CZ20012965A3 - Benzaldehyde derivatives that are suitable for use as antitumor preparations - Google Patents

Abstract

The present invention relates to benzaldehyde derivatives which are useful as anticancer agents. Some of the compounds of this invention are novel per se.

Description

Technical field

The invention relates to benzaldehyde derivatives useful as antitumor agents. Some of the compounds are novel.

BACKGROUND OF THE INVENTION

Most currently used anticancer drugs have a cytotoxic effect. Although these compositions have had positive results in the treatment of some cancers such as lymphoma, leukemia and testicular cancers, they often have severe and unacceptable side effects limiting their potential for effective treatment. Furthermore, some types of cancer, such as solid tumors (carcinomas), have so far shown that chemotherapy has limited scope because current cytostatics rarely improve patient prognosis. The ability of tumor cells to develop resistance to cytotoxic agents is one of the main reasons for their failure to treat solid tumors.

Thus, there is a great need in the art for new antitumor agents having fewer side effects and having more selective effects on malignant cells.

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

The aldehydes react with various nucleophilic groups containing O, S or N, such as hydroxy, thiol and amino groups to form carbonyl condensation groups

products such as acetals, mercaptals, aminals, etc. However, they usually react with primary amines to form adducts, Schiff's bases (imines). It is notorious that the formation of Schiff's bases in vivo is involved in key biochemical processes such as transamination, decarboxylation and other amino acid-modified reactions mediated by pyridoxal phosphate, aldolase or fructosydiphosphate in glycolysis and condensation of retinal with rhodopsin in the vision process. It is also known that carbonyl condensation reactions are involved in transmembrane signaling processes, for example in the generation of an immune response.

The formation of imines takes place via a two-step mechanism: the addition of an amine nucleophile to the carbonyl group results in a carbinolamine (aminohydrin) intermediate in which in the subsequent dehydration step a C = N double bond is formed. Both steps are reversible, the reaction being supported by pH. Thus, the reaction proceeds in a manner where the pH / rate dependency has a characteristic bell-shaped profile with the highest rates of reaction in a slightly acidic environment.

// ° T ^H

R-C + H ₂ N-R .....- R-C-N-R '- R-C = N-R' + HO \ i ^{11 H} H Η Η aldehyde! amine · carbinoiamine imine ·:

However, it is also known that Schiff's bases also easily arise under physiological conditions and that many carbonyl condensation reactions take place in vivo (E. Schauenstein et al., Aldehydes in biological systems, London, Pion Ltd., 1977).

The Schiff bases are reactive in themselves, tending to participate in other reactions leading to the addition of nucleophilic double bonding agents. Of certain sulfur-containing amines, especially amino acids, cysteine, methionine, as well as glutathione, the initially formed Schiff base may undergo reversible internal cyclization in which the sulfhydryl group joins the imine to form a thiazolidine carboxylate (M. Friedman, The Chemistry and Biochemistry of Sulfhydryl Group in Amino Acids, Peptides and Proteins, Oxford, Pergamon Press, 1973).

R = C

Α / R — CH

H ₂ O aldehyde

SH cysteine

OH thiazolidine-carboxylic acid

The occurrence of reactions occurring between carbonyl compounds and the free amino groups of proteins forming the reversible bonds of Schiff's bases has been described by G.E.Means and R.E. Feeney (Chemical Modification of Proteins, pp. 125-138, San Francisco, Holden-Day, 1971). Aromatic aldehydes are generally more reactive than saturated aliphatic aldehydes, and can form Schiff bases even without removing the water formed during the reaction. (R. W. Kayer, Chem: Rev.63 (1963), 489-510). This fact is important in assessing the possibility of creating Schiff bases under physiological conditions. Zaugg et al. (J. Biol. Chem. 252 (1977), 8542-8548), using hemoglobin as the amino group source, have shown that aromatic aldehydes have two- to three-fold increased reactivity for the formation of Schiff's bases compared to aliphatic aldehydes. The explanation for the limited reactivity of alkanals lies in the fact that in aqueous solution at neutral pH

a very large excess of free aldehyde is required for Schiff base formation (E. Schauenstein et al., Aldehydes in Biological Systems, London, Pion Ltd., 1977).

Benzaldehyde and salicylaldehyde readily form imine Schiff bases with amino groups of membranes, and high equilibrium constants have been found for the benzaldehyde reaction by amines (JJešek and JHFrost, Org.Magnet.Res., 8 (1976), 173-176; JNWilliams Jr.). and RMJacobs, Biochim. Biophys.Acta, 154, (1968), 323-331).

The inventors have previously shown by radioactive labeling that benzaldehyde does not penetrate the cell but adheres to the cell membrane (Dornish J.M. and Pettersen E.O .: Cancer Letters 29 (1985) 235-243). This finding is consistent with an earlier study demonstrating that benzaldehyde interacts with E.coli membrane proteins (K. Sakaguchi et al., Agric. Bi. Chem., (1979), 43, 1775-1777). It has also been found that both pyridoxal and pyridoxal-5-phosphate protect cells against the cytotoxic and antitumor agent cis-DDP. CisDPP is an agent acting on the nucleus of a cell. While pyridoxal can basically penetrate the lipophilic cell membrane, in pyridoxal-5-phosphate, this possibility is blocked by the ionically attached phosphate group. Thus, pyridoxal-5-phosphate must exert its protective effect by acting on the cell membrane externally. The adduct formation of the Schiff base formed between the aldehyde and the amino groups of the cell membrane also corresponds to the concomitant observed spectral shift of pyridoxal-5-phosphate absorbance towards lower wavelengths (JMDornish and E.0.Pettersen, Cancer Lett., 29, (1985), 235 -243).

These findings suggest that aldehydes bind to amines and other nucleophilic groups on the cell membrane to form 9 99 99 9 19 9

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Schiff's bases and other condensation products. It is also known that stimulation of cell growth is mediated by a cascade of processes affecting the cell membrane from outside. In the same way, the derivatives of the present invention can trigger intracellular triggering of cell membrane adducts relevant to cell growth parameters such as protein synthesis and mitosis, and to the expression of tumor suppression and immune response genes. Because condensation reactions are reversible, effects on the cell can be modulated by shifting the equilibrium in relation to the binding components. The presence of this existing equilibrium at the chemical level is consistent with the reversible and non-toxic effect observed with benzaldehyde derivatives.

Inhibition of Protein Synthesis Induced by Benzaldehyde Derivatives The research group of the inventors has been extensively studying in vitro. In solid tumors, reduction of protein synthesis can result in a lack of vital proteins leading to cell death. In normal cells, the potential protein synthesis capacity is greater than in most solid tumor cells. This can be demonstrated by comparing the cell cycle time of normal stem cells, which is often below 10 hours, and is therefore shorter than most solid tumor cells where the time is typically 30-150 hours (see Gustavo and Pileri, The Cell Cycle and Cancer. Ed.: Baserga, Marcel Dekker Inc., NY, 1971, p. Since cells on average double the protein during their cell cycle, this means that the accumulation of protein is higher in growth-stimulated normal cells than in most tumor cell types.

In addition to knowing the above difference between normal and tumor cells, there is another difference of similar significance. While normal cells respond to regulatory ·

growth stimuli, the tumor cells have said response decreased or absent. Thus, while normal normal growth conditions have a reserve in growth potential, tumor cells have said reserve little or no. If inhibition of protein synthesis is applied for the same longer period to both normal cells and tumor cells, the two cell types may react differently. Normal tissue can use its growth reserves to maintain its normal cell formation. However, tumor tissue has little or no reserve. At the same time, the rate of protein accumulation in most tumor cells can be assessed as low (ie, protein synthesis is only slightly higher than degradation). Therefore, inhibition of protein synthesis may be a sufficient factor to induce an imbalance in tumor tissue with respect to protein accumulation, thereby causing a negative shift in the balance of certain proteins. Thus, in continuous treatment for several days, cell inactivation and tumor tissue necrosis can be achieved while normal tissue remains intact.

Most reviewed compound inducing reversible protein synthesis inhibition and displaying anti-tumor efficacy is currently 5,6-benzylidene-dl-ascorbic acid [zilascorb ^(2H)]. This known protein synthesis inhibiting compound is described in detail in Petterson et al., (Anticancer Res., Vol.11, p.10771082, 1991) and in EP-0283139. Zilascorb ^(2H) induces tumor necrosis in vivo in human tumor xenografts in nude mice (Pettersson et al., J. Cancer, Vol.67, str.650656, 1993). In addition to zilascorb ^(2H) is the nearest previously known compounds in the field of cancer treatment

4,6-O-benzylidene-D-glucopyranose (Compound 1). These two compounds are known to have general antitumor activity, and have already been evaluated in clinical trials for multiple cancer. However, no proposed treatment of organs or

tumor tissue suggested to be suitable using said compounds and the commercial development of these compositions have not yet been approved.

The present inventors have now surprisingly found that benzaldehyde derivatives of hexose-type sugars (including

Unexpectedly, 4,6-O- (benzylidene-di) -D-glucopyranose, compound 2), has a potent effect on tumors in certain organs or tissues. It is not yet possible to explain the mechanism of the above selectivity, but is believed to be related to the sugar moiety of said derivatives acting on certain cells or tissues. For instance Compound 2 (the glucose derivative of deuterated benzaldehyde) have a surprisingly better effect on the tumor in the liver than zilascorb ⁽² H) (a derivative of deuterated benzaldehyde and ascorbic acid) (see Example 6). Also in experiments on cells PANC1 originating from human pancreatic adenocarcinoma has surprisingly been found that Compound 2 induces stronger inhibition than prosteinu zilascorb ⁽² H) (see Exemplary unsubstituted 2, fig.3). Common to these tissues is that they contain high levels of urine glucose transporters and their receptors. In the literature no mention predicting better treatment of liver tumors or pancreatic Compound 2 as compared to zilascorb ^(2H). Conversely, based on the experiments described in EP-A-0283139 would be expected that the compound 2 should have similar or less effect than zilascorb ^(2H).

The inventors have also found in their studies that deuterated analogs of said compounds are more effective than their corresponding proton analogs. This difference is very obvious in the cell adhesion assay (see Example 3 as well as Example 7). When replacing a hydrogen atom with a doubly heavy atom with a deuterium isotope, the kinetic properties of the molecule change in a ratio in which the rate of C-D bond disruption is reduced.

to the rate of C-H bond breaking. It is known, among others, by M.I.Blake et al., J.Pharm. Sci., 64 (1975), 367-391, that deuteration of drugs can alter their pharmacological function.

It is also known in the art (EP 0 283 139 and Anticancer Res. 15: 1921-1928 (1995)) that in replacement of the acetal proton in

4,6-O-benzilidene-D-glucopyranose deuterium (Compound 1 versus Compound 2) affects both the protein synthesis and the surviving cell fraction determined in vitro. The inventors assume that one possible explanation is that the effect of D-isotope at the chemical level is related to slowing the oxidation of deuterated benzaldehyde to inactive benzoic acid, which also leads to a longer half-life of the deuterated active ingredient at the cellular level. However, to demonstrate a significant difference in effects on the surviving fraction of NHIK 3025 cells exposed to Compounds 1 and 2, respectively, the drug concentration applied must be greater than 6 mM. The difference in effects inhibiting protein synthesis is very small when cells are exposed to concentrations of 1-10 mM.

The inventors have carried out a completely different experiment. The experiment involves measuring the adhesion force between NHIK 3025 cells and the substrate after preincubation of the cells in solutions of compounds 1 and 2 (see Example 3). Even at concentrations of 1 mM, the experiment using D-isotope showed surprising results. Using compound 2 there was a significant reduction in adhesion force to 1/3 relative to the control, while the compound did not significantly reduce adhesion force. The inventors hypothesize that Compound 2 may interfere with integrin biosynthesis, reducing the ability of cells to attach to the substrate. Integrins are structural trans-membrane proteins critical for the binding of cells to the extracellular matrix and for cell-cell interactions. Inhibition of integrin function would thus ♦ 4 44 ** · · ·

443 4 4 · 4 ·

4444 444 ··· ·· 4 · Φ could indirectly affect the ability of tumor cells to metastasize. This experiment suggests that integrins could be particularly sensitive to inhibition of protein synthesis. Accordingly, Compound 2 could thus have a useful use in preventing the spread of metastases in cancer development.

The effect of Compounds 1 and 2 was compared in an in vivo model where human liver cancer colorectal tumor cell line C170HM2 was injected i.p. nude mice treated. In animals treated with compound 2, the incidence of liver tumors was surprisingly less than in animals treated with compound 1 (see Example 7).

In the experiments described above, it has been shown that the effect of Disotopes in treatment with aldehyde derivatives could lead to more effective treatment of tumors than previously known results.

From the two experiments considered together (Example 3 and Example 7), it follows that Compound 2 and similar drugs could be particularly useful for the treatment of primary and secondary liver tumors.

The use of compound 1 (4,6-O-benzylidene-D-glucopyranose) among others for the treatment of liver tumors is described in US-4,882,314. According to Experiment 2 in US 4,882,314, a colon cancer patient who metastasized to the liver was thus treated. However, in trial 5 of the aforementioned US patent, a patient with a primary liver turner died 4 months after treatment.

Later G.Tannum et al., Am.J.Clin.Oncol. (CCT), 13 (2), 1990, pp.161-163 concluded that Compound 1 was not effective for the treatment of colorectal tumor. The drug was in that study

administered once daily for 2 months and the effect was evaluated by measuring tumor size.

In an experiment performed by the inventors on chemically induced liver tumor in Wistar rats treated for 10 days by intravenous drug administration, it was surprisingly found that 2 out of 5 animals treated with compound 2 developed massive necrotic areas, whereas animals treated with compound 1 experienced this. none of the five treated animals occurred.

The present inventors conducted another study in which nude mice were injected with C170HM2 cells and treated with Compound 1 and Compound 1. The C170HM2 tumor line is a human cell line derived from the patient's primary colorectal tumor. At the end of the experiment, the liver was removed and the visible liver tumors and their total sectional area were counted. The effect of Compound 2 on invasion of human colorectal C170HM2 tumor in the liver was much better than Compound 1 (Example 7).

Furthermore, the inventors have surprisingly found that compound 2 has a significantly better effect on liver tumor development in rats than zilaskorb (H). After initial administration of nitrosamine to young rats and partial hepatoectomy, the animals develop a liver tumor within 3 to 6 months. After an additional 11 months of treatment with either zilaskorb ( ² H) or compound 2, both the number of animals with liver cancer development and liver tumor tissue size were found to be several-fold reduced in animals treated with compound 2 compared to animals treated with zilaskorb ( ² H) (see Example 6).

• ·

9 9 • 4,999,999,999,999,999

Thus, it has been found that Compound 2 provides a surprisingly better therapeutic effect on liver tumors (both primary and on liver metastases) compared to the results of previously known therapies.

In the inbred Wistar strain rats (see Eker R., Acta Path. Microbiol.Sand., 34, (1954), 554-562), renal tumors were induced by the autosomal dominant gene. At 11 months of age, the animals were operated on to determine whether they had developed a tumor (50% of the animals had a kidney tumor). Animals with a tumor diameter of 2-4 mm were included in the experiment based on the experience that there are no necrotic areas in these small tumors. These animals were injected with isotonic saline (placebo) or saline containing zilaskorb (Η), a non-deuterated zilaskorb analogue (H) (sodium salt) for 10 days.

5,6-O-benzylidene-ascorbic acid) or Compound 2. While after 10 days treatment, the tumors of animals who received undeuterated analogue of zilascorb ⁽² H) as well as placebo showed little or no necrosis, tumors of animals treated with zilascorb ⁽² H ) or Compound 2 were half necrotic (see Example 8).

Chemically induced carcinogenesis (see Example 6) has a similar mechanism to carcinogenesis induced by certain types of viruses such as hepatitis B and C viruses, certain papilloma viruses, certain herpes viruses, etc. This is particularly the case with liver cancer in patients infected with hepatitis B and C. Therefore, it is believed that prophylactic treatment of these patients with the compounds of the invention could prevent or delay the development of liver cancer. Also, the fact that said compositions have low toxicity makes them suitable for use in said treatment.

• φ · φ · ♦ · ♦ · «·« · · · · · · · φ · φ · · · · · ·

In the immunological recognition process, a foreign protein fragment is captured in a MHC class II proetin notch on the surface of an antigen presenting cell (APC). The T-helper lymphocyte receptor is also attached to this MHC antibody complex. At least two signals are required to activate T-helper lymphocyte: the primary signal is provided by the antigen itself through the MHC class II complex, and CD4 co-receptors are amplified. The second signal provides a specific plasma membrane bound signaling molecule on the surface of the APC. The ligand co-receptor protein is located on the surface of the T-helper lymphocyte. Both signals are required for T cell activation. Once activated, they stimulate intrinsic proliferation by secretion of interleukin growth factors and synthesis of ligand surface cell receptors. Binding of interleukins to these receptors then directly stimulates T-cell proliferation.

In the 1980s, it was found that an inclusion cyclodextrin-benzaldehyde inclusion complex can stimulate the immune system by enhancing lymphokine-activated killer cells in a mouse model (Y.Kuroki et al., J.Cancer

Res.Clin.Oncol. 117, (1991), 109-114). In vitro studies carried out later revealed the nature of the chemical reactions at the APC-donor / T-cell receptor interaction sites responsible for the second co-stimulatory signal, and that these reactions take the form of carbonyl amine condensations (formation of Schiff's bases). In addition, it has been found that the reactions can be imitated by synthetic chemical components. These findings open new therapeutic possibilities through artificial potentiation of the immune system. WO 94/07479 discloses the use of certain aldehydes and ketones that form Schiff's bases and hydrazones with amino groups on the surface of T-lymphocytes. In EP 0609606A1, an immunostimulatory 4 4,999,999 is preferred

9 9 99 99 99 999

9

9

9999 999 999 999 99 compound referred to 4- (2-formyl-3-hydroxyphenoxymethyl) benzoic acid (tukaresol), a compound originally designed to treat sickle cell anemia. This compound is administered orally and is systemically bioavailable. Possible therapeutic options for tukaresol that include multiple diseases with groups including bacterial, viral and protozoal infections, autoimmune-related diseases and cancer are currently under investigation (H.Chen and J.Rhodes, J.Mol.Med. (1996) 74: 497504), and the current drug development includes combination administration strategies, wherein tukaresol is co-administered with a vaccine for the treatment of chronic hepatitis B, HIV and malignant melanoma.

It has also been determined by determining in vitro immunologically significant values that the dose / response curve is bell-shaped (H. Chen and J.Rhodes, J. Mol.Med. (1996), 74: 497504). This otherwise unusual dose / response course can be explained by the assumption that high concentrations of the aldehyde drug saturate the co-stimulatory ligands required for effective APC binding to the T cell, and therefore act inhibiting. The optimal dose appears to be sufficient to achieve dynamic equilibrium to achieve co-stimulation without blocking intercellular ligation.

In general, aldehydes are themselves unstable due to oxidation. The 4- (2-formyl-3-hydroxyphenoxymethyl) benzoic acid (tukaresol) described in EP-0609606 is significantly more stable in vivo than in vitro. The reason for this may be the 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 administer them as such, and although they have a proven in vitro effect as antitumor drugs, they are irritating to direct application in vivo and are unsuitable for such administration. In the biotic system, the carbonyl group of the aldehyde reacts rapidly with nucleophilic groups that are primarily present in all body fluids. These undesirable side reactions can lead to rapid drug metabolism and difficult control of serum levels of the active ingredient. Control of drug concentration at the cellular level within a narrow range is critical to achieving effective immunopotentiation. Tucaresol is administered orally as unprotected aldehyde, and there is a suspicion that drug breakdown and problems in controlling its pharmacokinetics occur.

Benzaldehyde derivatives of 4,6-benzylidene-D-glucose and their deuterated analogues (compounds 1 and 2) have been shown to have high bioavailability either at i.v. administration or p.O. administration. The bioavailability determined by drug concentration in the serum following oral administration of compound 2 to BALB mice was found to be 93-99%. (CBDunsaed, JMDornish and E.0.Pettersen, Cancer Chemother.Pharmacol. (1995), 35: 464470. In addition, the glucose moiety can exhibit affinity for receptors present on the cell surface and thereby improve drug availability at the cellular level. Free aldehyde it can be easily released by hydrolysis of acetal, thereby releasing the carbonyl group, which becomes available for the formation of Schiff's bases on targeted ligands.

According to the present patent application, aldehydes are derivatized with biologically acceptable carbohydrates such as glucose, galactose and others to form acetals. The sugar component contributes to improved stability and also increases the bioavailability of the aldehyde functionality for the target cells. Surprisingly, the efficacy of condensation reactions increases and their pharmacokinetics are more easily controlled in comparison with the compounds known to date.

To compare compound 2 with tukaresol in terms of their effects on cell inactivation and protein synthesis, the two effects were determined in the presence of the same concentrations of both drugs. As shown in Figures 1 and 2, Compound 2 was shown to be more potent than tukaresol in both endpoints.

The immunostimulatory effect of the compounds of the invention can also be used to treat certain viral diseases in combination with other antiviral therapies such as using antivirals or vaccines. Many types of viruses are incorporated into the cell nucleus after the first infection and remain inactive for a long time. Oncogenic viruses such as hepatitis B and C viruses, certain retroviruses and certain papilloma viruses can induce the development of cancer. In these latent periods, it is very difficult to treat viral infection. As a consequence of immune responses, said viruses are often released to form viremia, and at this stage they can be removed from the body. The ability of the benzaldehyde derivatives to elicit an immune response can be used to develop treatments for said diseases in combination with antivirals or vaccines.

The main object of the invention is to provide novel compounds for the prevention and / or treatment of cancer.

Another object of the invention is to provide compounds suitable for the prevention or treatment of cancer without toxic side effects.

A third object of the invention is to provide compounds for the effective and beneficial prevention and / or treatment of liver tumors (primary liver tumors or metastasis of other tumors such as colorectal tumor). Preventive treatment may also have 9 9 99

9 9 · · 9 99 • 9 9999 999 999 * · 999 of great importance for preventing liver tumor development in patients infected with hepatitis B or C.

A fourth object of the invention is to provide compounds for the effective and beneficial prevention and / or treatment of tumors of tissues and cells containing receptors with an affinity for the corresponding sugar components.

A fifth object of the invention is to provide compounds for the effective and beneficial prevention and / or treatment of renal tumor.

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

Said achieved and other achieved objects of the invention are the subject matter of the appended claims.

SUMMARY OF THE INVENTION

The invention relates to the following compounds:

4,6- (O) - (benzylidene-di) -D-glucopyranose (compound 2),

4,6- (O) - (benzylidene) -L-glucopyranose (compound 3),

4.6- (O) - (Benzylidene-di) -L-glucopyranose (Compound 4), or a pharmaceutically acceptable salt thereof.

Compounds 3 and 4 are novel compounds.

changed sheet

DETAILED DESCRIPTION OF THE INVENTION

The invention is further elucidated by the following examples and tables and attached figures.

Description of the figures in the attached drawings

Figure 1 shows the results of an experiment in which NHIK 3025 cells were treated with compound 2 (Δ) or tukaresol (·) for 20 hours at 37 ° C to bind them to plastic petri dishes. Surviving fraction means the fraction of cells forming a macroscopic colony after treatment. Each point represents the mean number of colonies from 5 parallel treated plates. Standard errors are smaller than the size of the symbols used.

Figure 2 shows the results of protein synthesis rate determination in an experiment in which NHIK 3025 cells were treated with compound 2 () or tukaresol (*) for 1 hour at 37 ° C relative to untreated control cells. Protein synthesis rate was determined by determining the amount of [ ³ H] -valine incorporated during the first hour of drug treatment. Protein synthesis rate was determined relative to the total protein content of the cells. The results represent a four-fold run of one experiment. Standard errors are recorded if they exceed the size of the symbols used.

Figure 3 shows the results of protein synthesis rate relative to control in an experiment in which Panc-1 cells were treated with compound 2 () or zilaskorb ( ² H) Protein synthesis rate was determined by determining the amount of [ ³ H] -valine incorporated during first hours of drug treatment. Protein synthesis rate was determined relative to the total protein content of the cells. Standard errors are smaller than the size of the symbols used.

Figure 4 shows the median adhesion forces of cells exposed to various benzaldehyde derivatives. Cells were exposed to 1 mM concentrations of Compounds 1 and 2.

Fig.5 Peripheral blood mononuclear cells and Superantigen in Ex Vivo 10 was subjected to exposure with either benzaldehyde deuterovanému benzaldehyde, Compound 2 or zilascorb ^(2H). Proliferation of peripheral mononuclear blood cells was determined by incorporation of tritiated thymidine at various drug concentrations.

6 NMRI mice were infected i.p. administration of Frienderytroleukemia virus attacking the spleen. Infected and uninfected mice were treated i.p. with a daily dose of 5 mg / kg of Compound 2. After treatment for 19 days, the spleens were removed and weighed.

Figure 7 shows the proportion of animals with tumor tissue detected at necropsy.

Figure 8 shows the mean amount of tumor tissue in the liver in tumor-bearing animals.

Fig. 9 shows growth curves based on body weight calculated per animal in each group. For better representation of the curves, standard deviations are shown in only a few cases.

·· 9 • 9 « • • • • » ·· ·· • · • · • • • • • • • • • * • • • · • • • • © • • • • ···· ·· »· ··· ··

Figure 10 shows both liver frequency and size on the same curve using a logarithmic scale on the x-axis.

Figure 11 shows the effect of Compounds 1 and 2 on invasion of human colorectal C170HM2 tumor in the liver.

Fig.12. Protein synthesis rate in human cervical carcinoma NHIK 3025 cells treated with Compound 1 or Compound 3 was determined by determining the amount of [ ³ H] valine incorporated either within a period of one hour after immediate administration of the test compound (dark symbols) or within one hour at time from 2 hours later (bright symbols).

Fig.13. Protein synthesis rate in human cervical carcinoma NHIK 3025 cells treated with Compound 2 or Compound 4 was determined by determining the amount of [ ³ H] valine incorporated either within a period of one hour from the immediate administration of the test compound (dark symbols) or within one hour at time from 2 hours later (bright symbols).

Figure 14 shows the survival of human cervical carcinoma NHIK 3025 cells, determined by colony forming ability, after treatment for 20 hours with either Compound 1 (·) or Compound 3 (0).

Figure 15 shows the survival of human cervical carcinoma NHIK 3025 cells, determined by colony forming ability, after treatment for 20 hours with either Compound 2 (0) or Compound 4 (*).

• · · ·

Figure 16 shows cell survival as determined by the ability of T47-D human lung carcinoma cells to form colonies after treatment for 20 hours with either L-glucose (·) or compound 3 (0).

Description to tables

Table 2: Histological results found in normal and tumor tissue. Group 1: untreated control group.

Table 3: Histological results found in normal and tumor tissue. Group 2: placebo

Table 4: Histological results found in normal and tumor tissue. Group 3: 85 mg / kg / day of compound 2

compound C. chemical structure name 1 H OH OH 4,6-O-benzylidene- -D-glucopyranose 2 D OH OH 4,6-O- (benzylidene-dl) - -D-glucopyranose

• ·

compound C. chemical structure name 3 H ^Ho 4,6-O-benzylidene- -L-glucopyranose 4 D HO 4,6-O- (benzylidene-di) - -L-glucopyranose

Preparation

It is known that aldehydes react with alcohols in condensation reactions supported by the presence of acid to form acetals. At the same time, water is formed as a by-product. This reaction is reversible and an equilibrium mixture of aldehyde / alcohol and acetal / water is formed in the solution. The equilibrium state is determined primarily by the reactivity and concentration of each of the reactants. To move the reaction towards its complete course, one of the reaction products (acetal or water) is usually removed from the reaction mixture.

According to the invention, D (+) or (L) -) glucose is condensed with benzaldehyde or a benzaldehyde equivalent compound to form the corresponding glucose benzylidene acetals. Particularly preferred is a reacetalization strategy wherein instead of benzaldehyde itself, benzaldehyde protected in the form of dimethyl acetal is used. Methanol is formed as the co-product of the reaction. Once methanol is formed, the reaction mixture is warmed slightly under reduced pressure to remove methanol. In most cases, the equilibrium shifted continuously in favor of acetal under the reaction conditions.

Acetalisation of sugars usually results in a mixture of regio- and steroisomers. Also, contraction ring transformations can occur resulting in the formation of a mixture of pyranos and furanos and in some cases the formation of diacetal adducts. As a result, if a protection strategy is not used, a very complex reaction mixture is formed. However, the processing described below, in particular using liquid chromatography, gave surprisingly pure proportions of pure products, as was the case for large-scale preparation using crystallization methods. The identity of the products is performed by GC-MC spectroscopy and various NMR methods.

The specific reaction conditions used, the solvent and the catalyst may vary according to the person skilled in the art. As the catalyst, a mineral acid such as sulfuric acid, an organic acid such as paratoluenesulfonic acid, an acidic ion exchange resin such as Amberlyst 15, a Lewis acid in the form of a mineral clay such as Montmorillonite K-10 or a superacid on a carrier resin such as Nafion NR 50. The reaction may conveniently be carried out in a dipolar aprotic solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, dimethoxyethane or the like. The most preferred and most commonly applied reaction conditions include embodiments with para-toluenesulfonic acid in dimethylformamide.

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The deuterated compounds may be prepared as described above, starting from benzaldehyde-di-dimethylacetal starting material. The preparation of deuterobenzaldehyde can be carried out by modified Rosenmund reduction using D ₂ gas in deuterated solvent as described in EP 0 283 139 B1.

Possible preparations of the compounds of the invention are illustrated by the examples below.

DETAILED DESCRIPTION OF THE INVENTION

Compound 1

4.6-O-benzylidene-D-glucopyranose

Said compound known in the art is prepared as described for compound 2 using undeuterated benzaldehyde dimethylacetal as starting material. Identity was confirmed by ^X H NMR spectroscopy in DMSO-d₆:

δ relative to TMS: 7.58-7.29 (5H, m, Ar-H), 6.83 (0.4H, d, ΟΗ-1β), 6.60 (0.6H, d, OH-1) -α), 5.61 (1H, s + s, acetal-H), 5.25 (0.4H, d, OH-3-β), 5.21 (0.4H, d, OH-2- β), 5.62 (0.6H, d, OH-3a), 5.00 (0.6H, Hla), 4.82 (0.6H, d, OH-2-ot), 4.49 ( 0.4H, t, H-1-β), 4.18-4.02 (1H, m, Η-β'-α + β), 3.89-3.77 (0.6H, m, H -5a), 3.75-3.57 (1,6H, m, Η-6-α + β, and H-3-a), 3.45-3.27 (2.5H, m, H- 3-β, H-4-α + β, H-5-β and H-2-a) and 3.11-3.00 (0.4H, m, H-2-β).

Compound 2

4.6- O- (benzylidene-di) -D-glucopyranose

• · · · · · · · · · · ·

According to the process described in EP 0 283 139 B1 benzaldehyde-di is prepared and converted to benzaldehyde dimethylacetal-di. The preparation of 4,6-O- (benzylidene-di) -D-glucopyranose is also described in EP 0 283 139 B1 but for preferential reasons to achieve high purity, in this case it is prepared by the alternative method described below.

D (+) glucose (706 g, 3.92 mol), benzaldehyde dimethylacetal-di (571 g, 3.73 mol), dry DMF (1) were mixed in a dry distillation apparatus connected via a reflux condenser to a vacuum pump. , 68 kg) and para-toluenesulfonic acid (4.5 g, 24 mmol). The mechanically stirred mixture was heated to a maximum of 69 ° C (30 Torr) to distill off methanol, and after 2 hours 235 g was removed. The reflux condenser was then turned off and the temperature raised to about 73 ° C to distill DMF. After a further 2 hours a further 1385 g was collected and then distillation was discontinued.

The residue was cooled to about 40 ° C and ice / water (2.9 L) was added over 5 minutes. Then the temperature is slowly lowered to below 0 ° C whereupon a precipitate precipitates partially in large clusters. The mixture was then transferred to a beaker and an additional 8-9 L of ice / water was added to disintegrate and form a slurry. Then, the suspension is filtered on two suction filters and the resulting two filter cakes are left overnight on the filters under suction with a water pump, purging with N ₂ atmosphere where nitrogen is introduced through an inverted funnel. The filter cakes were then spread on two plates and dried in a vacuum oven for 20 hours at 32 ° C. The vacuum is first set to 1300 Pa (13 mbar) and then increased to 100 Pa (1 mbar).

The crude product is recrystallized (to remove dibenzylidene acetals) and washed with water (to remove DMF and glucose) until the contaminants are removed. The crude product (500 g) was then dissolved in hot dioxane (800 mL) and the resulting solution was added through a pleated filter to boiling chloroform (9 L). The solution was then allowed to cool to room temperature first and then cooled in an ice bath overnight. The precipitate was filtered off, dried on the filter for 2 hours (with N 2 washing as described above) and further dried at 31 ° C under vacuum on a rotary evaporator. The product (142 g) was then suspended in ice / water (1 L), filtered on the filters by suction (washing with 200 ml of ice / water) and dried overnight on the filter as described above. The product is then milled, sieved (mesh size 0.5 mm) and dried on a rotary evaporator at 31 ° C for 5 hours. Then, the product is suspended again in ice / water (500 ml), filtered (washed with 150 ml ice / water) and dried (7 hours with N2 wash). Finally, grind in a mortar, sieve (0, 5 mm) and dried in a vacuum oven.

The product was a white, finely divided powder of high purity and analyzed by HPLC. Yield: 95%, 10% of theory. The ratio of anomers to β detected by DMSOd ₆ is about 7: 3.

¹ H- and ¹³ C-NMR (DMSO-d ₆ ) δ relative to TMS: 7.55-7.28 (5.00H, m, Ar-H), 6.85 (0.27H, d, OH- 1-β), 6.58 (0.71H, d, OH-1-α), 5.24 (0.27H, d, OH-3-β), 5.19 (0.28, d, OH -2-β), 5.61 (0.71H, d, OH-3-α), 4.99 (0.72H, H-1-α), 4.82 (0.71H, d, 0H- 2a), 4.48 (0.29H, t, H-1-β), 4.20-4.04 (1.04H, m, Η-6'-α + β), 3.88-3, 73 (0.78H, m, H-5-a), 3.73-3.56 (1.72H, m, Η-6-α + β and H3-α), 3.46-3.21 ( 2.61H, m, H-3-b, H-4-α + β, H-5-β and H-2-α) and 3.09-2.99 (0.28H, m, H-2 -β); 137.881, 128.854, 128.042, 126.435 (Ar-C), 100.462 (acetal-C), 97.642 (C-1-α), 93.211 (C-1-α), • ·

81, 729 (C-4-alpha), 80,897 (C-4-beta), 75, 796 (C-2-beta), 72,906 (C-2-alpha)

C-3-β), 69.701 (C-3-α), 68.431 (C-6-α), 68.055 (C-6-β), 65.810 (C-5-β) and 62.032 (C-5-α) ).

Compound 3

4,6-O-benzylidene-L-glucopyranose

L (-) - glucose (5.0 g, 27.8 mmol), benzaldehyde dimethylacetal (4.66 g, 30.6 mmol) and paratoluenesulfonic acid (32 mg, 0.17 mmol) were mixed in the distillation apparatus in dry DMF (20 mL). A water vapor pump for short-path distillation is then connected and methanol and DMF are removed by vacuum distillation. Within 1/2 hour at 55 ° C, the colorless suspension dissolves and the resulting solution is stirred for 1/2 hour at 120 mbar while gradually raising the temperature to 65 ° C. The vacuum was then increased to maximum and the reaction mixture evaporated for a further 45 minutes. At the end of the distillation, the reaction temperature was raised to 75 ° C. The residue is a slightly yellowish syrup which is neutralized by the addition of NaHCO ₃ (29 mg) and allowed to cool.

The crude product was dissolved in methanol (10 mL) and purified by RP-8 reverse phase chromatography using 1: 1 methanol / water. The fractions containing the product were combined and evaporated to remove methanol. The remaining solution was further diluted with water and freeze-dried. Three separate experiments yielded a white flocculent solid in a total amount of 2.42 g corresponding to 32.5% of the theoretical yield.

The results of GC chromatography of silylated samples show that the product contains two isomers (aa and β anomer) in a ratio of 35/65. ¹ H NMR shifts in DMSO-d ₆ are similar to 4,6-O-benzylidene D-glucopyranose shifts: 7.51-7.30 (5H, m, Ar-H), 6.86 (0.6H, broad s,

• 4 · 4 · 4 · 4 e 4 «

0Η-1β), 6.58 (0.3Η, wide s, 0Η-1-α), 5.58 (0.9Η, s + s, acetalΗ-α + β), 5.23 (0.7Η, d, OH-3-β), 5.20 (0.6Η, d, OH-2-β), 5.11 (0.4Η, d, 0Η-3-α), 5.00 (0.4Η , H-1-α), 4.82 (0.3Η, d, 0Η-2-α), 4.47 (0.7Η, d, H-1-β), 4.21-4.08 ( HH, m, Η-6'-α + β), 3.87-3.73 (0.4Η, m, H-5-α), 3.73-3.59 (1.3Η, m, Η) -6-α + β and H-3-α), 3,463.22 (3.7Η, m, H-3-β, H-4-α + β, H-5-β and H-2-α) and 3.09-2.99 (0.6Η, m, H-2-β).

Compound 4

4,6-O- (benzylidene-dx) -L-glucopyranose

L-glucose (5.14 g, 28.6 mmol) was heated in DMF (20 mL) at 95 ° C until a clear solution was formed. The flask with the reaction mixture was then placed in a water bath at 65 ° C and paratoluenesulfonic acid (33 mg, 0.17 mmol) was added. Benzylidene-dimethylacetal-di (4.7 mL, 31 mmol) was then added dropwise over 20 minutes to a stirred solution of glucose under 80 mbar using a water pump. The DMF was then removed in vacuo (200 Pa (2 mbar)) at 65 ° C to give a very pale yellow oil, to which was added NaHCO 3 (345 mg) and stirred for 5 minutes. To the oil at 65 ° C was added hot water (67 ° C, 15 ml) with stirring (magnetic bead) and the contents of the flask were shaken in a water bath until the oil was clearly dissolved. The flask with the reaction mixture was then placed in a stream of cold water for about 5 minutes. After about one or two minutes, an amorphous solid is formed. The aqueous mixture was then placed in an ice-water bath and left for 40 minutes. The precipitate was collected by vacuum filtration (after decantation of the amorphous material), washed with cold water (25 mL) and then with cold isopropanol (5 ° C, 2 x 5 mL) and dried under a stream of nitrogen to give 1.85 g of dry white powder. . The silylated product is then analyzed by gas chromatography to give 99% purity of the desired product.

¹ H NMR δ (DMSO-d ₆ ) δ relative to TMS: 7.55-7.25 (5H, m,

Ar-H), 6.85 (0.48H, β-1β), 6.55 (s, 0.33H, OH-1-α), 5.25 (d, 0.48H, OH-3-β) ), 5.20 (d, 0.49H, d, OH-2-β), 5.10 (d, 0.35, OH3-α), 4.98 (d, 0.35H, H-1-) α), 4.82 (d, 0.34H, OH-2-α), 4.48 (d, 0.51H, H-1-β), 4.20-4.05 (m + m, 0) 53H + 0.42H, Η-6'-α + β), 3.85-3.73 (m, 0.44H, H-5-α), 3.72-3.57 (m, 1, 27H, m, Η-6-α + β and H-3-α), 3.45-3.20 (m, 7.8H, H-3-β, H-4-α + β, H-5 -β and H-2-α) and 3.10-2.98 (m, 0.56H, H-2-β).

Biological examples

Example 1

Biological materials and methods used to demonstrate effects

Cell culture techniques

Human cells, NHIK 3025, originating from cervical cancer in situ (Nordbye, K., and Oftebro, R., Exp. Cell Res. 58: 458, 1969; Oftebro, R., and Nordbye, K-. Exp. Cell Res. 58: 459-460, 1969) were cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 15% fetal calf serum (Gibco BRL, Ltd.). Human breast cancer cells, T-47D (Keydar, I. et al., Eur. J. Cancer, Volume 15, pp. 659-670, 1979) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 0, 2 U / ml insulin, 292 mg / ml L-glutamine, 50 U / ml penicillin, 50 mg / ml streptomycin. Cells were cultured in a conventional manner as monolayers at 37 ° C in tissue culture tubes. For

• » 9 9 * • 99 • 9 • * • · • 9 • · 9 9 • • 9 9 9 4 • • • 9 9 • * «· • 4 ·· 9 9 9 • 99 • 9 β ·

keeping the cells in continuous exponential growth, the cells were treated with trypsin and recultivated three times a week.

Cell survival

Cell survival was measured by colony-forming ability. Prior to seeding, exponentially growing cells were trypsinized, suspended as single cells, and seeded directly onto 5 cm plastic discs. The number of inoculated cells was selected so that the number of viable cells was approximately 150 per disc. After incubation for 2 hours at 37 ° C, the cells were bound to the bottom of the disks. Thereafter, drug administration was initiated by replacing the medium with a medium containing the desired drug concentration. After drug administration, cells were washed once with warm (37 ° C) Hank balanced salt solution and then fresh medium was added. After 10-12 days at 37 ° C in a CO ₂ incubator, cells were fixed with ethanol and stained with methylene blue, and then colonies were counted.

Giant. 1 shows that compound 2 induces greater cell inactivation than tukaresol.

Example 2

Protein synthesis

Protein synthesis rate was calculated as previously described (Ronning, OW et al., J. Cell. Physiol. 107: 47-57, 1981). Briefly, the cellular protein was saturated with a labeling reagent during at least two days preincubation with [ ¹⁴ C] valine of constant specific radioactivity (0.5 Ci / mol). High concentrations of valine (1.0 mM) were used in the medium to keep the specific radioactivity constant. At this valine concentration, dilution of [ ¹⁴ C] valine with intracellular valine and proteolytically produced valine is negligible (Ronning, O. et al., Exp. Cell Res. 123: 63-72, 1979). Protein synthesis rate is calculated from incorporation of [ ³ H] valine relative to total [ ¹⁴⁰ C] radioactivity in the protein at the beginning of the respective measurement and expressed as a percentage per hour (Ronning, OW et al., J. Cell. Physiol. 107: 47 57, 1981).

Figure 2 shows that Compound 2 induces better inhibition of protein synthesis than tukaresol.

From Fig. 3 it can be seen that Compound 2 induces stronger protein synthesis inhibition than zilascorb ^(2H). The cells used in this experiment are Panc-1 cells derived from human pancreatic adenocarcinoma (Lieber et al., Int. J. Cancer, 15: 741-747, 1975) grown in E2a medium.

Example 3

Measurement of cell adhesion

Cell adhesion forces were measured using a handling force microscope (G. Sagvolden, Manipulation force microscope, Ph.D. thesis, University of Oslo, 1998, and G. Sagvolden, I. Giaver and J. Feder, Characteristic protein adhesion forces on glass and polystyrene substrates by atomic force microscopes, Langmuir 14 (21): 5984-5987, 1998). Briefly, NHIK 3025 carcinoma cells were cultured in a CO 2 -independent medium containing 15% fetal calf serum. These cells were exposed to a 1 mM concentration of Compound 1 or Compound 2 for 20 hours before being released from the cell culture tubes by trypsin. The cells were kept in suspension and placed in medium with compound 1 or compound 2 on the polystyrene tissue culture substrates 90 minutes after the trypsin reaction was complete. The adhesion forces between the cells and the substrate were measured by removing the cells using an inclined microscope console for measuring the force acting as a force sensor. One cell was removed at a time and each cell was removed only once.

The maximum force found for each cell was recorded as a function of time since the cells were placed on the substrate. The median force for group 19 of measurements is shown in Figure 4 as a function of the mean time for cells exposed to compound 1 or compound 2, together with the adhesion forces of cells not exposed to these compounds. Compound 2 has significant effects in reducing the adhesion force at this concentration, while Compound 1 has no significant effects. The effect of the compound is mainly to reduce the adhesion strength of the cells, not during the adhesion.

The decreased ability to bind to the substrate may be related to blocking integrin-mediated cell anchoring. It has been shown that such blocking can induce programmed cell death in hepatoma and melanoma cells (Paulsen, PE, Hall, KS, Rugstad, HE, Reichelt, KL, and Elgjo, K.). 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. Royal Soc., Med 89 (1992), 393-395).

The adhesion force between NHIK 3025 cells and the substrate was measured after pre-incubation of the cells in the solution of compounds 1 and 2.

3/9 9 9 9 9 9 ·

A surprising effect of the D-isotope was observed at a concentration of 1 mM. Surprisingly, Compound 2 significantly reduced the adhesion force to 1/3 compared to control, while Compound 1 did not lead to a significant decrease. The inventors believe that compound 2 may interfere with integrin biosynthesis, which reduces the ability of cells to bind to the substrate. Integrins are structural transmembrane proteins essential for the binding of cells to the extracellular matrix and for intercellular interactions. Thus, inhibition of integrin function may directly affect the ability of metastasis of tumor cells. The experiment has shown that integrins may be particularly sensitive to inhibition of protein synthesis. Compound 2 can be used to prevent metastasis in tumor development.

Example 4

Compound 2 experiments on NMRI mice infected with FRIEND erythroleukemia virus (FLV)

Virus: Eveline cells were obtained from prof- Gerharh Hunsman, Munich. The inventors have shown that this virus, which was originally used as a source of Friend helper virus, contains a defective virus of the same size as Spleen Focis Forming Virus (SFFV), which induces erythroleukemia in NMRI mice after 48 weeks.

Mice: NMRI mice were obtained from Old Bomholt Farm, Denmark, and purchased through SIFF. Mice were obtained 6.5. and 11.5. Mice were infected intraperitoneally with 50 μΐ of supernatant from Eveline culture. Treatment was started after 24 hours. Compound 2 and compound 5 were dissolved in a sterile isotonic glycerol solution in · ♦

• 9 ···· 9

99

9 ·

Concentration equivalent to 5 mg per kg for intraperitoneal administration of 50 μΐ.

The experiment was performed as follows:

mice - uninfected mouse control - infected mouse control - uninfected, treated with compound 2 mice - infected, treated with compound 2

Mice were injected intraperitoneally once daily for 19 days. Od 1.6. no treatment was given until June 16, when the mice were sacrificed. 16.6. mice were sacrificed. Blood was collected (for subsequent analysis). Spleens were removed and weighed (see Table 3). Part of the spleen was frozen with nitrogen to prepare thin sections and part was fixed with formalin.

Table 1

Not infected Infected Compound 2, Compound 2, control control uninfected infected 125 154 266 151 160 240 143 153 94 214 106 168 146 212 153 145 118 165 117 149 120 171 157 127 115 190 63,284 131 103 204 170 130 203 127 147 148 148 125.8 190.1 157 146, 9 20.5 24.5 63 15,228

• ·

The results are also shown in Figure 6.

As can be seen, there is a significant difference in spleen weight in infected animals compared to uninfected controls. The weights in uninfected animals treated with Compound 2 are higher than the weights of uninfected controls, although the difference is not statistically significant. It can be said that the infected animals treated with compound 2 had indeed a lower average spleen weight compared to the uninfected animals treated similarly (here it is assumed that the result stems from one animal in the control group that had a significantly large spleen ).

Histological examination showed that uninfected controls had normal spleen anatomy. All animals in the infected untreated group had invasion of pathological leukemia cells into the red marrow. Spleens from uninfected animals treated with Compound 2 had hypertrophic germinal centers that are interpreted as a result of immunostimulation. No leukemia changes in spleens from infected animals treated with compound 2 could be found.

These results are promising considering the aggressive nature of FLV in mice and also when compared to the effects of azidothymidine and other antiviral treatments.

Example 5

Proliferation of peripheral blood mononuclear cells

The inventors conducted an experiment in which peripheral blood mononuclear cells were exposed to superantigen together with benzaldehyde, deuterated benzaldehyde, compound 2 or zilaskorb ( ^2). H). Superantigen has been used as a very active standard for T-cell proliferation and is presented to T-cells by antigen-presenting cells.

The experiment demonstrated (see Figure 5) that the addition of benzaldehyde, deuterated benzaldehyde or compound 2 statistically significantly increased peripheral blood mononuclear cell proliferation in a dose-dependent manner with a bell-shaped curve, while very little effect was observed for zilaskorb ( ² H). The fact that we were able to amplify the proliferation signal from the superantigen suggests that the compounds have an additional costimulatory effect on T cells.

Example 6

Anti-tumor effects in nitrosamine-induced liver tumors in rats

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

Materials and methods

Wistar Kyoto rats from the Versuchtierzucht Institute, Hannover were used. Partial hepatectomy (removal of 70%) was performed at Radium Hospital in rats 4 weeks prior to intraperitoneal administration of diethylnitrosamine (DENA) and 4 weeks of phenobarbital administration. Liver cancer occurred in most animals within 10 months after this initiation of carcinogenesis. Six and a half months after initiation of carcinogenesis, ie before onset

Treatment of any tumor was initiated at the National Institute of Occupational Health.

animals were randomized into 4 groups of 14 animals each and received the following doses of i.v .:

Group 1: control, no injections

Group 2: placebo, saline only injection Group 3: 85 mg / kg of compound 2

Group 4: 100 mg / kg zilaskorb ( ² H)

Treatment was administered in 5-week cycles: first 3 weeks daily i.v. injection, then a 2-week pause. A total of 9 complete 5-week cycles were applied. The total time from the first treatment to the end of treatment and autopsy was 11 months.

At necropsy, an approximate evaluation of liver tumor volume was performed, but without thorough liver incision. After fixation in formalin, Professor Jahn M. Nesland, Head of Pathology at Norwegian Radium Hospital, first performed an accurate evaluation of the tumor mass by incision and macroscopic evaluation of the tissue, followed by histological examination of both tumor tissue (which was present mainly in the liver) and normal tissue from all relevant organs and tissues.

Multiple foci as well as pre-malignant nodules were examined histologically for each animal.

Results

The antitumor effect of the drug was analyzed by frequency of animals with liver tumors at autopsy and it was found that a statistically significantly lower number of animals with liver tumors were found in Compound 2 group compared to the placebo group. Analysis by a one-sided Fischer exact test gave a p value of 0.05. When the two control groups were pooled together, 25 animals out of 28 developed liver tumors, whereas in Compound 2, 7 animals out of 14 developed liver tumors. In this case, the number of animals is quite high for the chi-square test, which shows that the difference is statistically significant at p - 0.015. In the group treated with zilascorb ⁽² H), the liver tumors developed in 11 of 14 animals. This is only one animal less than in the control group and the difference is not statistically significant.

Liver tumor size is best analyzed in Figures 7 and 8. Compound 2 shows a convincing effect: while 50% of the animals in the placebo group had liver tumors greater than 10 cm ³ , no animal in the compound 2 group it did not have such large tumors (chi-square test: p = 0.0038). Furthermore, while only 3 animals (ie 21%) in the Compound 2 group had tumors larger than 1 cm ³ , 10 animals (ie 71%) in the placebo group had tumors greater than this threshold (Fisher's exact test: p = 0.0002). Thus, the anti-tumor effect for Compound 2 is even more pronounced when considering the size of the tumors together with the frequency of tumor development.

Giant. 9 shows the mean body weight measurements for each group of animals over the entire period from partial hepatectomy and nitrosamine treatment (time 0). Both body weight measurements and histological examinations of normal tissues clearly indicate the complete absence of side effects. The fluctuation in body weight curves (Figure 9) is undoubtedly due to treatment, but not to medication. The animals seem to affect the injections themselves, as saline-treated animals have essentially the same saline solution and medication-treated animals' body weight variation. .

That animals treated with 210 i.v. injections over 11 months, have a change in body weight, not surprising. At each injection, the animals were warmed slightly under electric heating and then immobilized in a specially prepared fixation device for injection. Although this procedure was carried out in a quiet room and by an experienced worker trained to calm the animals, it is not surprising that it can induce a biological response in the animals.

One aspect of the side effect test is histological examination of normal tissues from various organs of the body. This extensive study is summarized in Tables 2 to 4 (attached). In no case were any abnormalities that could be associated with drug treatment detected. It should be noted that the tails of rats, in the region where they were i.v. injections given daily for a long time were not damaged by treatment.

An interesting conclusion can be drawn from the frequencies of preneoplastic lesions in the liver. According to Tables 2 to 4, all animals developed multiple foci. These lesions are considered to be an early stage in the process of liver malignancy and do not appear to be affected by treatment. Furthermore, the development of preneoplastic nodules, although limited to several animals, is present in all groups, therefore the data does not indicate any effect of drugs on these lesions, further reinforcing the view that compound 2 has only tumor-specific effects in the liver.

In Fig. 10, liver tumor frequencies and sizes are plotted on the same curve, using a logarithmic scale on the x-axis.

«4 ·· • · · · • • · · · · · · · · • · · 9 · ··· < •

·· · • • • · • • • • · • • • • • • ·· ·

Frequency of liver tumors. The number of animals in the control and placebo groups developing liver tumors was 13 and 12, respectively, out of 14 animals in each group (Tables 2 and 3). In Group 3 (85 mg / kg Compound 2), only 7 animals out of 14 developed liver tumors (Table 4). When testing the difference between Group 2.

(placebo) and 3 cases are too small for the chi-square test. However, a one-sided Fisher's exact test can be performed and shows that the two groups are statistically significantly different (p = 0.05) in terms of the frequency of liver tumors. However, this difference is even more statistically significant if we also included untreated control animals (group 1) in the control group. In this case, there are 28 control animals, 25 of whom have developed liver tumors, and the chi-square test is acceptable. In this case, the difference between group 3 and controls is statistically significant with p = 0.015.

In group 4 (100 mg / kg zilascorb ^(2H)), 11 of 14 animals developed liver cancer. This is only one animal less than in the placebo group and the difference is not statistically significant. Frequency analysis of liver tumor formation must be concluded that the treatment with Compound 2 significantly reduced the formation of liver tumors, while zilascorb ⁽² H) has no such effect.

Size of liver tumors. The size of liver tumors is best shown in Figure 10 and also shown in Tables 2 to 4. Here Compound 2 has a convincing effect: while 50% of animals in the placebo and control groups had liver tumors greater than 10 cm ³ , no animal did not have such large tumors in the Compound 2 treatment group (chi-square test: p = 0.0038). Furthermore, while only 3 animals (ie 21%) in the Compound 2 treatment group had tumors larger than 1

cm ³ , 10 and 13 animals (ie 71% and 93%) in the placebo and control groups, respectively, had tumors greater than this threshold (Fisher's exact test: p = 0.0002 and p = 0.011, v respectively).

For compound 2, the antitumor effect is even more pronounced when considering the size of the tumors together with the frequency of tumor development. In fact, it is so strong that at the time of autopsy it was difficult to detect tumors by macroscopic examination of the liver.

For animals treated with zilascorb ⁽² H), the effect is much weaker than for animals treated with Compound 2. even when considered in tumor size. The number of animals treated with zilaskorb ( ² H) having a liver tumor greater than 1 cm ³ is 7. The significance test against the control and placebo groups as performed for Compound 2 treated animals showed p values of 0.036 and 0.44, respectively. . Thus, the difference is statistically significant compared to the untreated control, whereas it is not statistically significant compared to the placebo group. Considering that 3 of the animals in the placebo group had tumors of less than 1 cm ^3, and if the comparison were made for 0.5 cm ³ , there would be a statistically significant difference also for the placebo group (see Figure 10). . However, the present analysis shows that the effect of zilaskorb ( ² H) is weak and probably at the borderline of statistical significance. Furthermore, for animals having tumors of 3-5 cm ³ is no difference between the group treated with zilascorb ^(2H) and placebo or control group.

In a separate experiment in which the zilascorb ^(2H) and the compound 2 is administered to rats for only 10 days, histological examination of tissue showed no difference in the zilascorb-treated group, whereas in 2 of 5 animals treated with compound 2 has been observed in tumor necrosis.

Table 2: Results of histological examination of normal and tumor tissue: Group 1 - untreated control group

animal C. tissue outside the liver liver pituitary hart, aorta, thymus lung, thyro-, pancreas testes, prostate žalu- dek, columns spleen, bone marrow tail, paw ledv. L / R necrosis -za * preneopl. nodule lo- žiska tumor necrosis 3 0 / + 0 MF No 11 NF NF NF NF NF 0 MF (+) 22nd NF NF NF NF NF NF NF 0 MF + 24 NF NF NF NF NF NF NF Yes MF + 25 NF NF NF NF NF NF NF Yes MF No 27 Mar: NF NF NF NF 0 MF + C + 31 NF NF NF NF 0 MF + 33 NF NF NF NF NF NF 0 MF + 35 NF NF NF kare. ++ 0 MF + 38 NF NF NF NF NF NF NF Yes MF tumor 51 NF NF NF NF NF 0 MF + 54 NF NF NF NF NF kare. No 0 MF No 56 NF NF NF NF NF 0 MF + 60 TUne NF NF NF 0 MF +

Abbreviations: NF - result of histology, normal finding,

MF - multiple lesion no - no signs of necrosis

Table 3: Results of histological examination of normal and tumor tissue: Group 2 - placebo

animal C. tissue outside the liver liver pituitary hart, aorta, thymus lungs, thyro-, pancreas testes, prostate žalu- dek, columns spleen, bone marrow tail, paw ledv. L / R necrosis -za * preneopl. nodule lo- žiska tumor necrosis 4 NF NF NF 0 MF tumor 18 kare. NF NF NF kare. 0 MF No 19 Dec adeno NF NF NF NF NF NF 0 MF No 23 NF NF NF NF NF NF Yes MF + 26 NF NF NF NF NF 0 MF + 34 NF NF NF NF NF NF 0 MF No 39 NF NF NF NF NF 0 MF + 44 NF NF NF NF NF NF NF 0 MF tumor 45 0 MF + 46 NF? NF NF NF NF NF 0 MF No 50 NF NF NF NF 0 MF No 52 NF NF NF NF NF NF NF 0 MF + 55 NF NF NF NF NF NF NF 0 MF + 59 NF NF NF MF No

Abbreviations: NF - result of histology, normal finding,

MF - multiple lesion no - no signs of necrosis

Table 4: Results of histological examination of normal and tumor tissue: group 3-85 mg / kg / day of compound 2

animal C. tissue outside the liver liver hypophysis hart, aorta, thymus lungs, thyro-, pancreas testes, prostate žalu- dek, columns spleen, bone marrow tail, paw ledv. L / R nekró -for* preneopl. nodule bearings tumor necrosis 5 NF NF NF NF NF NF NF 0 MF + 7 NF NF NF NF NF NF NF 0 MF (+) 13 NF NF NF NF NF Yes MF tumor 14 NF NF Yes MF tumor * 15 NF NF NF NF NF NF Yes MF + 17 NF NF NF NF NF NF Yes MF tumor * 20 NF NF NF NF NF NF kare. + 0 DFF tumor 29 OLIGL NF 0 MF tumor 30 NF GRANU NF NF 0 MF + 32 NF NF NF NF NF NF 0 MF + 37 NF NF NF NF NF NF 0 MF tumor 42 tumor NF NF NF NF NF NF 0 MF + + 49 tumor 53 NF NF NF NF NF NF NF 0 MF +

Abbreviations: NF - result of histology, normal finding,

MF - multiple bearing

DFF - diffusion bearing change

OLIGL - Oligodendroglioma

GRANU - Granulomas * Animal # 15 had squamous cell carcinoma of the trachea. He was cystic and did not necrotize outside cystic to some extent

• (·) · (·) area (+). Animal # 20 had cystic degenerative changes in the liver.

Example 7

Effect on colorectal liver cancer metastases in nude mice

Material and procedures

The cell line used, C170HM2, is a known human colorectal carcinoma line (SA Watson et al., Eur. J. Cancer 29A (1993)), 1740-1745) and originates originally from the patient's primary tumor. C170HM2 cells were cultured in vitro in RPMI 1640 culture medium (Gibco, Paisley, UK) containing 10% (v / v) heat inactivated fetal calf serum (Sigma, Poole, UK) at 37 ° C in 5% CO ₂ and humid atmosphere. Cells from semi-confluent monolayers were obtained with 0.025% EDTA and were washed twice in the culture medium described above.

C170HM2 cells obtained from semi-confluent monolayers were resuspended at 1x10 ⁶ / ml in sterile phosphate buffered saline, pH 7.4 (PBS) and injected in a 1 ml volume into the peritoneal cavity of 20 MFI male nude mice (bred in Cancer Studies) Unit at the University of Nottingham). Mice were identified by an electronic system (RS Biotech DL2000 Datalogger). On day 10 after cell injection, mice were randomized into control groups (placebo, n = 2 mice) and experimental groups (n = β / group):

Group 1: Compound 1 5 mg / kg (n = 2 mice) mg / kg (n = 2 mice) 90 mg / kg (n = 2 mice)

Group 2:

Compound 2

mg / kg (n = 2 mice) mg / kg (n = 2 mice) mg / kg n = 2 mice)

Drugs were administered intravenously (i.v.) from day 10 to discontinuation of therapy. The experiment was terminated at day 40 after cell implantation. Mice were weighed at regular intervals during the pilot study.

At the end of the test, liver was harvested and visible liver tumors were counted and sectional areas were measured. Tumors were also photographed. No tumor liquefaction was observed and therefore all tumors were excised from normal liver tissue, weighed and fixed in formaldehyde saline. Peritoneal nodes were removed and their incision area and weight were measured. A detailed pathological evaluation of the tumors was performed.

The effect of compounds 1 and 2 on hepatic invasion of human colorectal C170HM2 tumor is shown in Figure 11.

Example 8

Necrotizing effect on rat primary kidney adenoma cells

In a non-hereditary rat renal adenoma experiment (Eker and Mossige, Nature 189 (1961), 858-859), extensive tumor necrosis was found after 10 days i.v. injection of 85 mg / kg body weight of compound 2 in two animals. Little or no necrosis was observed in animals treated with saline.

Example 9

Biological effects of compounds 3 and 4 compared to compounds 1, 2 and L-glucose

Protein synthesis

Giant. 12 shows the rate of protein synthesis in human cervical cancer cells, NHIK 3025, as measured by the amount of [ ³ H] -valine incorporated during a 1-hour pulse period that begins immediately after addition of test compound (solid symbols) or 2 hours later ( blank symbols). Test compounds, Compound 1 and Compound 3, were present from time 0 to the end of the pulses. Cells were pre-labeled with [ ¹⁴ C] -valine for at least 4 days to saturate all cellular proteins with a labeling reagent. The amount of [ ³ H] incorporated was relative to the amount of [ ¹⁴ C] incorporated so that protein synthesis was calculated as a percentage of the total amount of protein in the cells. Protein synthesis rate is reported as a percentage of rate relative to untreated control. The protein synthesis graph represents the mean of 4 simultaneous and similarly treated wells. Standard deviations are indicated by vertical bars in all cases where they exceed the symbol size. The data show that Compound 1 induces an inhibition of protein synthesis that increases linearly with increasing drug concentration, while for Compound 3 little or no effect was observed.

Giant. 13 shows protein synthesis rate in human cervical carcinoma cells, NHIK 3025, as measured by the amount of [ ³ H] -valine incorporated during a 1 hour pulse period that begins immediately after addition of the test sample.

• compounds (filled symbols) or 2 hours later (empty symbols). Test compounds, compound 2 and compound 4, were present from time 0 to the end of the pulses. Cells were pre-labeled with [ ¹⁴ C] -valine for at least 4 days in order to saturate all cellular proteins with a labeling reagent. The amount of [ ³ H] incorporated was relative to the amount of [ ¹⁴ C] incorporated so that protein synthesis was calculated as a percentage of the total amount of protein in the cells. Protein synthesis rate is reported as a percentage of rate relative to untreated control. The protein synthesis graph represents the mean of 4 simultaneous and similarly treated wells. Standard deviations are indicated by vertical bars in all cases where they exceed the symbol size. Data show that compound 2 and compound 4 induce effective inhibition of protein synthesis of approximately the same level for both compounds. Both of these deuterated compounds are more potent than the respective non-deuterated compounds as shown in Figure 12.

Cell survival

Giant. 14 shows cell survival, as measured by the ability of cells to form colonies, for human cervical cancer cells, NHIK 3025, after 20 hours of treatment with Compound 1 (·) or Compound 3 (o). Cells were treated in open plastic Petri dishes incubated in CO ₂ incubators at 37 ° C. The survival value graph represents the average of 5 simultaneous and similarly treated plates. Standard deviations are indicated by vertical bars in all cases where they exceed the symbol size. Dose-response curves having different shapes for the two compounds show that compound 3 is more potent than compound 1 in cell inactivation at low compound concentrations. The difference in the shape of curves • «♦ · · · · ^w • ···· ··· ··· indicates a different mechanism of these two compounds in cell inactivation.

Giant. 15 shows cell survival, as measured by colony forming capacity, for human cervical cancer cells, NHIK 3025, after 20 hours of treatment with compound 2 (o) or compound 4 (Δ). Cells were treated in open plastic Petri dishes incubated in CC> 2 incubators at 37 ° C. The survival value graph represents the average of 5 simultaneous and similarly treated plates. Standard deviations are indicated by vertical bars in all cases where they exceed the symbol size. Compound 4 is more potent than compound 2 in cell inactivation, especially at low doses. For example, cell survival is reduced to 50% after treatment with 0.5 mM Compound 4 and 4 mM Compound 2, indicating an 8-fold higher inactivation potency of Compound 4 compared to Compound 2 at this level of effect. With 10% survival, this difference is much smaller.

Giant. 16 shows cell survival, as measured by colony forming capacity, for human breast cancer cells, T47-D, after 20 hours of treatment with L-glucose (·) or compound 3 (o). Cells were treated in open plastic Petri dishes incubated in CO ₂ incubators at 37 ° C. The survival value graph represents the average of 5 simultaneous and similarly treated plates. Standard deviations are indicated by vertical bars in all cases where they exceed the symbol size. The data show that L-glucose has little or no effect on survival at concentrations up to 10 mM, the highest dose tested. Compound 3 also has a weak effect on these cells at concentrations up to 2 mM, but induces a significant inactivation effect at higher concentrations, and only 1 in 1000 cells survives for 20 hours in the presence of 8 mM of this compound.

• · · · ·

Conclusion

Both tested L-glucopyranose derivatives (compounds 3 and 4) inactivate cells with higher potency than the respective D-glucopyranose derivatives (compounds 1 and 2). L-glucose alone, however, does not induce any statistically significant cell inactivation at the concentrations used herein. This results in a better effect of L-glucose for benzylidene derivatives compared to D-glucose.

Submission

The pharmaceutical compositions of the invention may be administered for the treatment of tumors.

For the above purpose, the compounds of the invention may be administered to a patient in any suitable form, either alone or in admixture with pharmaceutically acceptable carriers or adjuvants.

Especially suitable are systemic therapy formulations, either in the form of preparations for oral administration or for parenteral administration.

Suitable formulations for enteral administration include tablets, capsules, for example, soft or hard gelatin capsules, granules, granular powders or powders, syrups, suspensions, solutions or suppositories. Said dosage forms are prepared by methods known in the art by mixing one or more compounds of the invention with one or more non-toxic, inert, solid or liquid carriers.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Suitable formulations for parenteral administration containing the compounds of the invention include solutions for injection or infusion.

For topical administration, the compounds of formula (I) may be administered in the form of a lotion, ointment, cream, gel, elixir, spray or the like, wherein said preparations contain the compounds of formula (I) in admixture with nontoxic, inert, solid or liquid carriers. commonly used for topical preparations. Particularly suitable is the use of a formulation in which the active ingredient is protected from air, water and the like.

Said compositions may contain inert or pharmacodynamically active ingredients. For example, tablets or granules may contain various binders, fillers, carriers and / or diluents. The liquid preparations may, for example, be in the form of a sterile solution. The capsules may contain, in addition to the active ingredient, a filler or a thickening agent. Additionally, flavor enhancers and additives commonly used as preservatives, stabilizers, moisture scavengers and emulsifiers, salts for affecting the osmotic pressure, buffers and other additives can be used.

Dosages of the preparation to be administered may vary depending on the indication, the mode of use and the route of administration, as well as the requirements of the patient. The usual daily dose for systemic therapy of an average adult patient is about 0.01500 mg / kg body weight administered once or twice daily, preferably about 0.5-100 mg / kg body weight administered once or twice daily, and most preferably is about 1-20 mg / kg body weight, administered once or twice daily.

If desired, the medicament containing the compound of formula (I) may contain an antioxidant, for example tocopherol, N-methyl-tocopheramine, butylhydroxyanisole, ascorbic acid or butylhydroxytoluene.

Claims (6)

7Ψ Loúdmi PATENT CLAIMS Use of 4,6-O- (benzylidene-di) -D-glucopyranose, 4,6-O-benzylidene-L-glucopyranose, and / or 4,6-O- (benzylidene-di) -L-glucopyranose, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the prevention and / or treatment of liver tumor, renal tumor and pancreatic tumor. Use according to claim 1 for the preparation of a medicament for the preventive treatment of tumors induced by viruses such as hepatitis B and C viruses, oncogenic papilloma viruses and other oncogenic viruses. A benzaldehyde derivative suitable as a medicament, wherein said benzaldehyde derivative is 4,6-O- (benzylidene) -L-glucopyranose and / or 4,6-O- (benzylidene-di) -L-glucopyranose, or a pharmaceutically acceptable salt thereof. salt. A pharmaceutical composition comprising a benzaldehyde derivative according to any one of the preceding claims and a pharmaceutically acceptable carrier, diluent and / or excipient. A process for preparing a pharmaceutical composition comprising the step of combining a benzaldehyde derivative according to any preceding claim with a pharmaceutically acceptable carrier, diluent and / or excipient. changed sheet A benzaldehyde derivative which is 4,6-O-benzylidene-L-glucopyranose, 4,6-O- (benzylidene-di) -L-glucopyranose, or a pharmaceutically acceptable salt thereof.