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  • C07H15/18—Acyclic radicals, substituted by carbocyclic rings

Definitions

• the present invention relates to benzaldehyde derivatives which are useful as anticancer agents. 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.
• 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 carboxy late (M. Friedman, The chemistry and biochemistry of the sulfhydryl group in amino acids, peptides and proteins, Oxford, Pergamon Press, 1973).
• 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.
• zilascorb( 2 H) 5,6-benzylidene-d ⁇ -ascorbic acid [zilascorb( 2 H)].
• the protein synthesis inhibiting activity of this prior art compound is described in detail by Pettersen et.al. (Anticancer Res., vol. 11, pp. 1077-1082, 1991) and in EP-0283139.
• Zilascorb( 2 H) induces tumour necrosis in vivo in human tumour xenografts in nude mice (Pettersen et al., Br. J. Cancer, vol. 67, pp. 650-656, 1993).
• zilascorb( 2 H) the closest prior art compound related to cancer treatment is
• 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. For instance Compound 2 (the glucose-derivative of deuterated benzaldehyde) gives an astonishingly better effect on cancer in the liver than zilascorb( 2 H) (the derivative of deuterated benzaldehyde and ascorbic acid) (see Example 6).
• the inventors of the present invention further made a study where nude mice injected with C170HM2 cells were treated with Compound 1 and Compound 2.
• Tumour line C170HM2 is a human colorectal cell line derived from a patient's primary tumour. At termination the liver was exposed, and visible liver tumours were counted and their total cross-sectional area measured. The effect of Compound 2 on the liver invasion of the human colorectal tumour, C170HM2 was far better than Compound 1 (Example 7).
• Compound 2 surprisingly gives far better therapeutic effect of cancers in the liver (primary as well as metastatic) as compared to previously known effects.
• the chemical induced carcinogenesis (like in Example 6 ) 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 (for example Compound 2 ) would make them suitable for such a treatment.
• 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
• aldehydes are intrinsically unstable due to oxidation.
• 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 compounds for prophylaxis or treatment of cancer not giving toxic side-effects.
• a third object of the invention is to provide compounds for effective and favourable prophylaxis and/or treatment of cancers in the liver (primary liver cancer as well as liver metastases from other cancers like colorectal cancer). Prophylactic treatment might also be of great importance to prevent the development of liver cancer in persons with Hepatitis B or C infection.
• 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 effective and favourable prophylaxis and/or treatment of renal cancer.
• a sixth object of the invention is to provide compounds for effective and favourable prophylaxis and/or treatment of lung cancer.
• An seventh object of the invention is to provide compounds for effective and favourable prophylaxis and/or treatment of pancreatic cancer.
• the compounds of the present invention are 4,6-O-(benzylidene-d ⁇ )-D-gluco ⁇ yranose (Compound 2), 4,6-O-benzylidene-L-glucopyranose (Compound 3) and
• Fig. 1 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. 2 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. 3 The rate of protein synthesis relative to untreated control of Pane- 1 cells treated with Compound 2 ( ⁇ ) or zilascorb( 2 H) (A). The rate of protein synthesis was measuered by amount of [ 3 H]-valine incorporated during the first hour after start of treatment. Protein synthesis rate was measured relative to the total amount of protein in the cells. Standard errors are smaller than the size of the symbols.
• Fig. 4 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. 5 Peripheral blood mononuclear cells and Superantigen in Ex Vivo 10 medium were exposed to either benzaldehyde, deuterated benzaldehyde, Compound 2 or zilascorb( 2 H). The proliferation of peripheral blood mononuclear cells was measured as incorporation of tritiated thymidine at different drug concentrations.
• Fig. 6 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 Compound 2. After treatment for 19 days, spleens were dissected out and weighted.
• Fig. 7 Fraction of animals having liver tumour tissue at the time of autopsy.
• Fig. 8 Mean amount of tumour material per liver in those animals showing a tumour take.
• Fig. 9 Growth curves representing the mean body weight per animal for each group.
• the time scale represent the age of the animals. In order to make the curves clear standard deviations are indicated only in a few of the measurements.
• Fig. 10 In this figure both frequency and size of liver tumour are plotted in the same curve, using a logarithmic scale on the size axis.
• Fig. 11 The effect of Compound 1 and 2 on the liver invasion of human colorectal tumour, C170HM2 is shown.
• Fig. 12 Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, treated with Compound 1 or Compound 3 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. 13 Rate of protein synthesis of human cervix carcinoma cells, NHIK 3025, treated with Compound 2 or Compound 4 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. 14 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 3 (O) is shown.
• Fig. 15 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 4 (A) is shown.
• Fig. 16 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 3 (O) is shown.
• Table 2 Histological observations in normal and cancer tissue. Group 1: Untreated control.
• Table 3 Histological observations in normal and cancer tissue. Group 2: Placebo.
• Table 4 Histological observations in normal and cancer tissue. Group 3: 85 mg/kg day Compound 2.
• 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.
• D(+) or L(-) glucose condensed with benzaldehyde or benzaldehyde equivalents to form the corresponding benzylidene glucose acetals is particularly preferred.
• a re-acetalisation strategy where benzaldehyde protected as its dimethyl acetal is used instead of benzaldehyde 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 product 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, complex reaction mixtures are often encountered.
• the specific reaction conditions, solvent and catalyst maybe subject to variation according to the preferences of the experimentalist.
• the catalyst may be a mineral acid, e.g. sulphuric acid, an organic acid, e.g. /? ⁇ ra-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.
• R r ⁇ -toluene sulfonic acid in dimethyl formamide constituted the preferred and most applied reaction condition.
• the deuterated compounds may be prepared as described above, but starting with the dimethyl acetal of benzaldehyde-di.
• 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.
• Benzaldehyde-di was prepared and converted to benzaldehyde dimethylacetal-di as described in EP 0 283 139 B 1.
• the preparation of 4,6-O-(benzylidene-d ⁇ )-D-glucopyranose is also described in EP 0 283 139 Bl, but the compound was this time prepared according to an alternative procedure with priority given to achieving high purity:
• 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.
• 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.
• 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 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).
• Fig. 3 From Fig. 3 it can be seen that Compound 2 induces stronger protein synthesis inhibition than zilascorb( 2 H).
• the cells used in this experiment are Panc-1 cells originating from a human pancreatic adenocarcinoma (Lieber et al., Int. J. Cancer, 15: 741-747, 1975) grown in medium E2a.
• NHIK 3025 carcinoma cells were cultured in CO 2 -independent medium containing 15% fetal calf serum. The cells were exposed to a 1 m-M 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. 4, 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 was dissolved in sterile isotonic glycerol solution in a concentration corresponding to 5 mg per kg when giving 50 microlitres intraperi tonally.
• mice were given injections intraperitonally once daily for 19 days. From June 1st till
• 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.
• Group 1 Control, no injections
• Group 2 Placebo, injections of saline only.
• Group 3 85 mg/kg Compound 2
• Treatment was given in periodic cycles of 5 weeks: first 3 weeks of daily i.v. injection, then 2 weeks pause. A total of 9 complete 5 week cycles of treatment was given. The total period from the first treatment to end of treatment and autopsy was 11 months.
• Tumour drug effects analysed by means of frequency of animals having liver tumours at the time of autopsy show that there is a significantly lower frequency of animals having liver-tumours in the Compound 2-treated group as compared to the placebo-treated group.
• the zilascorb( 2 H)-treated group 11 out of 14 animals developed liver cancer. This is only one less than in the control group, and not significant.
• the size of the liver tumours are most easily analysed from Fig. 7 and Fig. 8.
• Fig. 9 represent mean body weight measurement of each group of animals over the whole period from partial hepatectomy and nitrosamine treatment (time 0). Both the body weight measurements and the histological evaluation of normal tissues seem clear in indicating a total absence of side effects.
• the tooth-edged shape of the body weight growth curves (Fig. 9) are undoubtedly due to the treatment, but not to the drugs. Rather it is the injections itself that affect the animals since animals treated with saline only had exactly the same characteristic body weight variations as those treated with saline and drugs.
• That animals treated with 210 iv injections over an 11 months period show signs of body weight affection is not surprising: For each injection the animals were warmed up slightly under an electrical heater bulb, and were afterwards immobilised in a specially constructed holder so that injection could take place. Although this procedure took place in a quiet room and was performed by a skilled person, trained to calm down the animals, it is not surprising that it may create a biological reaction in the animals.
• Fig. 10 both frequency and size of liver tumour are plotted in the same curve, using a logarithmic scale on the size axis.
• NF Histologic examination performed, Normal findings.
• MF Multiple foci, no: means no necrosis.
• Table 3 Histological observations in normal and cancer tissue.
• Group 2 Placebo
• NF Histologic examination performed, Normal finding. MF: Multiple foci. no: means no necrosis.
• Table 4 Histological observations in normal and cancer tisses. Group 3: 85 mg/kg day Compound 2
• NF Histologic examination perfo rmed, Normal finding.
• 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.
• 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. At termination the liver was exposed, and visible liver tumours were counted and their total cross-sectional area measured. The tumours were also photographed. No liquefaction of the tumours had occurred, 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.
• Fig. 12 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 3 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.
• Fig. 13 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 4 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.
• Fig. 14 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 3 (O).
• 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 size of the symbols. From the data the dose response curves follow different shapes for the two compounds, indicating that Compound 3 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. 15 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 4 (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 errors are indicated by vertical barrs in all cases where they exceed the symbols.
• Compound 4 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 4 and 4 mM of Compound 2 respectively, indicating an 8-fold higher inactivating efficiency of Compound 4 compared to Compound 2 at this particular effect level. At a survival level of 10 % the difference is much smaller.
• Fig. 16 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 3 (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 errors are indicated by vertical barrs 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 3 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.
• compositions according to the present invention may be administered in anti-cancer treatment.
• the compounds according to the present invention may be formulated in any suitable manner for administration to a patient, either alone or in admixture with suitable pharmaceutical carriers or adjuvants.
• formulations for systemic therapy are 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 the invention with non-toxic, inert, solid or liquid carriers.
• Suitable parental preparations of the compounds according to this invention are injection or infusion solutions.
• the compounds When administered topically the compounds may be formulated as a lotion, salve, cream, gel, tincture, spray or the like containing the compounds 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 preferred 1-20 mg/kg weight once or twice a day.
• the pharmaceutical preparation of the compound can contain an antioxidant, e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.
• an antioxidant e.g. tocopherol, N-methyl-tocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

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