25 min. and then cooled to 10°C (ice/water).
• the syrup was dissolved in a warm solution of 100 mg NaHCO 3 in 20 ml methanol and 8 ml water and precipitated by adding 100 ml ethylacetate.
• the precipitate was isolated from the mother liquor by filtration, washed with cold water (4 x 15-20 ml) and transferred to a rotavapor flask. Humidity was removed by adding ethylacetate and evaporating twice. The product was finally dried under high vacuum. More precipitate was filtered off from the mother liquor, washed and dried to give a second crop. The two crops were combined to give 1.94 g pure product, 13 % of the theoretical yield.
• the syrup was dissolved in slightly alkaline (NaHCO 3 ) methanol/water 60/40 and purified on a Merck LiChroprep RP-8 reversed phase column, eluting with methanol/water 60/40. Product fractions were concentrated to remove methanol and freeze dried to give a white, fluffy solid. Products from four separate runs were combined to give 3.18 g, 21 % of the theoretical yield.
• Benzaldehyde-di was prepared and converted to benzaldehyde dimethylacetal-di as described in EP 0 283 139 Bl.
• Benzaldehyde dimethylacetal-d i (8.7 g, 56.8 mmol), N-acetyl-D-glucosamine (10.0 g, 45.2 mmol), dry DMF (30 ml) and p ⁇ r ⁇ -toluene sulfonic acid (88 mg, 0.46 mmol) were mixed under N 2 to give a white suspension.
• the reaction mixture was stirred at 50°C for 45 min, then a vacuum pump was connected through a vertical condenser and the reaction continued for 2 hours at 55°C/60-70 mbar.
• the apparatus was rebuilt to remove DMF through a short path and the distillation continued at 55-60°C, max. vacuum for 1 more hour. The residue was a yellow-white, soft solid.
• Volatile material (CH 3 OH + HCOOCH 3 ) was distilled off in a water jet, the distillation interrupted and a vacuum pump connected. The distillation was then continued and a yellow oil distilled off at 93 - 97.5 °C/20 mbar.
• the oil was identified by NMR to be 3-nitrobenzaldehyde dimethylacetal of high purity. The yield was 127 g, 97.6 % of the theoretical.
• the water phase (forming a 5 cheese-like suspension) was re-extracted twice with chloroform and filtered, washing several times with water and ether.
• the product was dried in vacuo to form a slightly brownish powder.
• the yield was 570 mg, 7 % of the theoretical.
• Benzaldehyde-di was prepared and converted to benzaldehyde dimethylacetal-di as described in EP 0 283 139 Bl.
• the syrup was dissolved in slightly alkaline (NaHCO 3 ) methanol/water 60:40 and purified on a Lobar C RP-8 column, eluting with methanol/water 60:40.
• Product fractions were evaporated (to remove methanol), freeze-dried and combined to give 5 g of a white, fluffy solid.
• the product was re-purified eluting with methanol/water 40:60 to give the title compound of sufficient purity.
• the yield was 3.3 g, 12 % of the theoretical.
• H-6'- ⁇ + ⁇ 3.36-3.16 (m, 1.39H, H-4- ⁇ + ⁇ , H-5- ⁇ + H 2 O), 2.05-1.86 (m, 0.93H, H-2- ⁇ + ⁇ + EtOAc) 1,63-1.47 (m, 0.51H, H-2'- ⁇ ) and 1.47-1.32 (m, 0.48H, H-2'- ⁇ ); 154.71 and 154.66 (Ar-C-OH), 130.09, 127.88, 127.78, 124.54, 124.48, 118.96, 118.93, 115.67 (Ar-C , 97.06 and 97.00 (acetal-C), 94.36 (C-l- ⁇ ), 91.71 (C-l- ⁇ ), 84.52 and 83.70 (C-4),
• the crude product was dissolved in methanol (10 ml) and purified on a reversed phase RP-8 column, eluting with methanol/water 1:1. Product fractions were combined and evaporated to remove methanol. The residual solution was further diluted with water and freeze dried. White, fluffy solid from three separate runs were collected to yield a total of 2.42 g, 32.5 % of the theoretical.
• the DMF was then evaporated under vacuum (2 mbar) at 65 °C to give a very pale yellow oil to which was added NaHCO 3 (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.
• 2-Acetoxybenzaldehyde is prepared, either by acetylation of 2-hydroxybenzaldehyde or by reduction of 2-acetoxybenzoyl chloride.
• a catalytic amount of p r ⁇ -toluene sulfonic acid is added to an equmolar mixture of 2-acetoxybenzaldehyde and trimethyl ortoformate. After stining for 1 h., an equal molar amount of D-glucose and DMF are added and the mixture heated to about 60 °C. The conversion is followed by TLC chromatography and when an equilibrium is reached, the reaction is quenched by the addition of a small amount of pyridine. Most of the DMF is evaporated at reduced pressure and the residue applied on silica as described previously. This is purified by chromatography, the product fractions isolated and evaporated and the compound analysed by NMR spectroscopy.
• EAM Eagel's Minimal Essential Medium
• foetal calf serum Gibco BRL Ltd
• Human breast carcinoma cells, T-47D, (Keydar, I. et al., Eur. J. Cancer, vol 15, pp. 659-670, 1979) were cultivated in medium RPMI-1640 supplemented with 10% foetal calf serum, 0.2 u/ml insulin, 292 mg/ml L-glutamine, 50 u/ml penicillin, 50 mg/ml streptomycin.
• the cells are routinely
• 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.
• Fig. 1-3 show cell surviving fraction for NHIK 3025-cells treated for 20 hours with either Compound 8 and 9 (Fig. 1), Compound 5 and 7 (Fig. 2) or Compound 12 (Fig. 3).
• the data indicate that all compounds induce cell inactivation in a drug dose range similar or better to that of zilascorb( 2 H) (Pettersen et al., Anticancer Res. vol. 11, (1991), pp. 1077-1082).
• the rate of protein synthesis was calculated from the incorporation of [ 3 H] valine related to the total [ 14 C]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).
• 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 mm 3 ) were randomly assigned to drug-treated or control groups, with the average tumour size among the groups being approximately equal.
• RV relative tumour volume
• tumour volume doubling time was determined from the fitted curve (loge2/&, 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.
• tumour volume doubling time (TD) in human tumour xenografts grown in nude mice, treated daily i.v. with drugs and doses as indicated, are shown.
• Fig. 6 mean tumour growth curves of the tumour line SK-OV-3 ovarian carcinoma xenograft implanted in nude mice, where the mice were treated daily with 1 mg/kg and 7.5 mg/kg of Compound 8 are shown. The curves show a significant growth inhibitory effect for both doses.
• Fig. 7-12 show microscopic photographs of tumours from each group. These photographs indicate a general finding of this compound, namely that there is a difference with respect to tumour cell necrosis between control tumours and those treated with Compound 8. Since the treated tumours are necrotized by the treatment the drug effects are in reality even stronger than that shown by the growth curves.
• Spheroids were initiated by transferring suspended single cells to a 25 cm 2 tissue culture flask containing 12 ml of medium.
• the flask was then placed on a tilting board (MIXER 440, Swelab Instrument) inside a walk-in incubator room at 37 °C. Tilting rate was adjusted to 10 tilts per 18 s.
• Tilting rate was adjusted to 10 tilts per 18 s.
• the suspended cells were prevented from attaching to the bottom of the flask. Instead many cells were able to attach to each other forming small aggregates of cells containing typically 50-100 cells each after about 24 h tilting.
• the small aggregates were then transferred to another 25 cm 2 tissue culture flask. In this case the bottom of the flask was on beforehand covered (i.e.
• spheroids When spheroids had reached a size of about 400 ⁇ m in diameter (after 2 to 3 weeks cultivation) they were transferred to small micro wells, with 1 spheroid per well together with 1 ml medium. It was made sure that all selected spheroids were of about the same size.
• the wells were also coated with agar in order to avoid attachment of spheroids to the bottom of the wells.
• the various wells were supplemented with new medium containing the test substance in the chosen concentration and thereafter the diameter of each individual spheroid was measured once each day. This was done by microscopy, using phase contrast optics and a grating of known line separation in one of the oculars (distance between two neighbouring lines.
• Relative spheroid volume (volume at day n divided by volume at day 1) was calculated for each individual spheroid each day and growth cures were plotted with mean relative volume for all spheroids in a group as a function of time after start of treatment.
• T-47D spheroid volume doubling time (TD) treated for 259 hours with Compound 8 at doses indicated are shown.
• Compound 8 is shown to increase the spheroid doubling times (i.e. inhibiting spheroid growth) in a dose-dependent manner, since the effect is clearly stronger with 1.0 mM than with 0.1 mM of the drug.
• Fig. 13 mean spheroid volume growth curves of cell line T-47D breast carcinoma where the spheroids were treated with 0.1 mM and 1.0 mM Compound 8 dissolved in medium, are shown.
• Fig. 14 shows microscopic photographs of sections of 3 differently treated NHIK 3025 cell spheroids, one untreated control (A), one treated with 0.1 mM Compound 8 for 4 days (B) and one treated with 1.0 mM Compound 8 for 4 days (C). Spheroids were fixed in 4% formaldehyde and embedded in paraffin before 6mm thick sections were made and stained with haematoxylin and eosin.
• T-47D cells express functional pRB, the retinoblastoma protein, which is a normal tumour suppressor gene that is important in regulating cell-cycle progression in normal cells. This gene is often defect in cancer cells, and NHIK 3025 cells are among those with a defect pRB-function.
• pRB may be activated to arrest cells under conditions of stress even when cells have entered the S-phase of the cell cycle, indicating that this gene may protect cells against the inactivating effects of a stress in combination with DNA-synthesis (see Amellem, Sandvik, Stokke and Pettersen, British Journal of Cancer 77 (1998) 862-872).
• the benzaldehyde derivative acts as a growth-inhibitory stress influence.
• the extracted nucleii were fixed in 4% paraformaldehyde for lh before pRB was bound with the PMG3-245 monoclonal antibody (Pharmingen) which recognise both the under- and hyperphosphorylated forms of the protein.
• the pRB antibody was streptavidin-FITC-stained and DNA was stained with Hoechst 33258.
• the nucleii were measured in a FACStart plus flow cytometer (Becton-Dickinson) equipped with two argon lasers (Spectra Physics) tuned to 488 nm and UV respectively.
• Fig. 15-18 show the fraction of nuclei within each of the interphase stages, Gl, S and G2, having the RB -protein bound in the nucleus following treatment with Compound 8.
• pRB is considered to regulate cells out of the cell cycle, i.e. to take over cell-cycle control, (see Amellem, 0., Stokke, T., Sandvik, J.A. & Pettersen, E.O.: The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phase during hypoxic stress.
• Exp. Cell Res. 227 (1996) 106-115.)
• Data are shown for two types of human breast cancer cells, MCF-7 (Fig.
• Fig. 20 it is shown that protein synthesis following 24h treatment with either 1.5 or 2.5 mM Compound 8 is 75 or 50% respectively, but increases back to normal in about 6h after removal of the drug. Possibly cell-cycle inhibition that inevitably follow as a result of the reduced protein synthesis inhibition (see R ⁇ nning, 0.W., Lindmo, T., Pettersen, E.O. & Seglen, P.O.: Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells. J. Cell Physiol. 107 (1981) 47-57.) is in itself over-ruling the regulatory effects of pRB at concentrations in the range 1.5 to 2.5 mM.
• NHIK 3025 carcinoma cells were cultured in CO 2 -independent medium containing 15% fetal calf serum. The cells were exposed to a 1 mM concentration of Compound 1 or Compound 2 for 20 hours before they were released from the cell culture flasks using trypsin.
• the cells were kept in suspension, and seeded in medium with Compound 1 or Compound 2 on polystyrene tissue culture substrates 90 minutes after the trypsin reaction had been stopped.
• the cell-substrate adhesion forces were measured by displacing cells using an inclined atomic force microscope cantilever acing as a force transducer. One cell was displaced at a time and each cell was displaced only once. The maximal force exerted on each cell was recorded as a function of the time since the cells were seeded on the substrate.
• the median force of a group of 19 measurements is shown as a function of the mean time for cells exposed to Compound 1 or Compound 2 in Fig. 21, together with the adhesion forces of cells not exposed to these compounds.
• Compound 2 shows a large effect in reducing the adhesion force at this concentration, while Compound 1 shows no significant response.
• the effect of the compound is mainly to reduce the adhesion force of the cells, but not the time course of adhesion.
• the reduced ability to attach to the substrate may be related to the blocking of integrin-mediated anchorage of the cells. It has been shown that such blocking may induce programmed cell death in both hepatoma and melanoma cancers.
• Paulsen JE, Hall KS, Rugstad HE, Reichelt KL and Elgjo K The synthetic hepatic peptides pyroglutamylglutamylglycylserylasparagine and pyroglytamylglutamylglycylserylaspartic acid inhibit growth of MH1C1 rat hepatoma cells transplanted into buffalo rats and athymic mice. Cancer Res. 52 (1992) 1218-1221. and Mason MD, Allman R, and Quibell M, "Adhesion molecules in melanoma - more than just superglue?” J. Royal Soc. Med. 89 (1992) 393-395.)
• Virus Eveline cells were supplied by prof. Gerhard Hunsman, Kunststoff. We have shown that this virus, which originally was used as a source of Friend helper virus, contain a defect virus of the same size as Spleen Focus Forming Virus (SFFV) which induces erythroleukaemia in NMRI mice after a delay of 4-8 weeks.
• SFFV Spleen Focus Forming Virus
• mice came from old Bomholt Farm, Denmark, and were purchased via SIFF. The mice were received on May 6th and entered into the experiment on May 11th. They were then infected with 50 microlitres supernatant from Eveline culture, intraperi tonally. After 24 hours the treatment was started. Compound 2 and Compound 5 were dissolved in 15 sterile isotonic glycerol solution in a concentration conesponding to 5 mg per kg when giving 50 microlitres intraperi tonally.
• mice uninfected, treated with Compound 5 25 10 mice infected, treated with Compound 5
• mice were given injections intraperitonally once daily for 19 days. From June 1st till June 16th, when they were sacrificed, no treatment was given. On June 16th, the mice were sacrificed. Blood was withdrawn (for future analysis). The spleen was removed and 30 weighed (see table 3 below). One bit of the spleen was frozen in nitrogen for the purpose of cutting thin slices and one bit was formaline fixated. Table 3:
• the inventors performed an experiment where peripheral blood mononuclear cells were exposed to Superantigen together with benzaldehyde, deuterated benzaldehyde, Compound 2 or zilascorb( 2 H).
• Superantigen is used as a very active standard for proliferation of T-cells and is presented via antigen presenting cells to T-cells.
• C170HM2 The cell line evaluated, is an established human colorectal cell line (S.A.Watson et al., Eur.J.Cancer 29A (1993), 1740-1745) and was derived originally from a patient's primary tumour.
• C170HM2 cells were maintained in vitro in RPMI 1640 culture medium (Gibco, Paisley, UK) containing 10% (v/v) heat inactivated foetal calf serum (Sigma, Poole, UK) at 37°C in 5% C0 2 and humidified conditions. Cells from semi-confluent monolayers were harvested with 0.025% EDTA and washed twice in the culture medium described above.
• C170HM2 cells harvested from semi-confluent cell monolayers were re-suspended at lxl0 6 /ml of sterile phosphate buffered saline, pH 7.4 [PBS] and injected in a 1 ml volume into the peritoneal cavity of 20 MF1 male nude mice (bred within the Cancer Studies Unit at the University of Nottingham). Mice were identified by an electronic tagging system (RS Biotech DL2000 Datalogger). On day 10 following cell injection, the mice were randomly assigned to alter a placebo control group or experimental groups;-
• Group 1 Compound 1 5 mg/kg 30 mg/kg
• the drugs were dosed intravenously (iv) from day 10 and continue until therapy termination. The experiment was terminated at day 40 post cell implantation. Mice were weighed at regular intervals throughout the pilot study.
• liver tumours were counted and their total cross-sectional area measured.
• the tumours were also photographed. No liquefaction of the tumours had occuned, thus they were dissected free from the normal liver tissue, weighed and fixed in formal saline. Peritoneal nodules were dissected free and the cross-sectional area and weight measured. Detailed pathological assessment of the tumours was performed.
• Cell Survival Fig. 25 shows cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or Compound 13 (•).
• Cells were treated in open plastic Petri dishes incubated in CO 2 -incubators at 37°C.
• the plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard enors are indicated by vertical bars in all cases where they exceed the size of the symbols.
• the data indicate that Compound 13 induce roughly a 10 times stronger inactivating effect than Compound 1 on a dose basis.
• Fig. 26 show cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (O) or Compound 14 (•).
• Cells were treated in open plastic Petri dishes incubated in CO 2 -incubators at 37°C.
• the plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard enors are indicated by vertical bars in all cases where they exceed the size of the symbols. The data indicate that the two compounds induce similar inactivating effect over the whole concentration range up to 12 mM.
• Fig. 27 show rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, as measured by amount of incorporated [ 3 H]-valine during a pulse period of lh starting either immediately following addition of test compound (closed symbols) or starting 2h later (open symbols).
• Test compounds, Compound 1 and Compound ' 21, were present from time zero to the end of the pulses.
• Cells were pre-labeled with [ 14 C]-valine for at least 4 days in order to have all cellular protein labeled to saturation.
• Incorporated amount of [ 3 H] was related to incorporated amount of [ 14 C] so that protein synthesis was calculated as per cent of the total amount of protein in the cells. 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 enors are indicated by vertical bans 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 while little or no effect is seen by Compound 21.
• Fig. 28 show rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, as measured by amount of incorporated [ 3 H] -valine during a pulse period of lh starting either immediately following addition of test compound (closed symbols) or starting 2h later (open symbols).
• Test compounds, Compound 2 and Compound 21 were present from time zero to the end of the pulses.
• Cells were pre-labeled with [ 14 C]-valine for at least 4 days in order to have all cellular protein labeled to saturation.
• Incorporated amount of [ H] was related to incorporated amount of [ 14 C] so that protein synthesis was calculated as per cent of the total amount of protein in the cells. 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 enors are indicated by vertical bans in all cases where they exceed the symbols. The data indicate that both Compound 2 and Compound 22 induces an effective inhibition of protein synthesis at about the same level for both compounds. Both these two deuterated compounds are more effective than the corresponding undeuterated compounds shown in Fig. 27.
• Fig. 29 show cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 1 (•) or Compound 21 (O).
• Cells were treated in open plastic Petri dishes incubated in CO 2 -incubators at 37°C.
• the plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard enors are indicated by vertical bans in all cases where they exceed the size of the symbols. From the data the dose response curves follow different shapes for the two compounds, indicating that Compound 21 is more effective than Compound 1 in inactivating cells at low compound-concentrations. The differences in curve shapes indicate different mechanisms of cell inactivation for these two drugs.
• Fig. 30 show cell survival as measured by colony-forming ability for human cervix carcinoma cells, NHIK 3025, after treatment for 20h with either Compound 2 (O) or Compound 22 (A).
• Cells were treated in open plastic Petri dishes incubated in CO 2 -incubators at 37°C.
• the plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard enors are indicated by vertical bans in all cases where they exceed the symbols.
• Compound 22 is more effective than Compound 2 in inactivating cells, particularly in the low-dose region. For example is cell survival down to 50% following treatment with 0.5 mM of Compound 22 and 4 mM of Compound 2 respectively, indicating an 8-fold higher inactivating efficiency of
• Fig. 31 show cell survival as measured by colony-forming ability for human breast carcinoma cells, T47-D, after treatment for 20h with either L-glucose (•) or Compound 21 (O).
• Cells were treated in open plastic Petri dishes incubated in CO 2 -incubators at 37°C.
• the plotted survival values represent mean values from 5 simultaneously and similarly treated dishes. Standard enors are indicated by vertical bans in all cases where they exceed the symbols.
• the data indicate that L-glucose has little or no effect on cell survival for concentrations up to at least 10 mM, the highest dose tested.
• Compound 21 also has little effect on these cells for concentrations up to 2 mM, but induces considerable inactivating effect for higher concentrations and only one of 1000 cells survive 20h in presence of 8 mM of this compound.
• mice of 6 weeks old were sensitized with 7 i.p. injections of 10 ⁇ g ovalbumin in 0,5 ml saline on alternate days.
• the mice were exposed to 8 ovalbumin ( 2 mg/ ml ) or 8 saline aerosols on consecutive days, 1 aerosol per day for 5 minutes.
• Two days prior to the first ovalbumin alt. saline injection treatment with Compound 2 was started, 5 mg per kg given daily i.p, for a period of 10 days, nine animals given ovalbumin and nine animals given saline, the control groups were given saline.
• the number of cells (Fig. 32) in saline treated or ovalbumine sensitized mice were not affected by Compound 2 treatment, and there was no difference in the count of the macrophages, the lymphocytes or the eosinophils between the two groups.
• the benzaldehyde derivatives of this invention react with certain groups on the cell surface, e.g. with free amino groups to form Schiff s bases. As many cell processes, like protein synthesis, cell cycle, 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 benzaldehyde 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 possibly also infections of other microorganisms. We have found that the hexose derivatives of benzaldehydes are surprisingly more effective than derivatives of other carbohydrates in treating cancer in certain organs like liver, kidney and lung. We believe that this phenomenon is connected with receptor affinity of these organs to the sugar moiety of the derivatives.
• 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.
• This pharmaceutical compositions may also be administered as immunopotentiators.
• 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 caniers or adjuvants.
• 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.
• the compounds of formula (I) 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.
• 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.
• 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 0.5-100 mg/kg body weight once or twice a day, and most prefened 1-20 mg/kg weight once or twice a day.
• 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 hydroxy toluene.
• an antioxidant e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxy toluene.

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  • 2000-02-18 CZ CZ20012965A patent/CZ20012965A3/cs unknown
  • 2000-02-18 RU RU2001123124/15A patent/RU2001123124A/ru unknown
  • 2000-02-18 IL IL14489500A patent/IL144895A0/xx unknown
  • 2000-02-18 PL PL00350520A patent/PL350520A1/xx not_active Application Discontinuation
  • 2000-02-18 BR BR0008302-0A patent/BR0008302A/pt not_active Application Discontinuation
  • 2000-02-18 MX MXPA01008347A patent/MXPA01008347A/es not_active Application Discontinuation
  • 2000-02-18 HU HU0105281A patent/HUP0105281A2/hu unknown
  • 2000-02-18 WO PCT/NO2000/000059 patent/WO2000048609A1/en not_active Application Discontinuation
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  • 2000-02-18 HU HU0105280A patent/HUP0105280A2/hu unknown
  • 2000-02-18 AU AU28341/00A patent/AU2834100A/en not_active Abandoned
  • 2000-02-18 BR BR0008297-0A patent/BR0008297A/pt not_active Application Discontinuation
  • 2000-02-18 CZ CZ20012966A patent/CZ20012966A3/cs unknown
  • 2000-02-18 SK SK1160-2001A patent/SK11602001A3/sk unknown
  • 2000-02-18 IL IL14489600A patent/IL144896A0/xx unknown
  • 2000-02-18 EP EP00906781A patent/EP1150689A1/en not_active Withdrawn
  • 2000-02-18 CA CA002363670A patent/CA2363670A1/en not_active Abandoned
  • 2000-02-18 SK SK1161-2001A patent/SK11612001A3/sk unknown

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